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hemifacial spasm is a disorder of the seventh cranial nerve , which is characterized by irregular , involuntary and recurrent tonic and clonic contractions of the ipsilateral facial expression muscles . this disorder affects mainly adults , and there are few cases reported in childhood . the main etiologies are vascular problems , although tumors are an important cause of hemifacial spasm via a direct or an indirect mass effect . we report a 6-year - old girl who presented with right hemifacial spasm . magnetic resonance imaging showed a lesion in the cerebellopontine angle , extending from the midbrain to the pons with a slight mass effect on the fourth ventricle . the histological examination revealed a pilocytic astrocytoma .

hemifacial spasm is a disorder of the seventh cranial nerve , and is characterized by irregular , involuntary and recurrent tonic and clonic contractions of the ipsilateral facial expression muscles . each spontaneous motor paroxysm starts with eye twitching and progresses to involve the other muscles innervated by the facial nerve . the episode characteristically begins with a series of twitches that increase in frequency and intensity , followed by a sustained spasm . the disorder is typical during adulthood and is commonly attributed to vascular compression of the facial nerve emergence at the brain stem . altogether , posterior fossa or cerebellopontine angle tumors are rare causes of hemifacial spasm , reported in less than 1% of a large series of adult patients . in children , the association of hemifacial spasm with tumors is also rare , with very few cases reported . a 6-year - old girl without any significant past medical history was admitted with intermittent involuntary twitching of the right orbicular region ; the twitching had occurred for 18 months . over time , the spasms became more tonic , and after 4 months , the spasms also involved the right orbicularis oris area . she was noted to be in good overall condition , showing a normal physical examination without fever or any other signs of infection . her height was 133 cm , and she weighed 39 kg , with a body mass index of 22.4 . the neurological examination was noteworthy for dysarthria and right hemifacial spasm [ figure 1 ] . sustained right hemifacial spasm laboratory evaluations , including a complete blood cell count , electrolytes , blood urea nitrogen and liver enzymes were within the normal range . cranial magnetic resonance imaging ( mri ) revealed a right - sided heterogeneous lesion from the midbrain to the pons , extending to the cerebellopontine angle , with a slight mass effect on the fourth ventricle [ figure 2 ] . after the neuroimaging procedures , a cerebral biopsy was performed , which revealed a pilocytic astrocytoma . the hospital ethic commission approved this case report , and the patient 's parents gave informed consent for publication . ( a ) t2-weighted coronal and ( b ) flair axial mri images showing a right - sided heterogeneous high signal lesion from the midbrain to the pons , extending to the cerebellopontine angle , with a slight mass effect on the fourth ventricle . ( c ) t1-weighted aspect of the lesion , with heterogeneous low signal and ( d ) t1-weighted post - gadolinium sagittal image showing its enhanced pattern these causes include venous sinus thrombosis , masses of the fourth ventricle , pilocytic astrocytoma , congenital or acquired cholesteatoma , tuberculous meningitis , thickening of the arachnoid membrane , neurosurgical facial nerve injury , accommodative esotropia and mandibular prognathism . masses may directly impinge on the facial nerve depending on the size and location of the tumor . some studies described cases in which the facial nerve was compressed against bony structures or tethered to arachnoid adhesions . this case shows the importance of a thorough neurological evaluation because our patient presented with subtle clinical signs of hemifacial spasm as the initial manifestation of a pilocytic astrocytoma . children who present with hemifacial spasm , even if very mild , should undergo further investigation .

ischemic and hemorrhagic stroke are recognized complications of varicella zoster virus ( vzv ) infections , although uncommon and poorly documented . the authors report the case of a 31-year - old woman admitted with acute ischemic stroke of the right posterior cerebral artery and a history of a thoracic rash 1 month before . aspirin and simvastatin were prescribed , but the patient suffered a stepwise deterioration the following days , with new areas of infarction on brain imaging . despite no evidence of cardiac or large vessel embolic sources , anticoagulation was started empirically 6 days after stroke onset . one week later , symptomatic hemorrhagic transformation occurred . the diagnosis of vzv vasculopathy was then considered , and treatment with acyclovir and prednisolone was started with no further vascular events . cerebrospinal fluid analysis and digital subtraction angiography findings corroborated the diagnosis . the patient was discharged to the rehabilitation center with a modified rankin scale ( mrs ) score of 4 . on the 6-month follow - up , she presented only a slight disability ( mrs score 2 ) . in conclusion , vzv vasculopathy needs to be considered in young adults with stroke . a high index of suspicion and early treatment seem to be important to minimize morbidity and mortality . anticoagulation should probably be avoided in stroke associated with vzv vasculopathy .

varicella zoster virus ( vzv ) is a neurotropic dna alpha herpesvirus that infects more than 95% of the world population . usually , varicella results from primary infection . the virus then becomes latent in ganglion neurons along the entire neuroaxis , and after reactivation , vzv moves transaxonally to the skin resulting in zoster . less often , vzv reactivation leads to neurologic complications more common in immunocompromised patients , such as vzv vasculopathy . vasculitis has , however , been reported in patients with dermatomal herpes zoster infection without trigeminal nerve involvement . the average time from rash to neurologic symptoms and signs is 4.1 months , but sporadically the patient can present with rash and stroke simultaneously . nonetheless , approximately one third of patients have no history of rash . in recent years , the number of recognized vzv vasculopathies has grown , but the diagnosis is not easy because , frequently , clinical presentation and imaging features are not specific of this entity . antiviral and antithrombotic treatment should probably be considered in stroke associated with vzv vasculopathy , but data on which to base therapeutic decisions are lacking . a 31-year - old right - handed female was admitted to the emergency department because of headache associated with visual disturbance . she had no previous history of headache and no known vascular risk factors but she used an oral contraceptive pill . on admission , she reported a 9-hour progressive pulsatile headache , with nausea and photophobia , followed by a sudden left visual field defect . neurological examination a head computed tomography ( ct ) scan was judged to be normal ( fig . the next day , the patient was observed at an outpatient consultation and subsequently admitted in the neurology ward because the headache had worsened and she felt weakness and numbness of the left limbs . she also recalled a history of thoracic rash 1 month before and she reported having had varicella at the age of 7 years . neurological examination at that time revealed slight drowsiness , mild left hemiparesis , left hemihypesthesia and homolateral homonymous hemianopia . a new ct scan showed an ischemic infarct on the territory of the right posterior cerebral artery ( fig . two days after admission , the neurological picture worsened with acute onset of left central facial palsy , visual and tactile inattention and worsening of the left motor deficit . a new ct scan showed enlargement of the ischemic lesion with involvement of the right thalamus ( fig . 1c ) and new areas of infarction involving different arterial territories , further depicted on mri ( fig . mr angiography showed an occlusion of the right posterior cerebral artery 5 mm after its origin ( fig . 2c ) . transthoracic and transesophageal echocardiography and holter ecg were within the norms , as were the of laboratory blood tests ( including hiv antibodies ) . clinical deterioration continued with further motor compromise , and although no cardiac or large vessel embolic source had been documented , facing neurological worsening , acetylsalicylic acid was substituted by low molecular weight heparin ( 60 mg subcutaneously twice a day ) 6 days after admission . the patient remained stable for a week , but then a new episode occurred , with headache and slight drowsiness associated with hemorrhagic transformation involving the thalamus but sparing the cortical territory of the posterior cerebral artery ( fig . because of the medical history of recent vzv reactivation , it was then hypothesized that the stroke could be secondary to vzv vasculopathy . however , at that time , evidence of increased intracranial pressure contraindicated the lumbar puncture . acyclovir and prednisolone were started empirically ( acyclovir 750 mg three times daily , and prednisolone 60 mg once a day for 21 days ) , with slow but steady improvement . digital subtraction angiography ( dsa ) showed narrowing at the origin of the right posterior cerebral artery and demonstrated an irregular lumen of the posterior segment of the pericallosal artery with beading - like pattern appearance ( fig . she underwent a lumbar puncture 11 days after initiation of therapy ( 27 days after admission ) . cerebrospinal fluid ( csf ) protein content was slightly increased ( 48 mg / dl ) , glucose 59 mg / dl , and a discrete mononuclear pleocytosis was detected ( 6 cells ) . vzv - dna was not detected by polymerase chain reaction , but there was an increased csf / serum ratio of vzv igg ( 3.3 ) , confirming intrathecal production of anti - vzv antibodies . motor function improved , but the patient was discharged with persistent left homonymous hemianopia and spatial inattention , unable to walk without assistance [ modified rankin scale ( mrs ) score 4 ] . after antiviral therapy , she restarted acetylsalicylic acid 150 mg once a day . on the 6-month follow - up , only a mild to moderate paresis of the left limbs was present . the patient was able to walk unassisted and look after her own affairs ( mrs score 2 ) . recognition of vzv vasculopathy was not straightforward because this is an uncommon entity and strokes of other etiologies may present with a similar neurologic picture and imaging abnormalities . on the other hand , finally , vzv - induced central nervous system ( cns ) disease seems to be rare in immunocompetent patients . despite no previous history of headache and sudden presentation of the visual deficit , migrainous infarction could be considered in our patient on account of presentation with pulsatile progressive headache in a young female and the known predilection of this complication for the posterior circulation . cryptogenic stroke , which may be responsible for as much as 50% of strokes in young adults [ 6 , 7 ] , was also considered in the absence of findings that supported cardiac embolism , atherosclerosis , arterial dissection , inflammatory or drug - induced etiologies . although there was no evidence to support the decision , anticoagulation was considered because of the stepwise deterioration of the neurological status under aspirin , with new ischemic areas in different territories , and a mr angiography not indicative of vasculitis . looking backwards , anticoagulation may not have been the most suitable therapeutic choice and should probably be avoided with in - patients with stroke and recent vzv infection , as hemorrhagic transformation occurred probably in the context of vessel wall damage but may also have been triggered by anticoagulation . the biological mechanisms are probably multifactorial : inflammation associated with systemic infection may lead to endothelial dysfunction ; in addition , the vzv virus spreads along nerve fibers and directly involves the vessels . vasculitis has been described in patients with dermatomal herpes zoster infection without trigeminal nerve involvement , as in our case . the vasculopathy typically involves the anterior circulation , but vzv vasculitis involvement of the vertebrobasilar system has also been reported . these studies suggest that vzv may enter the cns not only from trigeminal ganglia but also from other pathways . as suggested in previous reports , this case highlights the usefulness of dsa and csf / serum ratio of vzv igg for the diagnosis of vzv vasculopathy . vzv dna was not present in csf , but several studies with serial analysis revealed its presence only during the first 2 weeks of disease , while anti - vzv igg antibodies become detectable during the second week after infection . the diagnostic value of detecting anti - vzv igg antibody in csf is greater than that of detecting vzv dna . the multifocal nature of vzv vasculopathy makes biopsy a test with low sensitivity and high morbidity . when the diagnosis of vzv vasculopathy was considered , a lumbar puncture was contraindicated due to increased intracranial pressure ; nevertheless , we began treatment with intravenous acyclovir and steroids . although there are no controlled trials to access the optimal treatment schemes , we continued treatment during 21 days . duration of antiviral treatment as well as acyclovir dosage and benefit of steroids are issues that deserve further research . in conclusion , our case underlines that vasculopathy and stroke related to vzv infection should be borne in mind when dealing with stroke in young adults . in fact , available data suggest that this complication of a frequent infection may be more common than usually reported , not only after herpes zoster or varicella but also in cases of a stroke of unknown origin [ 1 , 2 ] . dsa seems to be important to the diagnosis of vzv vasculopathy , which is confirmed by intrathecal synthesis of anti - vzv igg antibody . the authors declare that there are no conflicts of interest and that no funding was received for this report .

backgrounddue , in part , to family constraints in dealing with the economical burden of raising a family , a wave of street children is sweeping the developing world . such children are prone to both somatic and mental illnesses . this is the first ever study that has been conducted to explore the psychopathology among street children in the duhok governorate.methodsthe study was conducted between march 2004 and may 2005 in duhok city among street children who attended the zewa center the only center for street children in the region at the time of the study . among a total of 107 eligible children , 100 agreed to participate ( 93% response rate ) . a modified family map ( genogram ) was used to obtain demographic data from the children and their caregivers through semi - structured interviews . in addition , the mini international neuropsychiatric interview for children and adolescents ( mini - kid ) structured interviews were conducted with the children.resultsthe study found that 98% of children worked on the street because of the economic need and pressure on their families . there was high rate of parental illiteracy ( 90% of fathers and 95% of mothers ) , and 61% of respondents were shown to have at least one psychiatric disorder . a high percentage ( 57% ) of these children suffered from anxiety disorders including posttraumatic stress disorders ( 29% ) . ten percent had depression , and 5% had attention deficit hyperactivity disorder.conclusionstreet children in duhok seem to be working children due to their families needs .

this group of children has been identified as a highly vulnerable group that needs special attention and care . it has been reported that the number of street children range from 10 million to 100 million ; the majority of them are living in major urban areas in developing countries.1 in amman , jordan , it is estimated that 3% of children aged 1018 years were working , which is the highest percentage noted in the capital.4 according to the palestinian central bureau of statistics , 3.5% of palestinian children were working in 2001.5 more than one factor plays a role in driving children onto the streets . these include dropping out of school,2 family economic problems,3 child abuse and neglect,68 urbanization,7 broken families,9,10 peer pressure,11 as well as the role of media in promoting this phenomenon.7 the world health organization has identified that street children are prone to developing malnutrition disorders,12 sexually transmitted diseases,1 dental problems,13 psychiatric problems,14 cognitive and learning problems , unplanned pregnancies,1 as well as being prone to being trafficked.1 due to its longstanding situations of war and economic sanctions , this phenomenon is not unknown in iraq . however , there is no clear estimate of the size of the problem in iraq . since formal education is compulsory for the age group ( 612 years ) , the only clue to the magnitude of this problem is the school drop - out rate , which was found to be 1.9 million out of 4.3 million primary school - aged children.6,15 there is no evidence , neither in duhok nor in the kurdistan region , on the demographic characteristics or the psychopathology of street children in this country . this study aims to explore the demographic data and psychiatric morbidity among street children in duhok . a cross - sectional study was conducted from march 2004 to may 2005 at the zewa center for street children ( duhok , iraq)the only drop - in daycare center for street and working children in the governorate of duhok , which has about 1,300,000 inhabitants ; 60% are under 18 years of age . at the time of the study , the illiteracy rate among men was 11.6% , and among women it was 26.4%.16 almost all of the children found on the street in duhok were registered with the police directorate and social affairs directorate , and they were referred to this center for follow - up and support . this registration by these directorates only served an administrative purpose to enlist those children who worked on the street . the zewa center aimed at involving these children in educational programs , while providing social and psychological support . the focus of the center was to encourage children to attend the center on a voluntary basis . the activities in the center included drama , creative art , and music sessions that complemented the school curriculum . the center also provided medical care and follow - up services , as well as family visits . a total of 107 of children were eligible to participate ; all were included in the study except for seven children ( 6.5% ) who refused to complete the diagnostic interviews . the study was ethically evaluated by the ethical committee at duhok university ( duhok , iraq ) . a letter was sent by a social worker to the parents for consent before the child was included . the study instruments were administered by a trained social worker . a modified family map ( genogram)1719 was used to obtain the demographic and background information of the children , such as their age , gender , education level , work type , number of working hours , reasons for working ( they were asked whether family need / pressure or peer pressure were among the reasons that drove them to work on the street ) , number of years on the street , socioeconomic status , number of family members ( all members of family living in the same house were counted ) , and history of disabilities and medicosurgical problems . parental information regarding their own education ( never attended school or illiterate , or completed primary school , secondary / high school , or a university degree ) , work type , illness history , parent s death and reason of death , and child s age when the parent died was also collected . a composite score was used to determine the socioeconomic status ; this score was based on : 1 ) whether the father is employed or not ; 2 ) the house is owned , belongs to a relative , or is being rented ; 3 ) monthly income is below or above average , or no income ; 4 ) number of family members working in an income - generating activity ; and 5 ) other sources of income . every item mentioned above had a maximum score of 2 and a minimum score of 0 . a total score between 04 was considered low , 57 was average , and 810 was considered good . trained social workers were involved in the study , and they used the genogram , while also collecting information about each child s background characteristics . the children were given time to recall and answer questions ; if they were not able to answer , one of the parents or siblings was approached . children were not forced to answer questions , particularly about the death of their parents . the structured international neuropsychiatric interview for children and adolescent ( mini - kid ) tool was used to identify childhood psychiatric disorders.20 for every diagnosis assessed in the instrument , there is a key question that indicates the exclusion of the diagnosis if the child answers negatively . the mini - kid tool showed high interrater and test retest reliability , and it was good at screening for all psychiatric disorders except for dysthymia in children and adolescents aged 617 years.21 the harvard uppsala trauma questionnaire for children ( hutq - c)19 was administered to children to measure the exposure of traumatic events and their severity . children were asked to recall traumatic events , regardless of whether they were experienced , witnessed , or heard of . one score was given for every reported event , regardless of whether the child was exposed to , witnessed , or heard about the event . if the same type of event occurred more than three times during the same year , it was given a maximum score of 3 . the trauma level was determined by the sum of scores experienced , witnessed , and heard of . children were identified as being mildly traumatized ( scores 14 ) , moderately traumatized ( scores 59 ) , or severely traumatized ( scores > 9 ) . children who were found to have mental health problems were referred by the researcher to the mental health center in duhok for further management . the statistical package for the social sciences ( spss for windows , version 10.0 ; ibm corporation , armonk , ny , usa ) was used for the analyses . the study found that all of the children belonged to the children on the street category . the background characteristics of the studied street children revealed that most of them had parents who were illiterate ( 90% of fathers and 95% of mothers ) . fourteen percent of children had at least one parent that died , and 1% reported that both parents had died . ninety - eight percent reported that they worked because there was family need and/or pressure . the phenomenon was also common in children of low socioeconomic status ( 84% ) , and when there was a higher number of siblings . two - thirds of the children reported selling a variety of items ( table 1 ) . the mini - kid interviews indicated that 61% of the children had at least one psychiatric disorder ; 16% fulfilled the criteria for two or three diagnoses , and 8% for more than three diagnoses . fifty - seven percent of the children suffered from anxiety disorders , 29% fulfilled the criteria of posttraumatic stress disorder ( ptsd ) , and 10% for depression , 7% for conduct disorders , 5% for attention deficit hyperactivity disorder ( adhd ) , and 2% for tic disorders 1 ) . the study revealed no cases of psychotic disorders , drug abuse , or bipolar affective illnesses . eighteen percent of them were categorized as being mildly traumatized , 49% as moderately traumatized , and 29% as severely traumatized , as shown in figure 2 . the current study was the first to describe the phenomenon of street children in duhok . though the actual statistics pertaining to the number of street children in duhok was not known , the sample could be interpreted as being representative of the street children in the duhok governorate . at the time of the study , almost all street children who attended the zewa center were registered by the social affairs directorate and police directorate , which tracks working children . the study revealed an extremely high level of parental illiteracy when compared to what was reported in other studies conducted in developing countries . for instance , in bangladesh , only 70% of fathers and 76% of mothers of street children were illiterate.22 this high level of illiteracy , as well as the low socioeconomic status , observed among 48% of street children could explain why the children work on the street ; 98% of the children indicated that their reasons for being on the street included family need and pressure . similarly , in middle eastern countries such as sudan , syria , yemen , egypt , tunisia , and jordan , low socioeconomic status pushes children to work on the street.5 in addition , in duhok ( as is evident in other middle eastern countries ) , some cultures have dominating religions , such as islam . both these characteristics encourage children learn to take responsibilities at early ages for their family s survival and income - generating activities . the situation is different in north america , where two other background factors among street children prevail as the main causes that lead children to work on the street : low levels of parent support and increased feelings of rejection and hostility , as well as difficulties and failure at school.23 the high rate ( 61% ) of reported psychiatric problems has no relation to whether the reason for the child working on street was due to neglect , poverty , or family need and pressure.3,23,24 rather , the reasons why children work on the street are related to genetic predisposition or exposure to traumatic events , as experienced by these children . this is supported by the prevalence of ptsd among these children ; one - fourth of the children meet all of the criteria for ptsd.3,5,23,24 high levels of ptsd symptoms among the street children were consistent with the traumatized children that were noted in previous studies conducted in kurdistan.19,25,26 however , these findings have to be regarded as hypothesis - generating rather than conclusive . in addition , these children will be at risk of experiencing failures in nurturance , which are likely to be complicated by abuse and derogation on the street . furthermore , half of the street children suffered from anxiety disorders a rate that was much higher than that reported among the general population ( 10%15%),27 but this is consistent with what was reported in a previous study.24 rates of depression were also higher among the street children ( 10% ) when compared to the prevalence rate of 2%8% observed among the general population . literature evidence has indicated that there is a gender difference in depression rates , with a lower prevalence observed among males.29 since the study subjects in the current study were only comprised of boys , it is expected that the rate of depression would be closer to that of the general population , which is a rate that accounts for depression in both males and females . it was slightly higher than the prevalence of conduct disorders observed among the general population ( 5.6% ) . this may be due to high levels of stress experienced on the street , with bad friends , and due to low levels of social support.30 the study showed that 5% of children on the street had adhd , which is similar to the rate observed in the general population ( 3%5%).30 there was an absence of psychotic disorders , and this can be explained by difficulties of diagnosis or misdiagnosis with other pervasive disorder and social dysfunction of psychotic disorders in contrast to the selected sample . the nonreporting of bipolar affective illness among the studied sample could be partly due to its misdiagnosis as adhd.31 the study revealed that there was no substance abuse noted among the subjects , which was in contrast to our expectations . however , during the years of 20002003 , several cases of glue sniffing were identified among street children in duhok city , but the local authorities took active steps to prevent glue sniffing , which can partly explain the absence of glue sniffing cases among our sample.24 street children in duhok seem to be mostly comprised of children working on the street . the study concluded that family needs and financial pressures drive children to work on the street . lack of psycho - social support and a nurturing environment will lead to a child s exposure to many health problems , both mental and somatic . special attention to this risky group of children by their families , communities , schools , and the government is essential . provision of good environments at the family level , with the availability of community - based education to increase psycho - social awareness , can help decrease the amount of suffering experienced among street children . school support for street children , the provision of psychological support , as well as involving families and street children in family care programs through the adoption of hosting centers ( similar to the zewa center ) are urgently needed . however , further research is needed to test the hypotheses generated by this study , as the findings were not conclusive . it should be noted that this study was a descriptive study , and the findings were not representative of all street children and their families in duhok .

the pathogenesis of sepsis - induced multiple organ failure may crucially depend on the development of mitochondrial dysfunction and consequent cellular energetic failure . according to this hypothesis , interventions aimed at preventing or reversing mitochondrial damage may have major clinical relevance , although the timing of such interventions will be critical to both ensuring benefit and avoiding harm . early correction of tissue hypoxia , strict control of glycaemia , and modulation of oxidative and nitrosative stress may afford protection during the initial , acute systemic inflammatory response . the regulated induction of a hypometabolic state resembling hibernation may protect the cells from dying once energy failure has developed , allowing the possibility of functional recovery . repair of damaged organelles through stimulation of mitochondrial biogenesis and reactivation of cellular metabolism may accelerate resolution of the multiple organ failure syndrome .

sepsis is the systemic inflammatory response to infection and represents a major cause of morbidity and mortality in patients admitted to intensive care units ( icus ) . however , despite decades of research , the pathophysiology of sepsis remains incompletely understood . a critical limitation of tissue oxygen delivery due to macrocirculatory or microcirculatory failure may play a role , especially in the early phase of the disease process before resuscitation has been initiated . nonetheless , a growing body of evidence suggests that multiple organ failure ( mof ) may develop during sepsis mainly as a consequence of impaired cellular oxygen utilization . supportive data in patients include the following findings : total body oxygen consumption falls progressively with increasing severity of sepsis ; skeletal muscle tissue oxygen tension is abnormally high but normalizes during the recovery phase ; necrotic and apoptotic cell death is minimal , if it occurs at all , in most dysfunctioning organs ; and organs with limited regenerative capabilities , such as kidney , are usually able to recover to such an extent that long - term support is usually not needed . sepsis - induced mof may thus be related to a potentially reversible impairment in cellular function rather than any permanent structural damage . cellular energy production depends on three interconnected pathways : glycolysis within the cytoplasm , the krebs cycle and the electron transport chain within the mitochondria ( figure 1 ) . glycolysis is a sequence of reactions that degrade glucose to pyruvate . in the presence of oxygen , pyruvate and other fuel molecules such as fatty acids and amino acids enter the mitochondria , where they are completely oxidized within the krebs cycle . the reduced nicotinamide ( nadh ) and flavin ( fadh2 ) adenine dinucleotides transfer electrons to the respiratory enzyme complexes located in the inner mitochondrial membrane ( electron transport chain ) for the process of atp generation by oxidative phosphorylation . the electrons then flow via coenzyme q ( ubiquinone ) to complex iii , and are then transported via cytochrome c to reach complex iv ( cytochrome oxidase ) . at this final stage electron transfer through complexes i , iii and iv generates a proton gradient across the inner mitochondrial membrane that is used by atp synthase ( complex v ) to generate energy by phosphorylating adp . the complete oxidation of one molecule of glucose yields 3036 molecules of atp , two of which come from glycolysis and two from the krebs cycle . however , when oxygen is lacking , pyruvate can no longer be further oxidized within the mitochondria and is thus metabolized to lactate within the cytoplasm . glycolysis represents a much less efficient metabolic pathway compared with the krebs cycle and oxidative phosphorylation , because there is net synthesis of only two molecules of atp per molecule of glucose . because mitochondria utilize more than 90% of total body oxygen consumption to produce atp , the abnormalities in oxygen consumption described during sepsis are likely to be associated with evidence of mitochondrial dysfunction . studies conducted during the early phase of sepsis ( within the first few hours ) have produced conflicting results . nonetheless , mitochondrial structure and function were consistently shown to be impaired in a severity - dependent manner in animal models lasting at least 1216 hours . of note , atp levels were variably affected , depending on the balance between energy production and consumption , the model and possibly the tissue under investigation . in septic shock patients studied within 24 hours of icu admission , the degree of skeletal muscle mitochondrial dysfunction was associated with the severity of the disease . in this work , tissue atp levels were significantly lower in nonsurvivors than in an orthopaedic surgical control population , but they were maintained in those who survived sepsis . a reduction in energy consumption implies a reduction in cellular metabolism , which manifests clinically as organ dysfunction . rather than being viewed negatively as ' failure ' , an alternative paradigm may be advanced whereby this metabolic shutdown represents an adaptive cellular strategy . in the face of persisting mitochondrial dysfunction and reduced atp production , the cell may shift its focus to survival rather than aiming to continue normal functioning . nitric oxide ( no ) , reactive oxygen species and other inflammatory mediators are produced in excess and can directly inhibit mitochondrial respiration . no competes with oxygen in binding to cytochrome oxidase ( complex iv ) , thereby decreasing the activity of the enzyme . superoxide will react with no to generate peroxynitrite and other nitrogen species that are able to alter the structure and function of several other mitochondrial proteins , notably complex i . early cellular hypoxia may favour the competitive no - mediated inhibition of cytochrome oxidase , contributing to the earlier , if not greater , development of mitochondrial dysfunction . endocrine changes that occur during sepsis are also likely to play a role . among others , thyroid and sex hormones , insulin , glucocorticoids and leptin positively modulate mitochondrial energy production , protein synthesis and biogenesis [ 13 - 17 ] . increased incidences of the low tri - iodothyronine ( t3 ) syndrome , hypogonadism , insulin resistance , adrenal insufficiency and decreased circulating leptin levels in nonsurvivors compared with survivors have been reported during prolonged sepsis and critical illness . accordingly , depletion of respiratory complex proteins has been described in the diaphragm in a rat model of sepsis . a further mechanism could be represented by the down - regulated synthesis of new mitochondrial protein . in human volunteers , administration of bacterial endotoxin decreased blood leucocyte expression of mitochondrial respiratory chain complexes and atp synthase genes . assuming that the pathogenesis of mof during sepsis is contingent on development of mitochondrial dysfunction and cellular energetic failure , recovery is likely to occur when damaged organelles are repaired or replaced . preliminary results have shown an association between progressive improvement in mitochondrial respiration and organ function in patients who survive their episode of septic shock . strategies aimed at preventing or reversing mitochondrial dysfunction and cellular energetic failure may thus represent a new therapeutic option in the treatment of sepsis ( figure 2 ) . mitochondrial dysfunction in sepsis can occur even with aggressive fluid resuscitation and adequate tissue oxygenation . derangement in liver metabolism possibly due to mitochondrial damage was recently reported in a hyperdynamic , normotensive , mechanically ventilated , antibiotic - treated septic animal model , despite preserved microvascular perfusion . nonetheless , early cellular hypoxia can further limit aerobic production of atp and contribute to the development of energy failure . optimization of oxygen delivery can ameliorate cellular energetic failure provided that mitochondria retain their ability to produce energy . patients with severe sepsis or septic shock whose global oxygen delivery was optimized early after admission to an emergency room experienced better outcomes than did conventionally managed patients . conversely , no benefit or even harm was reported when a similar approach was adopted after admission to icu , when organ failure had already become established . the same intervention , performed at different time points , had very different clinical impacts . in the early phase , when the cellular energetic machinery is still likely to be functional and oxygen supply may represent a limiting factor , reversal of tissue hypoxia may ameliorate the impending cellular energetic failure and reduce the incidence / severity of organ dysfunction . in a later phase , when mitochondrial damage has occurred and the cell has become intrinsically unable to utilize oxygen to produce atp , a similar strategy may not provide any benefit . lack of improvement in oxygen consumption despite a re - established oxygen supply has been associated with unfavourable outcomes in patients with sepsis syndrome or septic shock . hyperglycaemia and insulin resistance are common among critically ill patients and represent an additional potential threat to mitochondrial integrity . acute hyperglycaemia can dramatically increase the production of reactive oxygen species in normal bovine aortic endothelial cells . maintenance of normo - glycaemia with intensive insulin therapy during critical illness has been demonstrated to preserve hepatocyte mitochondrial ultrastructure and function and improve outcome in both medical and surgical intensive care patients . reactive oxygen and nitrogen species are over - produced during sepsis , whereas mitochondrial antioxidants ( reduced glutathione and manganese superoxide ) are depleted . the membrane permeable glutathione ethyl ester can protect complex i from oxidative and nitrosative damage in an early phase . manganese - based superoxide dismutase mimetics may exert a similar protective effect , scavenging superoxide anions and preventing them from further reacting with no to generate peroxynitrite within the mitochondria . once permanent mitochondrial dysfunction has developed , cellular optimization of any residual ability to produce energy and/or reduce metabolic requirements may prevent the atp level from dropping below the threshold that stimulates initiation of cell death pathways . electron donors that are able to ' bypass ' defective components of the respiratory chain may help in attaining the former objective . within the inner mitochondrial membrane , complex ii works in parallel with complex i , albeit to a lesser extent , transferring electrons from fadh2 produced during the oxidation of succinate to coenzyme q. unlike complex i , the activity of complex ii is relatively preserved during sepsis . when complex i is inhibited , the administration of succinate may increase electron flow through the respiratory chain and thus increase generation of atp , provided that any inhibition of the electron transport chain distal to complex ii has not become rate - limiting . preliminary data from our laboratory confirm this action . in two different animal models of sepsis , the infusion of succinate dimethyl ester prevented the fall in liver atp content and prolonged survival time . another possible strategy that could be pursued in the face of a severe and extended impairment in mitochondrial energy production is to reduce cellular energetic expenditure . hibernating and aestivating animals reduce their metabolic rate in the face of climate change or drought . similarly , oxygen - conforming organisms such as turtles and frogs can tolerate prolonged periods of hypoxia by suppressing atp turnover . humans do not hibernate or aestivate and have only a limited tolerance to inadequate oxygenation . nonetheless , patients with chronic coronary artery disease frequently develop a myocardial contractile dysfunction termed myocardial hibernation that may represent an adaptive response to ischaemia , rather than depend on an ongoing energetic deficit , which will recover on restoration of adequate perfusion . carbon monoxide and no may mediate the active decrease in energy demand that occurs in cells that lack oxygen . the natural peptide ' hibernation induction trigger ' , its synthetic analogue [ d - ala , d - leu ] enkephalin ( dadle ) and other -opioids can also reduce cellular metabolism and protect organs against ischaemia . rapid induction of profound cerebral hypothermia in animals that do not normally hibernate may guarantee protection during prolonged cardio - circulatory arrest . mice exposed to hydrogen sulphide experience a dramatic decrease in their metabolic rate : within 6 hours , oxygen consumption and carbon dioxide production drop by around 90% , and body core temperature approaches that of the environment . such a suspended animation - like state fully reverses when the hydrogen sulphide is discontinued , without any permanent behavioural or functional damage . it is conceivable that , even during sepsis , induced hibernation may protect the organism from prolonged energetic failure and enable faster recovery on resolution of the inflammatory insult . for example , the hyperthermic response to infection activates the expression of cytoprotective heat shock proteins and may therefore be considered beneficial . premature stimulation of cellular metabolism before mitochondria have regained their ability to respond adequately in terms of energy production may lead to cellular compromise . examples of harmful therapeutic approaches that may be invoked are the use of high - dose dobutamine , thyroxine and growth hormone . repair and replacement of damaged mitochondria are probably controlled at a transcriptional level , but proximal steps in the signalling pathway still need to be elucidated . no was recently suggested to play a major role . long - term exposure to a low concentration of the gas triggered expression of transcriptional factors that regulate mito - chondrial proliferation and significantly increased mito - chondrial mass in different cells in culture . the large quantity synthesized by the inducible isoform of nitric oxide synthase ( nos ) during the acute inflammatory response to sepsis blocks mitochondrial respiration and can be cytotoxic . on the other hand , the smaller amounts of no produced by the specific constitutive endothelial nos may trigger mitochondrial biogenesis in a later phase . taken together , these results suggest that recovery from mito - chondrial dysfunction may depend on a no - dependent signalling pathway . specific inhibition of inducible nos during sepsis may represent a potential therapeutic strategy [ 52 - 55 ] , although dose selection will be critical . this is pertinent to the dose - related increase in mortality reported in a phase iii trial of a nonspecific nos inhibitor in septic shock patients . indeed , the overall negative outcome of this study camouflages the survival benefit seen with low doses . in contrast to the acute response , persistently low circulating levels of t3 during the prolonged phase of critical illness may be due to neuroendocrine dysfunction . replacement hormonal therapy given at the right time , when cells have regained the ability to both restore mitochondrial activity and increase metabolic rate , may beneficially arouse the cell and promote earlier organ recovery . however , as described above , thyroxine supplementation may prove dangerous , and so the right conditions must be in place . other hormones that could be considered in the treatment of sepsis are leptin and oestrogen . it regulates food intake and energy balance to maintain constancy of total body fat mass . in diabetic fatty rats , ectopic hyperleptinaemia triggered mitochondrial proliferation , transforming white adipocytes into mitochondria - rich , fat - oxidizing cells . administration of oestrogen or antiandrogen agents after trauma / haemorrhage also increased mitochondrial enzyme activities , protein synthesis and atp levels relative to those in sham - operated controls . this is a reversible , low - growth state well recognized in mycobacteria such as mycobacterium tuberculosis . micrococcus luteus can be aroused from its quiescent phase by an endogenous protein named ' resuscitation promoting factor ' . as mitochondria descend from a bacterial endosymbiont , the identification and application of a similar protein that can specifically stimulate mitochondrial activity may well yield beneficial results . mitochondrial dysfunction occurs during sepsis and may play a major role in the development of mof . prevention and correction of mitochondrial dysfunction and cellular energetic failure represent novel strategies that may improve clinical outcomes of septic patients . timing of any intervention appears to be critical and the possibly adaptive role of some changes currently viewed as pathological must be considered . the regulated induction of a hypometabolic state resembling hibernation may help the cell in facing a reduced capacity to generate energy . the stimulation of mitochondrial activity and biogenesis during the late phase of sepsis may accelerate the recovery process . this increasing insight into underlying mechanisms promises to be an exciting era of novel therapeutic developments . fadh2 = flavin adenine dinucleotide , reduced ; icu = intensive care unit ; mof = multiple organ failure ; nadh = nicotinamide adenine dinucleotide , reduced ; no = nitric oxide ; nos = nitric oxide synthase ; t3 = tri - iodothyronine . ms receives research funding from the medical research council and wellcome trust to undertake basic science research on mitochondria . electrons donated from nadh and fadh2 pass down the electron transport chain with oxygen being the terminal acceptor at complex iv . this movement of electrons results in a shift of protons across the inner mitochondrial membrane , generating the energy necessary for atp synthase to produce atp from adp . fadh2 , flavin adenine dinucleotide , reduced ; nadh , nicotinamide adenine dinucleotide , reduced .

we explored the impact of interfacial property changes on aggregation behavior and photoinduced charge separation in mixed metal oxide nanoparticle ensembles . tio2 and sno2 nanoparticles were synthesized by metal organic chemical vapor synthesis and subsequently transformed into aqueous colloidal dispersions using formic acid for adjustment of the particles surface charge . surface charge - induced heteroaggregation was found to yield blended nanoparticle systems of exceptionally high mixing quality and , after vacuum annealing , to extremely high concentrations of heterojunctions between tio2 and sno2 nanoparticles with dehydroxylated surfaces . for tracking charge transfer processes across heterojunctions , the photogeneration of trapped charge carriers was measured with electron paramagnetic resonance ( epr ) spectroscopy . on blended nanoparticles systems with high concentrations of sno2tio2 heterojunctions , we observed an enhanced cross section for interparticular charge separation . this results from an effective interfacial charge transfer across the interfaces and gives rise to substantially increased concentrations of electrons and hole centers . the here presented insights are key to the rational design of particle - based heterojunctions and mesoporous nanoparticle networks and help to engineer composite nanomaterials for photocatalysis and solar energy conversion .

an efficient photocatalyst minimizes recombination of photoexcited electron hole pairs and maximizes electron and hole transfer to the adsorbate upon consecutive surface reactions . in general , the photocatalytic activity of a given particle system depends on multiple factors . for a reliable photoactivity assessment , relative contributions from the particles bulk and interfaces have to be sorted out , and their impact to the overall performance requires a careful evaluation . a major limitation of any photocatalytic process originates from charge carrier recombination . an important approach to counteract charge carrier recombination in photocatalytic particle systems is based on the coupling of different semiconductor components with desirable matching of their electronic band structures . in the case of composite nanoparticle systems , synergistic properties and those that are enhanced with respect to the individual components can only emerge if the particles are mixed intimately enough . for that reason , researchers have explored efficient and cost - effective methods for combining different metal oxide nanoparticles . chemical ways could offer great control in this respect . while a variety of experimental approaches leading to the coupling of two semiconductors have been successfully employed for particles that were grown in colloidal solutions , there are very few reports about the charge separation properties of dry particle systems that feature corresponding types of interfaces . with this study we present a simple as well as versatile approach that involves surface charging of multiple particle systems inside the same aqueous dispersion medium to achieve blended nanoparticle systems of superior mixing quality and , thus , a maximum concentration of functional interfaces between different particle types . ( a ) schematic showing the principle of hydration - dehydration induced particle aggregation and solid solid interface formation . ( b ) adjustment of opposite surface charges on multiple particle types is expected to favor preferential attraction between different particles and yields good mixing qualities . we have shown in previous work that water - mediated aggregation of tio2 and zro2 nanoparticles and subsequent dehydration procedures under high vacuum conditions are effective in the generation of solid solid interfaces . the mechanistic steps of the underlying material transformation process are as follows ( figure 1 ) : in aqueous dispersion , mutual attraction and agglomeration of metal oxide nanoparticles takes place . upon vacuum treatment dehydration / dehydroxylation processes ( evaporation of liquid phase ) in conjunction with particle condensation lead to the formation of chemical interparticle metal oxygen bonds . annealing - induced aggregation of particles during thermal treatment results in interconnected particles but , according to n2 sorption measurements , does not reduce the specific surface area in comparison to the unprocessed nanoparticle powders . schematic energy diagram showing the positions of conduction and valence band edges of tio2 and sno2 . expected vectorial charge transfer directions are indicated for electrons ( top arrow ) and holes ( bottom arrow ) . for the exploration of surface charge - induced heteroaggregation , we chose the tio2sno2 system . both materials are central to photocatalysis , solar water splitting , and dye sensitized solar cells . as a result of composite formation upon generation of heterointerfaces , light - induced charge separation and vectorial charge transfer ( figure 2 ) the conduction band position of sno2 is lower than that of tio2 and such that it is incapable of reducing oxygen molecules to form superoxide anions . the band offsets between tio2 and sno2 will promote charge separation across the interfaces . using electron paramagnetic resonance ( epr ) spectroscopy , we address the vectorial transfer of separated charges to generate paramagnetic o radicals as well as paramagnetic ti sites . another reason for selecting this system has been the exclusion of sn ti o compound formation . for sno2tio2 core shell nanostructures , it was found that the phase boundary between the two oxides remains abrupt up to t = 1200 k without substantial chemical transformations in this region . thus , heteroaggregated sno2tio2 nanoparticle networks represent an ideal model system to explore the potential of surface charge - induced heteroaggregation with regard to the generation of heterojunctions that enhance the separation of photogenerated charges . tio2 and sno2 nanoparticles were prepared by metal organic chemical vapor synthesis ( mo - cvs ) based on the decomposition of either titanium(iv ) isopropoxide ( aldrich , 99.999% trace metals basis ) or tetra - n - butyltin ( aldrich , technical grade , > 93% ) vapor at t = 1073 k in a flow reactor system . for purification , the obtained powder samples were subjected to thermal treatment under high vacuum conditions . first , the powder sample was heated to t = 870 k using a rate of r 5 k min . subsequent oxidation with o2 at this temperature followed by cooling in o2 atmosphere was successfully applied to remove organic remnants originating from the precursor material , on the one hand , and to guarantee the stoichiometric composition of the oxide on the other . the average particle size determined by transmission electron microscopy ( tem ) and nitrogen sorption was 13 nm for tio2 and 10 nm for sno2 nanoparticles . for the preparation of mixed ensembles of tio2 and sno2 nanoparticles , 250 mg of the powder samples were dispersed in 100 ml of c = 10 moll formic acid solution for 30 min under ultrasonication ( hielscher sonifier 200s ) and simultaneous agitation by a magnetic stirrer . cooling with an ice centrifugation and subsequent drying in vacuum at room temperature resulted in the formation of monolithic pieces , which were transferred to a quartz glass cell and annealed to t = 873 k at p < 10 mbar prior to spectroscopic investigations . prior to liquid exposure zeta potentials and agglomerate size distributions of suspensions of 2.5 g oxide nanoparticlesl formic acid solution were derived from dynamic light scattering measurements on a malvern zetasizer nano zs . nitrogen sorption isotherms were obtained at t = 77 k using nitrogen adsorption / desorption ( quantachrome nova 4000e ) . samples were degassed for 2 h in the degas unit of the adsorption apparatus at t = 473 k under vacuum prior to analysis . the bet surface area sbet was evaluated using adsorption data in a relative pressure range p / p0 from 0.05 to 0.2 . scanning electron microscopy ( sem ) measurements were performed on a zeiss gemini ultra 55 microscope operating at 20 kv equipped with an energy dispersive x - ray emission ( edx ) detector . to characterize the mixing quality inside the nanoparticle networks we used edx to track compositional homogeneity changes in the range of a few hundreds of nanometers . the local resolution of chemical information for edx analysis is limited to the penetration depth and scattering of the primary electrons and therefore to the volume where characteristic x - rays are emitted of the sample . with acceleration voltages of 20 kv , the sampled volume in edx studies is on the order of a few cubic micrometers for bulk tio2 or sno2 samples . consequently , the determination of absolute ti and sn concentrations via edx at the nanometer scale is not feasible . nevertheless , by scanning the aggregate with a linescan , the change in the ratio between ti and sn can be tracked with a very high spatial resolution of approximately 100 nm . for epr measurements , the powder sample was contained within a suprasil quartz glass tube connected to an appropriate high vacuum pumping system with a base pressure p = 10 mbar . the light beam was passed through a water filter to exclude ir contributions from the excitation spectrum . it was held constant at pirr = 0.9 mwcm for the energy range 3.2 ev < e < 6.2 ev throughout all experiments . during uv exposure , the samples were held at a temperature between t = 90 k and t = 140 k in order to keep uv - induced heating effects constant . x - band epr measurements were performed on a bruker emx micro spectrometer using a bruker er 4119 hs resonator . for measurements in the temperature range between t = 90 k and t = 140 k , an er 4131 vt variable - temperature accessory was used . the procedure of particle aggregation as outlined by figure 1 aims at the conversion of aerosol metal oxide nanoparticle powders , of high purity and well characterized particle surface properties , into binary metal oxide nanoparticle networks of high mixing quality and therefore with high concentrations of solid solid heterointerfaces ( figure 1b ) . prior to the process , aggregation between particles must be avoided in the starting mixture in order to achieve nanoparticle networks of different and perfectly intermixed metal oxides . dynamic light scattering experiments on dispersions of vapor phase - grown tio2 nanoparticles with an average particle size of 13 nm as determined by tem clearly demonstrate that metal oxide nanoparticles that were grown by mo - cvs can be efficiently deagglomerated by ultrasonic treatment in aqueous dispersion ( figure 3 ) . as a consequence of ultrasonification , the agglomerate size distribution maximum shifts from 100 nm ( figure 3a ) to 20 nm ( figure 3b ) . after discontinuation of ultrasonic treatment , particles reagglomerate , and the agglomerate size distribution maximum shifts to approximately 40 nm ( figure 3c ) . size distribution profiles determined by dynamic light scattering experiments on dispersions of vapor phase - grown tio2 nanoparticles with an average particle size of 13 nm in c = 10 moll formic acid . ultrasonic treatment breaks up particle agglomerates and leads to the shift of the agglomerate size distribution maximum from ( a ) 100 nm to ( b ) approximately 20 nm . after 15 min of reagglomeration , which occurs after discontinuation of ultrasonic treatment , the maximum increases to approximately 40 nm ( c ) . zeta potential measurements reveal that both oxides carry a negative surface charge in neutral aqueous dispersion ( figure 4 ) . as a result of particle aggregation , tio2 and sno2 form a nonuniform network which has a significant fraction of tio2tio2 and sno2sno2 interfaces . self - assembly of oppositely charged particles in colloidal dispersion and , in consequence , the generation of desired tio2sno2 heterointerfaces is achievable via control of the surface charge . in comparison to inorganic acids , which potentially contaminate the particle surface with ions such as cl , carboxylic acids are eligible candidates for surface charge adjustment since they can be efficiently eliminated from the particle network via oxidation at elevated temperatures . the zeta potential dependence of tio2 and sno2 nanoparticles on the formic acid concentration ( figure 4 ) prompted us to choose a formic acid concentration of c = 10 moll ( indicated by gray arrow ) where the zeta potential of tio2 and sno2 particles is + 24 mv and 9 mv , respectively . zeta potentials of tio2 and sno2 nanoparticle samples in aqueous dispersion as a function of formic acid concentration . to zeta potential measurements , all metal oxide samples were subjected to vacuum annealing and subsequent oxidation treatment at elevated temperatures to eliminate unintended effects that could originate from surface impurities . surface charge adjustment for dispersed particles in combination with control over the ph in solution shows a complex functional dependence on the formic acid concentration . this is due to the formate adsorption equilibria at the metal oxide particle surfaces which , in turn , are subject to particle concentration in the dispersion and on the concentration of the acid and , concomitantly , on the ph . ( for further details please see supporting information , figure s1 . ) dehydration and dehydroxylation as well as oxygen treatment of the obtained particle networks at elevated temperatures were applied in order to eliminate organic surface groups that previously lead to surface charging and heteroaggregation . x - ray diffraction , tem , and n2 sorption measurements were carried out to identify potential process - induced structural changes that may result from hydration- and annealing procedures . all methods unambiguously reveal that primary particle properties size , structure , and morphology have been retained ( supporting information , figure s2 ) . changes in the adsorption desorption hysteresis of nitrogen sorption experiments ( left panel of figure 5 ) clearly demonstrate that loose nanoparticles transform into a mesoporous network , which are made up from interconnected particles and show a distinct pore size distribution ( right panel of figure 5b ) . sorption isotherms ( left ) and bjh pore size distributions ( right ) of sno2tio2 nanoparticle ensembles before ( a ) and after formation of heterointerfaces the sorption isotherms and pore size distributions are shifted on the ordinate scale for 300 cmg and 3 cmgnm , respectively , for clarity . we employed sem to characterize the composition and mixing quality inside the nanoparticle networks and used edx to track compositional homogeneity changes in the range of few hundreds of nanometers . a typical edx line scan on nanoparticle networks obtained from surface charge - induced heteroaggregation in aqueous formic acid solution ( c = 10 moll ) is shown in figure 6a and reveals that the ratio of the two elements should remain constant across the scanned distance ( gray line ) . the mixing quality and thus the concentration of sno2tio2 heterojunctions is expected to be significantly higher than in the water processed networks . here , deviations of the desired ti to sn ratio point to a nonuniform distribution of the two elements ( gray line in figure 6b ) and therefore to the low mixing quality of the network . characteristic edx linescans of tio2sno2 networks obtained by heteroaggregation in ( a ) aqueous formic acid solution ( c = 10 moll ) and ( b ) water . scanned paths are shown in the sem insets . the black and red lines show the distribution of ti and sn within the scanned distance . the here presented procedure provides a very good measure of the local mixing quality within the nanoparticle networks . to obtain statistical meaningful information throughout different nanoparticle network samples , we evaluated the deviation of the desired ratio of ti / sn for approximately 600 sample spots ( figure 7 ) . deviations of 0% indicate that the chosen value of one tio2 particle per sno2 particle has been achieved . it can be shown that the nanoparticle network that originates from processing in pure water displays a very broad distribution of ti / sn deviation , while the network which was prepared in aqueous formic acid exhibits excellent mixing quality , i.e. , relatively small deviations from desired ti / sn ratio . deviations of the desired ti / sn ratios within line scans of several micrometers across the particle networks are plotted in the diagram . the samples were prepared in c = 10 moll aqueous formic acid solution ( hcooh / high mixing quality ) and water ( h2o / low mixing quality ) . a good mixing quality of sno2 and tio2 nanoparticle mixtures represents a necessary requirement for a high concentration of heterojunctions inside the resulting particle network . to verify that these solid solid interfaces are truly suitable for interfacial charge transfer , we spectroscopically probed the photoelectronic materials properties . for this purpose we utilized epr to track the photogeneration of surface trapped hole centers o , unpaired electrons in shallow trap states ti or adsorbed o2 ions ( supporting informations ) . as demonstrated by previous studies this technique provides site specific information about charge trapping sites . band gap and conduction band edge energies determine the pathway of electrons or holes from one oxide to the other ( figure 2 ) . the relative positions of the conduction band minima favor electron transfer from tio2 to sno2 . on the other hand , the valence band positions of the two oxides are such that photogenerated hole centers become accumulated on tio2 nanoparticles . epr spectra of metal oxide samples after 30 min uv / vis light exposure ( pirr = 0.9 mwcm for the energy range 3.2 ev < e < 6.2 ev ) . ( a ) simulated epr spectrum of o and ti centers on tio2 nanoparticles . ( b ) experimental epr spectrum of o and ti centers on tio2 nanoparticles . all spectra were acquired at t = 90 k and p < 10 mbar using a microwave power of pmw = 6.32 mw . in figure 8a , a simulated epr spectrum of o and ti centers is shown in comparison to an experimental spectrum ( b ) , which was acquired on dehydroxylated tio2 nanoparticles after 30 min of uv / vis exposure under high vacuum conditions . the separation of photoexcited states ( eq 1 ) is followed by persistent trapping of electrons and holes upon formation of ti and o centers , respectively ( eqs 2 and 3).123 the corresponding epr spectrum of sno2 nanoparticles shows no significant signals after irradiation and is therefore not presented here . after irradiation of a nanoparticle network of low mixing quality , the corresponding epr spectrum reveals the presence of o and ti stabilized on tio2 nanoparticles ( figure 8c ) . however , the network prepared from surface charge induced heteroaggregation in aqueous formic acid solution shows a strong signal for o stabilized on tio2 nanoparticles but no ti centers ( figure 8d ) . their absence points to an efficient interfacial electron transfer from tio2 particles to sno2 particles ( figure 2 ) upon formation of epr silent states . for corroboration of this assumption , we exposed the samples after uv exposure to molecular oxygen ( p = 10 mbar ) . in the case of tio2 nanoparticles , photogenerated electrons transfer to o2 and form paramagnetic superoxide anions ( o2 ) ( eqs 4 and 5 ) , which remain stabilized on the particle surface and give rise to characteristic epr signal signatures ( table 1 , figure 9a).45 epr spectra of preirradiated metal oxide samples after oxygen exposure ( p(o2 ) = 10 mbar ) . ( a ) epr spectrum of o and o2 species stabilized on mo - cvs tio2 nanoparticles . the spectra were acquired at t = 90 k and p < 10 mbar using a microwave power of pmw = 0.2 mw . for better comparison the redox potential of o2/o2 ( figure 2 gray dotted line , e = 4.22 ev ) is slightly above the conduction band minimum of sno2 ( ecb = 4.5 ev ) , which inhibits the transfer of sno2 related conduction band electrons to molecular oxygen and , thus , the formation of surface adsorbed o2 ions . figure 9 shows epr spectra of the preirradiated metal oxide samples after 15 min oxygen exposure at t = 90 k and subsequent pumping to p < 10 mbar . in the case of tio2 nanoparticles ( a ) , additional epr resonances are observed on the network of low mixing quality ( b ) and attributed to o2 radicals , which are stabilized on sno2 surfaces ( table 1 ) . for the network of high mixing quality , no o2 stabilized on tio2 are observed in the epr spectrum ( c ) . from the absence of o2 ions we infer that all photogenerated electrons are drained into sno2 ( figure 2 ) . this is different from tio2 , where photogenerated electrons , irrespective of whether they localize in shallow trap states ( ti ) or whether they remain in the conduction band , readily transfer to molecular oxygen . for particle systems that were only in contact with pure water and obviously , related networks contain too many regions where tio2 or sno2 particles are aggregated in such a way that interfaces between identical metal oxide particles types prevail . corresponding solid solid interfaces were found to actually enhance the recombination of photogenerated charge carriers . the quantitative analysis of persistently trapped photogenerated charges on the three different powder samples shows no concentration increase of stabilized hole centers ( o radicals ) for the water prepared network with low mixing quality in comparison to the pure mo - cvs tio2 nanoparticles ( figure 10 ) . however , a strong enhancement of the concentration of o radicals was observed for the sample of high mixing quality . the metal oxide samples were irradiated for 15 min ( pirr = 0.9 mwcm for the energy range 3.2 ev < e < 6.2 ev ) at t = 90 k and p < 10 mbar . the quantitative analysis of the yield of photogenerated charges clearly shows that the adjustment of surface charge during particle network formation allows for the achievement of high mixing qualities and enables the realization of a high concentration of heterojunctions that are vital for the separation of photogenerated electrons and holes . the here presented results clearly underline the necessity to characterize and annihilate charge recombination on photoactive materials . surface charge - directed aggregation of different types of particles and the subsequent introduction of functional interfaces is a cheap and versatile particle engineering approach to generate high concentrations of heterojunctions inside particle - based devices for photochemical application and energy production . we intentionally introduced functional particle interfaces between two types of metal oxide nanoparticles and quantitatively determined the yield of photogenerated and trapped charges . to yield blended nanoparticle systems hosting high concentrations of heterointerfaces , it is vital to prepare nanoparticles that subsequently can be easily deagglomerated in colloidal dispersion and to control the process of interface formation via surface charge induced heteroaggregation . as shown in this study , mo - cvs - grown particles can be simply dispersed by use of ultrasonic treatment to the single particle level . controlled interface formation was achieved via heteroaggregation , i.e. , via the self - assembly of oppositely charged particles in colloidal dispersion using formic acid for the adjustment of surface charge . in comparison to mixed particle systems prepared in pure water via surface charge - directed aggregation - prepared mixed particle systems show a substantially increased yield for photogenerated hole centers . the extremely high concentration of sno2tio2 heterojunctions actually gives rise to an enhanced cross section for the separation of photogenerated charges , which results from an effective interfacial charge transfer across the particle particle interfaces .

objectives : to disseminate transesophageal ultrasound - guided fine needle aspiration ( eus - fna ) as an alternative to investigate mediastinal tumoral lesions because it is an underused modality that has been available in brazil for more than 15 years.methods:descriptive analysis of a single endoscopy service 's experience since 1997 in the accomplishment of eus - fna for mediastinal staging of previously known malignancies ( group 1 ) or diagnostic definition of suspect lymph nodes and masses ( group 2).results : eus - fna was performed in 51 patients between 26 and 87 years of age . the diameter of the lesions ranged between 1.1 and 9.8 cm ( mean 3.9 cm ) . their location corresponded to the following stations : higher paratracheal ( 4 cases ) , lower paratracheal ( 7 ) , aortic window ( 12 ) , para - aortic ( 6 ) , subcarinal ( 9 ) , paraesophageal ( 8) , and hilar ( 5 ) . in group 1 , 17 patients had previously diagnosed primary lung ( 9 ) , breast ( 4 ) , kidney ( 2 ) , colon ( 1 ) , and bladder ( 1 ) cancer . fifteen of these punctures were positive for malignity . two others were later submitted to mediastinoscopy , which identified metastases not detected by eus - fna . group 2 comprised 34 patients . among these patients , eus - fna diagnosed 22 neoplasms , five cases of tuberculosis and two duplication cysts . cytology was inconclusive or without a specific diagnosis in five other cases . mediastinoscopy identified two undiagnosed cases of oat - cell carcinoma , one lymphoma and one cryptococcosis , and confirmed one reactive lymphadenitis . there were no complications related to the method.conclusions:eus-fna obviated the need for surgical procedures in 86.3% of cases . therefore , oncologists , pulmonologists , and thoracic surgeons should always remember the technique 's potential and availability .

the repercussions and clinical impact of this minimally invasive technological advance have been broadly highlighted in the international scientific literature and more recently expanded to pulmonology and thoracic oncology . endobronchial ultrasound ( known as ebus ) has faced greater technical bottlenecks , related to the smaller diameters of the bronchoscope , its working channel , the patients ' airways and , especially , the interface between the ultrasound and air.1 the first sectorial echobronchoscope was launched on the international market only in the middle of the first decade of 2000 . gastrointestinal endosonography ( known as eus ) , on the other hand , has been in use as a routine procedure for more than 15 years at large hospitals performing high - complexity procedures , including those in brazil.2 - 4 its diagnostic and therapeutic range has been well established for pancreatic and pelvic diseases ; mediastinal lesions can also be approached through the intrathoracic esophagus.5,6 fine - needle aspiration ( fna ) of masses and lymph nodes through the esophageal wall has been performed at specialized centers , with minimal risks of infection or bleeding and without great technical difficulty.5 - 8 the importance and usefulness of eus for the mediastinal staging of primary lung cancer has been well known since 1996.6 the main limitation of eus is its inability to access the anterior mediastinum because of the interference of air present in the trachea.1,7,8 this study aims to assess the performance of eus - guided fna in diagnosing mediastinal tumor lesions ( including lymph node enlargements ) and to describe some advantages and particularities of the technique . this observational , retrospective , and cross - sectional experience analysis reports the experience of a single endosonography service linked to a private hospital in sao paulo city between february 1997 and january 2011 . all clinical data ( including copies of radiological and endosonography images ) were obtained from the service 's computerized database . the demands for eus for mediastinal assessment purposes were spontaneous because the patients ' own physicians referred them due to pathological findings on chest - computerized tomography ( ct ) and , in some more recent cases , on positron emission tomography ( pet ) scans . for the sake of this study , patients were classified into two groups according to the purpose of the examination : group 1eus - fna performed for mediastinal staging of previously known malignant tumors ; and group 2eus - fna performed for diagnostic definition of lymph nodes or suspected mediastinal masses . no technical or logistic differences occurred when the procedure was accomplished in both groups , which always followed the same service routine . all examinations took place in an outpatient setting , under general anesthesia , starting with conventional upper digestive endoscopy . then echoendoscopy was used to identify the mediastinal lesions previously detected on radiology exams . under a direct and real - time ultrasound view , one single lesion ( the largest in cases of multiple identified lesions ) was punctured with a dedicated 22-gauge endoscopic needle . once guided into the target lesion , the needle was moved back and forth within the mass while applying suction with a 20-ml syringe . frozen - section examination was not performed during the procedure in any of the cases . the aspirated material was fixed in formaldehyde and analyzed through the cell - block technique . in case of inconclusive cytopathology results , the patient 's physician - in - charge was asked for further information on clinical monitoring , other diagnostic methods , and the respective final diagnosis in each case . approval for this study was obtained from the local institutional review board in compliance with the national health council resolution 196/96 . out of 1,639 gastrointestinal endosonographies performed during the study period , 51 ( 3.1% ) looked for mediastinal lesions . this series involved 37 ( 72.5% ) men and 14 women between 26 and 87 years old ( median 65 years ) . out of these 51 patients , 23 ( 45.1% ) manifested thoracic symptoms ( dysphagia , dyspnea , thoracic pain ) , 22 ( 43.1% ) reported nonspecific signs and symptoms ( fever and weight loss ) , and 6 ( 11.8% ) were asymptomatic . the forwarding physicians included 22 ( 43.1% ) oncologists , 18 ( 35.3% ) clinical pulmonologists and thoracic surgeons , and 11 ( 21.6% ) others ( general clinicians , digestive surgeons , and cardiologists ) . it should be highlighted that out of the 51 eus performed for mediastinal assessment purposes , 23 ( 45.1% ) happened in the final four years of the research period , and these cases were mostly referred by pulmonologists and thoracic surgeons . endoscopic alterations ( extrinsic compression ) were found in 24 ( 47.1% ) patients , three of whom already displayed esophageal stenosis . in group 1 ( with previously known malignant disease , forwarded for mediastinal staging ) , 17 patients were included , 9 with primary lung tumors , 4 with breast tumors , 2 with kidney tumors , 1 with a colon tumor , and 1 with a bladder tumor . out of these 17 patients , a previous pet scan had been done in only 4 , all of whom were considered the diameter of the punctured lesions ranged from 1.1 to 6.8 cm , with an average of 3.7 cm . their location ( mountain , 1997)9 corresponded to stations 2r ( 2 cases ) , 2l ( 1 ) , 4r ( 1 ) , 4l ( 2 ) , # 5 ( 1 ) , # 6 ( 1 ) , # 7 ( 3 ) , # 8 ( 2 ) , 10r ( 1 ) , and 10l ( 3 ) . eus - fna demonstrated metastatic involvement in 15 out of 17 ( 88.2% ) patients in group 1 . the respective lymph node stations sampled by eus were the paraesophageal ( # 8 ) and the left hilar ( 10l ) . both cases were later submitted to classical cervical mediastinoscopy , which identified metastases in lower paratracheal lymph nodes ( # 4 ) that were previously undetected through eus . group 2 ( undiagnosed lymph node enlargements or mediastinal masses ) comprised 34 patients . the diameter of the punctured lesions varied from 1.6 to 9.8 cm ( average 4.0 cm ) . their location ( mountain , 1997)9 corresponded to stations 2l ( one case ) , 4r ( 3 ) , 4l ( 1 ) , # 5 ( 11 ) , # 6 ( 5 ) , # 7 ( 6 ) , # 8 ( 6 ) , and 10l ( 1 ) . among the 34 patients in group 2 , 22 ( 64.7% ) new tumors were diagnosed through eus - fna , including epidermoid carcinoma ( 10 ) , adenocarcinoma ( 5 ) , oat - cell ( 3 ) , lymphoma ( 2 ) , sarcoma ( 1 ) , and neuroendocrine carcinoma ( 1 ) . other diagnoses established in this group included tuberculosis ( 5 ) and duplication cyst ( 2 ) . cytology was not malignant ( but without a specific diagnosis ) in three cases and inconclusive in two others these five patients were later submitted to mediastinoscopy , which identified two other cases of oat - cell carcinoma , one non - hodgkin b - cell lymphoma , and one ganglionic cryptococcosis , in addition to confirming one case of non - specific reactive lymphadenitis . figure 1 displays a flow chart that summarizes the procedures and diagnoses in this study . figure 2a illustrates a clinical case from group 1 ; figure 2b illustrates a clinical case from group 2 . despite its technical and commercial availability , eus is still rather underused in the treatment of thoracic illnesses . aside from its well - established importance for lung cancer staging,6,10 its indication extends to other clinical situations , such as mediastinal lymph node enlargement of unknown causes or primary tumor masses and cystic lesions ( for diagnostic or symptom relief purposes).11 considering each patient 's final diagnosis as the gold standard , the general sensitivity of eus - fna in our study was 88.0% , with 11.7% false negative cases . these rates still apply if the sample is limited to the 17 cases in group 1 . in a recent meta - analysis6 restricted to lung cancer cases , the general eus - fna sensitivity was 84% for metastasis detection ( n2 and/or n3 ) , with a global false - negative rate of 19% . the only other brazilian publication found that addressed this issue12 assessed 25 eus - fna performed for the sake of diagnostic clarification of mediastinal masses and lymph nodes . normal lymphatic tissue was obtained in three cases ( 12% ) and , in four others ( 16% ) , insufficient material was sampled . no data are available on other complementary methods used to define the diagnosis for inconclusive cases . the comparison between different methods ( eus , ebus , and surgical mediastinoscopy ) in mediastinal staging for primary lung cancer has been a recurrent and widely discussed theme . this technical choice depends , among other factors , on the patient 's clinical condition , the degree of suspected mediastinal involvement , the location of the primary tumor , the histological type , diameter and level of the biopsied lymph nodes , the number of samples obtained and , most importantly , the availability of different methods at each institution , as well as the respective results the local team has achieved.6,13 a larger number of recent eus have been performed at the request of chest physicians we believe this change resulted from these specialists ' recent contact with the large - scale dissemination of ebus in the international literature , particularly regarding clinical repercussions . both eus and ebus are recommended by the main thoracic oncology guidelines on the invasive mediastinal staging of primary lung cancer.6,14,15 yet other facts and peculiarities should be reminded : any invasive sampling method is more specific than ct scan and pet scan alone.16 the association between eus and ebus in the same patient reaches accuracy levels of more than 95%.17,18 these rates are quite encouraging , but combining both sets of equipment , logistics , training , and the availability of human and technical resources can hardly be justified in commercial terms . mediastinoscopy continues to be an obligatory complementary method whenever the above techniques reveal a negative result.6,14,15 some authors defend the position that if the main goal is the diagnostic confirmation of suspected metastatic disease detected through ct or pet scan , then endosonography methods ( eus and/or ebus ) , if available , are an excellent alternative , with high sensitivity and low morbidity levels . however , if the main goal of invasive staging is to confirm the absence of mediastinal involvement , in most cases , surgical mediastinoscopy seems to be the best option.13 based on mountain 's former lymph node map9 ( which was the gold standard used during the study period ) , eus can assess and obtain samples from the upper and lower paratracheal levels ( stations # 2 and # 4 ) , aortic window ( # 5 ) , subcarinal level ( # 7 ) , paraesophageal level ( # 8) , inferior pulmonary ligament ( # 9 ) , and pulmonary hilum ( # 10 ) . it should be noted that eus also permits staging ( and biopsying ) of primary pulmonary lesions when located near ( or eventually invading ) the mediastinum.19 it has also been capable of detecting ( and biopsying ) metastatic disease in subdiaphragmatic lesions , such as those affecting the left adrenal gland , celiac lymph nodes , and liver.6,8 the experience reported here includes a considerable number ( six cases ) of samples obtained from the para - aortic level ( station # 6 ) , which deserves more careful and detailed analysis . strictly speaking , station # 6 corresponds to the lymph nodes anterior and lateral to the ascending aorta , between a line tangent to the superior border and another to the inferior border of the aortic arch.9 hence , although station # 6 can indeed be visualized through eus , it is rather difficult to obtain samples through the esophageal route , as that would imply transection of the pulmonary artery or the aorta itself with the puncture needle . this location may therefore have not been very precise under the eus view , so that some lymph nodes attributed to the para - aortic position ( if not all of them ) may include lesions from stations 4l , # 5 and even # 8 . because esophageal endosonography does not offer easy anatomical reference points , the endoscopist 's experience and knowledge of regional topography are fundamental for a successful examination . identifying and sampling lesions located at the subcarinal level ( # 7 ) , for example , will hardly represent any difficulty because of its central position , which is always anterior to the middle esophagus . it is known that fna of station # 7 guided by endosonography techniques ( both eus and ebus ) does not obtain better results than a simple , blind transtracheal puncture.20 the pulmonary hilar levels ( # 10 ) , on the other hand , are frequently mixed up with the inferior paratracheal stations ( # 4 ) , especially on the right . such inadequate staging can radically change therapeutic decisions this means a possible n2 false - positive result . in lung cancer patients , if the lymph nodes of stations 10 r / l ( classified as n1 ) are unintentionally interpreted as belonging to stations 4 r / l ( classified as n2 ) , a malignant aspirate may exclude the option for a radical surgical resection and the potential for cure.8,13 there are further issues related to the routine adopted at our service , which remains limited to the puncturing of a single suspect lesion ( the largest in case of multiple identified lesions ) . hence , it is recommended that , in all cases , samples be obtained from at least two lymph node zones as recently mapped by the international association for the study of lung cancer ( ialsc),21 always including the subcarinal zone ( station # 7 ) , to improve prognostic definitions . equipment costs ( especially disposable needles ) and the learning curve for use of the technique are highlighted as the main difficulties that ebus will still have to face before achieving greater availability and widespread use.1 international and brazilian experiences with eus - fna , on the other hand , have already demonstrated the method 's ability to avoid surgical procedures ( mediastinoscopy , videothoracoscopy or even exploratory thoracotomy ) in a considerable number of patients86.3% of cases in this study . not only endoscopists but also oncologists , pulmonologists , and thoracic surgeons should consider its reliable potential and current availability .

prostate specific antigen ( psa ) is currently used as a biomarker to diagnose prostate cancer . psa testing has been widely used to detect and screen prostate cancer . however , in the diagnostic gray zone , the psa test does not clearly distinguish between benign prostate hypertrophy and prostate cancer due to their overlap . to develop more specific and sensitive candidate biomarkers for prostate cancer , an in - depth understanding of the biochemical characteristics of psa ( such as glycosylation ) is needed . psa has a single glycosylation site at asn69 , with glycans constituting approximately 8% of the protein by weight . here , we report the comprehensive identification and quantitation of n - glycans from two psa isoforms using lc ms / ms . there were 56 n - glycans associated with psa , whereas 57 n - glycans were observed in the case of the psa - high isoelectric point ( pi ) isoform ( psah ) . three sulfated / phosphorylated glycopeptides were detected , the identification of which was supported by tandem ms data . one of these sulfated / phosphorylated n - glycans , hexnac5hex4dhex1s / p1 was identified in both psa and psah at relative intensities of 0.52 and 0.28% , respectively . quantitatively , the variations were monitored between these two isoforms . because we were one of the laboratories participating in the 2012 abrf glycoprotein research group ( gprg ) study , those results were compared to that presented in this study . our qualitative and quantitative results summarized here were comparable to those that were summarized in the interlaboratory study .

prostate cancer is one of the most common cancers diagnosed in men in the united states : there were 238 590 new cases and 29 720 deaths recorded in 2013 . prostate specific antigen ( psa ) is currently used as a diagnostic biomarker of prostate cancer . psa is an androgen - regulated serine protease and a member of the tissue kallikrein family . the concentration of psa is elevated in the blood serum of prostate cancer patients . this is due to the loss of basal cells , basement membrane , and lumen architecture , thus permitting propsa to have direct access to the peripheral circulation . the simplicity of psa testing , which measures the concentration of psa in blood serum , has prompted its wide use to detect and screen prostate cancer . however , psa testing in the diagnostic gray zone does not clearly distinguish between benign prostate hypertrophy and prostate cancer . to develop new candidate biomarkers for prostate cancer , a deeper understanding of the biochemical properties of psa glycosylation is one of the most common protein post - translational modifications ( ptms ) , and approximately 8% of psa by weight is composed of n - glycans that occupy a single glycosylation site at asn69 . several papers have reported a variable degree of sialylation of psa in sera between healthy subjects and those with prostate cancer . the major isoform , consisting of over 90% of psa , has an isoelectric point ( pi ) of 6.9 , whereas the minor one has a pi of 7.2 . the glycosylation pattern at high pi was observed to have a high level of sialylation . these properties were similarly observed in a study of psa isoenzymes from the serum of patients diagnosed with prostate cancer and the serum of patients diagnosed with benign prostate hyperplasia . psa n - glycans were also reported to be mostly core fucosylated and to have a minor presence of galnac residues with the increasing pi of the psa fraction . these different glycosylation patterns at different pi s could be employed to develop new diagnostic biomarker for detecting different prostate diseases . to effectively characterize the n - glycosylation of psa , a widely used method is liquid chromatography interfaced with mass spectrometry ( lc ms or lc ms / ms ) . the applicability of these methods to investigate aberrations in glycosylation associated with several diseases has already been demonstrated . enrichment techniques , such as lectin affinity chromatography or hydrazide chemistry , and peptide labeling have also been used in conjunction with lc ms / ms to characterize and quantify the glycosylation sites of glycoproteins . here , we report the comprehensive qualitative and quantitative glycoproteomic studies of two psa isoforms by lc ms / ms . the overarching goal is to effectively identify the different glycoforms of psa prior to prostate cancer - related glycoproteomic study . our results were compared with findings of 2012 abrf glycoprotein research group ( gprg ) study , of which we were one of the participating laboratories . the purpose of the 2012 abrf study was to establish a global overview of the approaches and methodologies in glycoproteomics using a consensus among interlaboratory data . we found a comparable pattern / level of glycosylation between our results and those of the abrf study . psa and psa - high pi isoform ( psah ) samples were obtained from lee biosolutions ( st . louis , mo ) . sodium chloride and disodium phosphate were obtained from mallinckrodt chemicals ( phillipsburg , nj ) . d , l - dithiothreitol ( dtt ) , iodoacetamide ( iaa ) , and ms - grade formic acid were purchased from sigma - aldrich ( st . louis , mo ) . hplc - grade solvents , including methanol and isopropanol , were purchased from fisher scientific ( pittsburgh , pa ) . mass spectrometry grade trypsin gold was obtained from promega ( madison , wi ) . a 20 g aliquot of psa and 5 g aliquot of psah were prepared in 50 mm pbs buffer ( ph 7.5 ) ( phosphate buffered saline containing 50 mm disodium phosphate and 150 mm sodium chloride ) . in both cases , denaturation was performed at 65 c for 1 h. the samples were then reduced by adding by adding a 1.25 l aliquot of 200 mm dtt prior to incubation at 60 c for 45 min . those reduced samples were then alkylated with the addition of a 5 l aliquot of 200 mm iaa and incubated at 37.5 c for 45 min in the dark . excess iaa was consumed through the addition of a second 1.25 l aliquot of 200 mm dtt . the reaction was allowed to proceed at 37.5 c for 30 min in the dark . trypsin was added into the samples using an enzyme / substrate ratio of 1:25 w / w , and the samples were subjected to overnight incubation at 37.5 c for 18 h. to complete the enzymatic digestion , samples were subjected to microwave digestion at 45 c and 50 w for 30 min before adding a 0.5 l aliquot of neat formic acid to the samples . finally , the samples were dried and suspended in 0.1% formic acid prior to lc lc ms / ms was carried out on dionex 3000 ultimate nano - lc system ( dionex , sunnyvale , ca ) interfaced with an ltq orbitrap velos mass spectrometer ( thermo scientific , san jose , ca ) equipped with a nano - esi source . the psa and psah digests were initially online - purified using a pepmap 100 c18 cartridge ( 3 m , 100 , dionex ) . a 2 g aliquot of psa and 0.4 g aliquot of psah digests were injected into the trapping cartridges . the purified peptides were then separated using a pepmap 100 c18 capillary column ( 75 m i.d . the separation was achieved at a 350 nl / min flow rate using the following gradient conditions : 010 min , 5% solvent b ( 98% acn with 0.1% formic acid ) ; 1040 min , ramping of solvent b from 5 to 45% ; 4045 min , ramping of solvent b from 45 to 80% ; 4550 min , maintaining solvent b at 80% ; 5051 min , decreasing solvent b to 5% ; and 5160 min , sustaining solvent b at 5% . the first scan event was a full ms scan of 3802000 m / z at a mass resolution of 15 000 . the collision induced dissociation ( cid ) and higher - energy collision dissociation ( hcd ) ms / ms were performed on the eight most intense ions seen from the first ms scan event . the second scan event was the cid ms / ms of parent ions selected from the first scan event with an isolation width of 3.0 m / z , a normalized collision energy ( ce ) of 35% , and an activation q value of 0.250 . the third scan event was set to acquire hcd ms / ms of the parent ions selected from the first scan event . the isolation width of hcd experiment was set to 3.0 m / z , and the normalized ce was set to 45% with an activation time of 0.1 ms . the protein sequence and purity of prostate specific antigen were confirmed using mascot and scaffold . proteome discoverer , version 1.2 , software ( thermo scientific , san jose , ca ) was used to generate a mascot generic format file ( * .mgf ) which was subsequently employed for database searching using mascot , version 2.3.2 ( matrix science inc . , parent ions were selected from a mass range of 30010 000 da with a minimum peak count of 1 . the parameters from mascot daemon were set to search against uniprotkb ( uniprot release 2014_06 ) . oxidation of methionine was set as a variable modification , and carbamidomethylation of cysteine was set as a fixed modification . tandem ms ions were searched within a 0.8 da mass tolerance , and the peptide sequences were identified within 10 ppm . the results from mascot were imported into scaffold 3 ( proteome software , inc . , the protein identifications were based on an ion score cutoff higher than 30 and a minimum number of peptides of two . the identification of psa and psah glycopeptides was achieved by glypid 2.0 and glycoseq with additional manual confirmation . glypid 2.0 used a combination of hcd / cid scoring and precursor mass - based searching to detect and characterize glycopeptides . because trypsin is well - known to produce incomplete cleavage at c - terminal lysine residues , we searched glycopeptides for theoretical psa sequon - containing peptides . three sequences , nksvillgr , avcggvlvhpqwvltaahcirnk , and avcggvlvhpqwvltaahcirnksvillgr , have valid identifications of glycopeptides in tryptic digest psa samples . a default glycan library was modified to consider sulfated / phosphorylated versions of glycans . it contains 413 glycan compositions with combinations of glcnac , man , fuc , and neuac . glycoseq ( available as open source software at http://sourceforge.net/projects/glycoseq/ ; manuscript in preparation ) was used to determine glycan sequences ( i.e. , topology or cartoon - graph representation ) using a de novo sequencing algorithm . briefly , starting with the y1 ion , the glycan topology is grown using cid fragmentation peaks while adhering to n - linked glycan synthesis rules . all identified glycopeptides and associated glycan structures were manually confirmed . this strategy was also applied for other glycoprotein impurities that are present in the samples . the findings of these searches are discussed in detail in the results and discussion . although psa possesses a single glycosylation site at asn69 , competitive ionization of co - eluting peptides still presents a challenge that limits the ms / ms identification of glycopeptides because a lower number of glycopeptides would be subjected to tandem ms . therefore , we created an inclusion list consisting of all theoretical m / z values possibly associated with three peptide backbones , thus ensuring the acquisition of a large number of tandem ms data to facilitate identification of more glycopeptides . the theoretical m / z values were based on every theoretical peptide backbone sequence using the glycomod tool from the expasy web site . these m / z values were used to create extracted ion chromatograms ( eics ) using xcalibur qual browser 2.1 ( thermo scientific , san jose , ca ) . a mass accuracy of 5 ppm or better was then applied to confirm ions prior to manual evaluation of tandem ms data . however , only 9% of the detected ions depicted a mass accuracy of 7 ppm . the mass range was set to full ftms scan with 7-point smoothing enabled and a mass tolerance of 10 ppm allowed . prostate specific antigen in the samples analyzed here was identified with sequence coverages of 85.6 and 86.5% from psa and psah samples , respectively ( data not shown ) . peptides containing the glycosylation sequon ( n69ks ) were not detected in the proteomics analysis , thus suggesting that the n69 residue is glycosylated . protein identifications in proteomics analysis were based on an ion score cutoff higher than 30 and a minimum number of peptides of two . according to spectral count data generated by scaffold software , spectral counts of psa accounted for 86% of the total spectral count data ( 15 identified peptides ) , whereas the spectral counts of psah accounted for 78% of the total spectral count data ( 17 peptides ) . other proteins that were detected in the case of the psa sample were triosephosphate isomerase ( 6% of total spectral counts , three peptides ) , prostaglandin - h2 d - isomerase ( 3% of the total spectral count data , two peptides ) , and growth / differentiation factor 15 ( 5% of total spectral count data , two peptides ) . other proteins detected in the case of the psah sample were triosephosphate isomerase ( 12% of the total spectral count data , eight peptides ) , prosaposin ( 5% of the total spectral count data , two peptides ) , prostaglandin - h2 d - isomerase ( 2% of the total spectral count data , two peptides ) , and growth / differentiation factor 15 ( 3% of the total spectral count data , two peptides ) . furthermore , glypid analysis using these proteins did not identify any valid glycopeptides associated with above - mentioned proteins , yet they all have the glycosylation sequences . therefore , all glycopeptides reported here are unique to psa and are not originating from the other proteins observed in the lc ms / ms analysis . additionally , the y1 ions reported here are unique to psa and are not from any contaminant proteins observed in the lc since psa is known to have only one n - linked glycosylation site at asn69 , it is adequate to interpret its glycosylations with cid or hcd tandem ms . if the cid or hcd spectra include glycan fragmentation or glycopeptide diagnostic ions in addition to the expected y1 ion ( peptide + glcnac ) , then the glycopeptides associated with asn69 were confirmed . for example , a y1 ion associated with asn69 detected at an m / z value of 602 or 601 corresponded to doubly charged nksvillgr + glcnac . it is observed in low abundance ( < 8% ) when y1 ion intensities are incredibly high . however , hexose rearrangement is not the case in this study because y1 ion intensities associated with the psa glycosylation site are not that high , as can be seen in cid ms / ms scans ( supporting information figure 1 ) . in cases when the theoretical m / z values of glycopeptides were used , they were confirmed by matching the retention times of the glycopeptides that were confirmed by tandem ms . using a 5 ppm cutoff mass accuracy allowed glycopeptides that have similar molecular weights for example , a glycopeptide possessing 2 fuc residues is only 1 da different from that possessing an neuac residue , which can be differentiated using this 5 ppm cutoff . moreover , their retention time differences were also used to distinguish them , since a glycopeptide containing neuac is retained longer by lc than a counterpart glycopeptide containing 2 fuc . overall , the use of the aforementioned criteria results in an overall averaged mass accuracy of 3.05 ppm for all identified glycopeptides . in total , tryptic digestion of the samples resulted in the formation of three peptide backbones containing the glycosylation site , namely , nksvillgr ( 1723 min ) , avcggvlvhpqwvltaahcirnk ( 2621 min ) , and avcggvlvhpqwvltaahcirnksvillgr ( 3136 min ) , as shown in figure 1a . there are 56 glycopeptides observed for psa , as summarized in table 1 . because 55 of the structures were identified on the nksvillgr backbone , the discussion will be mainly focused on this peptide backbone . one missing glycoform associated with the nksvillgr backbone is hexnac3hex3dhex1neuac1 , which was detected on the avcggvlvhpqwvltaahcirnk backbone . figure 1b d illustrates averaged full ms with observed n - glycans detected on the nksvillgr there were 11 neutral n - glycans observed at 17.5 min ( figure 1b ) , 36 monosialylated and 2 sulfated / phosphorylated n - glycans were observed at 19.5 min ( figure 1c ) , and 6 disialylated the m / z values ( charge state = 3 ) of these glycopeptides were listed with confirmed glycan structures if they were supported by tandem ms data . otherwise , the most likely structures were described as previously reported . there were 32 n - glycans confirmed by tandem ms data , as shown in table 1 . total ion chromatogram of psa digests with different retention times of three peptide backbones possessing the asn69 glycosylation site ( a ) . different retention times correspond to the detection of neutral glycoforms ( b ) , sulfated / phosphorylated ( s / p ) neutral glycoforms and monosialylated glycoforms ( c ) , and disialylated glycoforms ( d ) . the identifications of glycan structures were supported by ms / ms , which are included in supporting information figure 1 . for psah , similar retention times were detected with different types of n - glycans , as mentioned above ( figure 2a ) . in total , 57 n - glycans all of the n - glycans were detected on the nksvillgr backbone except hexnac6hex4dhex2neuac1 , which was detected only on the avcggvlvhpqwvltaahcirnk backbone . figure 2b d depicts an averaged full ms with identified n - glycans associated with the nksvillgr backbone . there were 15 neutral n - glycans observed at 17.5 min ( figure 2b ) , 29 monosialylated and 2 sulfated / phosphorylated n - glycans observed at 19.5 min ( figure 2c ) , and 10 disialylated n - glycans detected at 22 min ( figure 2d ) . manual annotations of the cid ms / ms of 37 glycopeptides are depicted in supporting information figure 1 . total ion chromatogram of psah digests with different retention times of three peptide backbones possessing the asn69 glycosylation site ( a ) . different retention times correspond to the detection of neutral glycoforms ( b ) , sulfated / phosphorylated ( s / p ) neutral glycoforms and monosialylated glycoforms ( c ) , and disialylated glycoforms ( d ) . the identifications of glycan structures were supported by ms / ms , which are included in supporting information figure 1 . assigning glycan structure from cid ms / ms is achievable because the mass difference between the glycan residues of glycopeptides , such as hex , hexnac , fuc , or neuac , is rather large . also , sequencing n - glycan structures follows some rules because specific glycosyltransferases / exoglycosidases are involved in trimming or attachment of each glycan residue during the biosynthesis of n - glycans . the precursor m / z values of glycopeptides were confirmed using a 5 ppm cutoff mass accuracy because the detection of precursor ions was achieved by an orbitrap ftms analyzer . the intriguing features of n - glycans associated with psa are the presence of an galnac residue instead of gal followed by glcnac and sulfation / phosphorylation . in this study , many n - glycans with these characteristics were detected , some of which were confirmed by tandem ms , as depicted in figure 3 . the glycan fragments of a glycopeptide with m / z 1034.1406 were assigned with diagnostic ions originating from glycan residues ( figure 3a ) . the annotation of this tandem ms demonstrates that this glycopeptide is core fucosylated and monosialylated followed by either a gal or glcnac residue . the core fucosylation was confirmed on the basis of the existence of a y1 ion ( peptide backbone + glcnac ) with fucose , which has an m / z value of 675.3 . one of the fragment peaks with an m / z value of 1303.95 represents the loss of a galnac residue . also , we confirmed that there are two co - eluting isomeric glycopeptides . in the low m / z region , diagnostic ions with m / z values of 495.16 and 698.35 confirmed galnac + neuac and glcnac + galnac + neuac glycan structures , respectively . in addition , the presence of a 657.19 m / z value affirms the presence of a glcnac + gal + neuac glycan structure . therefore , the sialylation followed by gal and galnac residues co - eluted , as confirmed in this single tandem ms . also , on the basis of the assignment of fragment ions of glycans , we distinguished between the attachment of galnac followed by antenna glcnac and bisecting structures . when a series of hexnac fragmentation were detected as associated with antenna glcnac without assignment of a glc residue , regarding bisection structures , the presence of 2glcnac + man + glcnac , 2glcnac + 2man + glcnac , and 2glcnac + 3man + glcnac was detected , which correspond to m / z values of 886 , 967 , and 1048 , respectively . two examples of identified glycoforms whose structures were confirmed by cid ms / ms . the glycan fragments of hexnac5hex4dhex1neuac1 glycopeptide with m / z 1034.1406 ( a ) support the presence of a galnac residue followed by a glcnac residue on its antenna . the annotation of a sulfated / phosphorylated ( s / p ) glycopeptide , determined as hexnac5hex4dhex1s / p1 glycopeptide with m / z 963.7589 ( b ) , suggests the occurrence of sulfation / phosphorylation on the glcnac residue before the galnac residue . figure 3b illustrates annotated tandem ms of a sulfated / phosphorylated glycopeptide corresponding to m / z 963.7589 . the sulfation / phosphorylation on a glcnac residue was determined on the basis of the detection of fragment ions at m / z 1303.87 and 1344.45 , the difference of which corresponds to a doubly charged sulfate or phosphate ion . because the peak at m / z 1344.45 originated from the loss of a galnac residue , this suggests that the occurrence of sulfation / phosphorylation is on a glcnac residue . also , the presence of m / z values of 1221.05 and 1262.04 supports the occurrence of sulfation / phosphorylation on a glcnac residue . the difference between these two assignments of sulfation / phosphorylation was 0.5 da , which fits well within the mass accuracy of cid ms / ms . on the other hand , no diagnostic ions representing a sulfated / phosphorylated glycan residue were observed from hcd ms / ms , as shown in supporting information figure 2 . overall , more of core - fucosylated and/or disialylated glycoforms were identified in psah than in normal psa . moreover , highly branched glycan structures such as tri- or tetra - antennary structures were more abundant in psah than in normal psa there was a difference in the number of identified n - glycans from the three peptide backbones , as shown in tables 1 and 2 . this might be due to the fact that the ionization efficiency of peptides becomes substantially higher as the mobile phase s organic content increases . as a result , some of the glycopeptides with minor intensities would not be subjected to tandem ms experiments . quantitative results of all identified glycopeptides associated with the three peptide backbones are illustrated in supporting information figure 4a , b for psa and psah , respectively . glycopeptides with relatively low intensities associated with the nksvillgr backbone were not detected in the other two peptides backbones , which is believed to be mainly due to competitive ionization . the quantitation of all identified glycopeptides associated with the three detected peptide backbones is summarized in tables 1 and 2 . average peak areas and standard deviation values ( std ) were measured from technical triplicates . figure 4 shows the quantitation results of n - glycans on the nksvillgr backbone . there are 66 common n - glycans with 68 total identified glycoforms that were quantitatively compared between psa and psah samples . the abundances of two glycopeptides associated with the peptides avcggvlvhpqwvltaahcirnk and avcggvlvhpqwvltaahcirnksvillgr were excluded because their ionization efficiencies might be different from those of other glycopeptides associated with the peptide nksvillgr . figure 4b depicts a separate comparison of two glycoforms that demonstrated a higher abundance for psah relative to that in all other structures . the most abundant glycoform for psa was determined to be hexnac3hex4dhex1neuac1 ( 13.3% ) . four glycopeptides possessing different glycan structures , namely , hexnac3hex6neuac1 ( 9.3% ) , hexnac4hex4neuac1 ( 7.9% ) , hexnac4hex4dhex1neuac1 ( 6.8% ) , and hexnac5hex5neuac1 ( 5.7% ) were detected as the next most abundant ions . on the other hand , glycopeptides with hexnac4hex5dhex1neuac1 ( 28.9% ) and hexnac4hex5dhex1neuac2 ( 27% ) were observed at higher intensities in the psah sample . glycopeptides possessing hexnac5hex5neuac1 ( 6.8% ) , hexnac5hex4dhex1neuac2 ( 6.6% ) , hexnac5hex5neuac2 ( 5.3% ) , and hexnac5hex4dhex1neuac1 ( 5.2% ) glycans were observed as the next most abundant ions . the glycoform of hexnac5hex4dhex1s / p was identified in both psa and psah with different abundances ( 0.52 and 0.28% , respectively ) . an interesting observation related to this structure is that the summation of the relative abundances of hexnac5hex4dhex1 and hexnac5hex4dhex1s / p from psah was determined to be comparable with the intensity of hexnac5hex4dhex1s / p from psa . on the other hand , quantitative values ( relative abundance ) of n - glycans on the nksvillgr backbone . ( b ) relative abundances of two glycoforms , which shows low - abundance glycoforms for psah by separating the extremely high abundances of hexnac4hex5dhex1neuac1 and hexnac4hex5dhex1neuac2 in psah . an image representing a glycoform was added to the side for which higher quantities were observed . the abundances of the 46 common n - glycans were compared between the psa and psah samples . for example , the glycopeptides possessing hexnac3hex6neuac1 ( > 21.7-fold ) , hexnac4hex4neuac1 ( > 13.8-fold ) , hexnac4hex3dhex1neuac1 ( > 8-fold ) , hexnac3hex4dhex1neuac1 ( > 6.5-fold ) , and hexnac5hex4neuac1 ( > 5-fold ) were detected with higher intensities in the psa sample compared with that of the psah sample . on the other hand , the glycopeptides with hexnac5hex4dhex1neuac2 ( > 9.2-fold ) , hexnac4hex5dhex1neuac2 ( > 4.8-fold ) , and hexnac4hex5dhex1neuac1 ( > 3.2-fold ) were observed with higher abundances in psah than that in psa . as one of the 26 global laboratories that participated in the 2012 abrf glycoprotein research group ( gprg ) study ( the 2012 abrf study ) , the goal of the 2012 abrf study was to provide a global overview of glycoproteomics methods using a consensus among interlaboratory data . as a result , 57 twenty six data sets collected from the participating laboratories were classified into four clusters from a to d based on data similarity . the major cluster is c , where the data were summarized from 21 laboratories , including our laboratory . according to abundances , n - glycans were grouped into three categories , including major , intermediate , and minor abundance structures . they were also an abrf - participating laboratory that identified 42 glycoforms of intact psa / psah glycoprotein using hr esi / tof ms . as shown in supporting information figure 5a , the three studies reported 85 total glycoforms associated with psa and psah , with 29 common glycoforms . seventeen additional glycoforms were commonly identified between the 2012 abrf study and this study . three glycoforms were reported by behnken et al . and were observed in our study but were not described in the abrf study . our study and ten glycoforms were described in behnken et al.s study but were not observed in our study , of which four were described in the abrf study . this discrepancy may be due to the use of different methods : bottom - up gycoproteomics in this study and top - down glycoproteomics in the behnken et al . study . as shown in supporting information figure 5b , the relative abundances of 18 major / intermediate n - glycans appeared to be comparable with that in the 2012 abrf study . in particular , the n - glycans hexnac3hex4dhex1neuac1 , hexnac4hex5dhex1neuac2 , hexnac5hex3dhex1neuac1 , and hexnac5hex4dhex1neuac1 were observed with a very high overlap in intensities . from minor n - glycans , we noticed that 6 of the 13 missing structures were sulfated / phosphorylated n - glycans with very low intensities . one sulfated / phosphorylated glycoform , hexnac5hex4dhex1s / p , was reported in the 2012 abrf study as being less than 1% , but it is present in psa at approximately twice the amount as that in psah . this particular glycoform was observed in our analysis and with a similar intensity profile variation . behnken et al . reported a single analysis of quantitation ; thus , no variation was inserted ( supporting information figure 5c ) . among the 32 common glycoforms that were observed in our study , most of them showed a comparable trend of intensities between psa and psah . with regard to the 18 major / intermediate glycoforms defined by the 2012 abrf study , 16 glycoforms were observed by behnken et al . , whereas 2 major / intermediate glycoforms were not reported , including hexnac2hex5 and hexnac4hex4dhex1 . most of the major / intermediate glycoforms show comparable abundance profiles except for hexnac4hex5dhex1neuac2 , in which its abundance in psa appeared to be 4.60-fold lower when comparing this study and the 2012 abrf study . the comprehensive qualitative and quantitative glycoproteomic studies of two psa isoforms were successfully achieved by lc , there were 56 n - glycans detected for the psa sample , whereas 57 n - glycans were observed for psah . in total , three sulfated / phosphorylated neutral n - glycans were identified , which were supported by tandem ms . moreover , several galnac residues that were monitored were followed by glcnac residues on the antenna . from the quantitative results , the levels of the glycosylation patterns the glycopeptide possessing hexnac3hex4dhex1neuac1 ( 13.3% ) was observed with the highest intensity from psa , whereas the ones with hexnac4hex5dhex1neuac1 ( 28.9% ) and hexnac4hex5dhex1neuac2 ( 27% ) were predominantly present from psah . a comparable pattern / level of glycosylation was observed between the cumulative results from the 2012 abrf study and those from a specific participating laboratory . these results can be further investigated with the aim of distinguishing glycosylation patterns between healthy and prostate cancer patients .

aims . preoperative diabetic and glycemic screening may or may not be cost effective . although hyperglycemia is known to compromise surgical outcomes , the effect of a diabetic diagnosis on outcomes is poorly known . we examine the effect of diabetes on outcomes for general and vascular surgery patients . methods . data were collected from the michigan surgical quality collaborative for general or vascular surgery patients who had diabetes . primary and secondary outcomes were 30-day mortality and 30-day overall morbidity , respectively . binary logistic regression analysis was used to identify risk factors . results . we identified 177,430 ( 89.9% ) general surgery and 34,006 ( 16.1% ) vascular surgery patients . insulin and noninsulin diabetics accounted for 7.1% and 9.8% , respectively . insulin and noninsulin dependent diabetics were not at increased risk for mortality . diabetics are at a slight increased odds than non - diabetics for overall morbidity , and insulin dependent diabetics more so than non - insulin dependent . ventilator dependence , 10% weight loss , emergent case , and asa class were most predictive . conclusions . diabetics were not at increased risk for postoperative mortality . insulin - dependent diabetics undergoing general or vascular surgery were at increased risk of overall 30-day morbidity . these data provide insight towards mitigating poor surgical outcomes in diabetic patients and the cost effectiveness of preoperative diabetic screening .

the national diabetes epidemic continues to expand , with about 1.6 million new cases each year and an overall prevalence of 23.6 million people . additional 57 million american adults are at high risk for type 2 diabetes mellitus ( t2 dm ) . the annual diagnosed diabetes incidence is projected to almost double from 8 cases per 1,000 in 2008 to about 15 in 1,000 by 2050 , with a potential total prevalence projected to increase one - fifth of the us population by 2050 . currently , 10% of americans have t2 dm , and 20% to 25% are considered prediabetic with impaired glucose tolerance and elevated fasting glucose measurements . the long - term complications of uncontrolled hyperglycemia in diabetics include neuropathy , nephropathy , retinopathy , and other chronic issues . the in - hospital morbidity of diabetics and new - onset hyperglycemics is well documented . . showed that for inpatients with new hyperglycemia or known diabetes there was a higher in - hospital mortality , increased length of stay , increased intensive care unit ( icu ) stay , increased transitional or nursing home care , a higher rate of infections , and more neurologic events compared to a normoglycemic control group . in addition , diabetes and inpatient hyperglycemia increase mortality for patient with an acute myocardial infarction [ 5 , 6 ] . the role of diabetes in outcomes of the critically ill , intensive care unit patient continues to be debated [ 710 ] . diabetics are increasingly undergoing elective and emergent surgery ; however , whether the presence of diabetes compromises surgical outcomes remains unclear . it is accepted that diabetics have an increased risk of cardiovascular disease compared to nondiabetics . have shown that diabetics undergoing general and vascular surgery with postoperative hyperglycemia exceeding 150 mg / dl had increased rates of infection . moreover , bower et al . , in a study of prospective patient registries , showed increased morbidity in known diabetic patients with and without malignancy and that diabetes was related to degree of postoperative complications in a quantitative fashion . in addition , the detriment of uncontrolled hyperglycemia in surgical patients has been investigated [ 14 , 15 ] . in specific surgical populations , particularly cardiac and vascular patients , the negative effect of diabetes on postoperative outcomes is clear . patients undergoing surgery are subject to altered carbohydrate metabolism , increased production of glucose , and increased resistance to insulin , leading to a stress - induced hyperglycemia . in cardiac surgery patients , maintenance of perioperative blood glucose levels between 125 and 200 mg dl resulted in fewer episodes of atrial fibrillation and recurrent ischemia , as well as a shorter length of stay ( los ) [ 16 , 17 ] . similarly , in the vascular population suboptimal hgb a1c levels in diabetics and non - diabetics predicted poor postoperative outcomes . diabetics undergoing lumbar spine surgery fared worse than non - diabetics for spine - specific outcomes postoperatively . however , the neurosurgical literature reveals only modest benefits linked to strict control of glycemic indexes during acute periods of brain ischemia and in specific surgical situations such as spine and tumor surgery . a diagnosis of diabetes has not been shown to be an independent risk factor for poor outcomes in general surgery . studies investigating overall surgical outcomes of patients with a known diagnosis of diabetes , irrespective of perioperative hyperglycemia , are sparse , and most of these are fraught with small sample sizes , are specific to only one surgical specialty , are or guided towards one specific outcome measure ( e.g. , surgical site infection ) . we asked the question of whether a diagnosis of insulin dependent or non - insulin dependent diabetics impacted overall postoperative morbidity and mortality in a large administrative general and vascular surgical population . we sought to answer this question through query of a comprehensive statewide surgical outcomes database . this study was categorized as exempt from review at both central michigan university ( cmu ) college of medicine and covenant healthcare . we used data from the michigan surgical quality collaborative ( msqc ) for this study . briefly , the msqc is a statewide quality improvement effort representing 52 member hospitals in the state of michigan . it is modeled after the american college of surgeons ' national surgical quality improvement program . over 200 variables are collected about surgical interventions using reliable and validated systematic sampling techniques to provide a representative sample of cases from each participating hospital . the msqc prospectively collects data on ( 1 ) thirty - day morbidity , ( 2 ) preoperative laboratory values , ( 3 ) readmissions and unplanned return to the operating room , ( 4 ) preoperative risk factors , ( 5 ) postoperative laboratory values and discharge variables , and ( 6 ) 30-day postoperative mortality . at larger hospitals , in addition , quarterly and twice - a year reports are generated for participating hospitals to aid in quality improvement . we included subjects from the msqc database from the years 2007 through 2011 who had general or vascular surgery and diabetes status recorded and were over 18 years of age . in addition , we created a secondary outcome variable , any major morbidity , from six variables in the msqc database detailing postoperative occurrences : wound , respiratory , urinary tract , central nervous system , cardiac , and other occurrences . subjects experiencing one or more of these post - operative occurrences were classified as having any major morbidity . we also used a range of variables from the msqc database to model our outcome variables . preoperative risk variables included history of diabetes , smoking , dependence on ventilator within 48 hours , copd , dialysis , steroid use , 10% weight loss within six months , and sepsis within 48 hours . subjects were classified as having a cardiac preoperative risk if they had any of the following : congestive heart failure ( chf ) within 30 days , history of myocardial infarction ( mi ) , previous percutaneous coronary intervention ( pci ) or percutaneous transluminal coronary angioplasty ( ptca ) , previous cardiac surgery , history of angina within 30 days , or on hypertensive medications . finally , we included or time , wound classification , asa class , whether cases were emergent , and type of surgery : general or vascular . chf within 30 days is defined by the msqc as chf , congestive heart failure , or pulmonary edema . a history of mi within past 6 months is defined as a history of a non - q wave or a q wave myocardial infarct in the six months prior to surgery . a history of previous pci / ptca includes balloon dilatation and stent placement ; however , it does not include valvuloplasty . previous cardiac surgery includes procedures classified as off - pump repair or utilizing cardiopulmonary bypass , valve replacement or repair , great thoracic vessel repair , cardiac transplant , repair of atrial or ventricular septal defects , left ventricular aneurysmectomy , insertion of left ventricular assist devices , and so forth ; however , it does not include insertions of pacemakers or automatic implantable cardioverter defibrillators . hypertension requiring medication is defined as a persistent elevation of systolic pressure over 140 mmhg or a diastolic pressure over 90 mmhg or requires an antihypertensive treatment , at the time the patient is being considered for surgery . emergency cases are defined as being performed as soon as possible and no later than 12 hours after hospital admission or onset of appropriate symptoms . steroid use is defined as the regular administration of oral or intravenous corticosteroids 30 days prior to surgery for a chronic condition and does not include topical , rectal , or inhalation corticosteroids or a course of short course steroids ( 10 days or less ) . we conducted binary logistic regression models for 30-day mortality ( table 2 ) for the entire sample ( model 1 ) , a subgroup of diabetics with general surgery ( model 2 ) , and a subgroup of diabetics with vascular surgery ( model 3 ) . similarly , we conducted binary logistic regression models for any morbidity ( table 3 ) for the entire sample ( model 4 ) , a sub - group of diabetics with general surgery ( model 5 ) , and a subgroup of diabetics with vascular surgery ( model 6 ) . all models were saturated models , that is , using all independent variables within each dependent variable ( mortality and morbidity ) to see which independent variables had the highest contribution to the desired outcome . all analyses were done using statistical product and service solutions ( spss ) version 20 ( ibm , armonk , ny ) . a complete demographic and variable summary of our sample is presented in table 1 . of 211,436 subjects , there were 177,430 ( 83.9% ) general surgeries and 34,006 ( 16.1% ) vascular surgeries . there were 175,639 ( 83.1% ) nondiabetics , 15,050 ( 7.1% ) insulin dependent diabetics , and 20,747 ( 9.8% ) diabetics who were dependent on oral medications . overall thirty - day mortality was two percent ( 4,083 , 1.9% ) and 13.0% ( 27,533 ) of cases were observed to have any type of major morbidity . in addition , 24% of subjects were smokers with the past 12 months , 6% had chronic obstructive pulmonary disease ( copd ) , and 50.8% had some level of cardiac risk . 84% of patients were either asa class 2 or 3 , and 52.7% of wounds were classified as clean . our goal was to determine if specific patient characteristics influenced mortality and morbidity and so we began by running regression analyses on these variables . interestingly , our preliminary analyses indicated that smoking is not associated with an increased risk of mortality . while this is unexpected based on most other studies that associate smoking with increased mortality , in our sample , there is a negative correlation between smoking and diabetes . in addition , smokers were more likely to have anesthesia types other than general anesthesia . in both cases ( diabetes and anesthesia other than general ) the risk of mortality is decreased and the association of smoking with these characteristics likely impacts the influence of smoking . due to the high multicollinearity of smoking with several other characteristics , we removed smoking as a variable from the regression analyses reported in section 3.2 . we regressed the 30-day mortality variable using three binary logistic regression models ( table 2 ) to determine the ability of different variables to predict mortality in diabetic subgroups of general and vascular surgery patients . model 1 uses the entire sample . in this case , those subjects who died within 30 days were less likely to be oral ( or = 0.78 , 95% ci 0.700.86 ) or insulin ( or = 0.83 , 95% ci 0.750.92 ) dependent diabetics than not diabetic . in our sample , diabetes does not increase the risk of 30-day mortality . vascular surgery ( compared to general surgery ) is a significant predictor of mortality ( or = 1.57 , 95% ci 1.421.72 ) for the entire group . due to our interest in the diabetic population , we conducted further subgroup analyses for 30-day mortality using only diabetic patients . model 2 ( table 2 ) is a subgroup analysis of diabetic , general surgery subjects . in this case model 3 ( table 2 ) is a subgroup analysis of diabetic , vascular surgery subjects . as with the general surgery subgroup , there is no difference between oral and insulin dependent vascular surgery , diabetics in survival . moving from the consideration of diabetes as a variable , we also included our other variables in the analyses . in general and as we would expect , ventilator dependence , copd , cardiac risk factors , current dialysis , steroid use , weight loss , sepsis , emergent cases , increased asa class , and any wound classification increase the risk of 30-day mortality . in summary , our models for 30-day mortality show that for our entire sample , the presence of diabetes does increase the risk of 30-day mortality . there do not appear be significant differences in variables that contribute to mortality , when comparing models for diabetic subgroups of general and vascular surgery and , as expected , ventilator dependence , copd , current dialysis , weight loss , sepsis , emergent cases , increased asa and any wound classification increase the risk of 30 day mortality in diabetic patients . we also regressed the any morbidity variable using three binary logistic regression models ( table 3 ) to determine the ability of different variables to predict any type of morbidity in different subgroups of surgery patients . subjects with any type of morbidity were more likely to be insulin dependent ( or = 1.11 , 95% ci 1.061.16 ) diabetics than oral dependent diabetics , or not diabetic . vascular surgery ( compared to general surgery ) is also a significant predictor of morbidity ( or = 1.69 , 95% ci 1.621.75 ) . model 5 ( table 3 ) is a subgroup analysis of diabetic , general surgery subjects . in this case , insulin dependent diabetics are more likely to have any morbidity ( or = 1.20 , 95% ci 1.121.29 ) when compared to diabetics who are dependent on oral medications . model 6 ( table 3 ) is a subgroup analysis of diabetic , vascular surgery subjects . again , we found that insulin dependent diabetics are more likely to have any morbidity ( or = 1.24 , 95% ci 1.121.38 ) when compared to oral medication dependent . again , considering other variables across all models for any morbidity , we found that in general , ventilator dependence , copd , cardiac risk factors , current dialysis , steroid use , weight loss , sepsis , emergent cases , increased asa class , and any wound classification increase the risk of any morbidity . in summary , models for any morbidity show that for our entire sample , insulin dependent diabetics have an increased risk for any morbidity ( or = 1.11 , 95% ci 1.061.16 ) when compared to non - diabetics . this increased risk is also present in subgroup analyses comparing insulin dependent diabetics to oral medication dependent diabetics for general ( or = 1.20 , 95% ci 1.121.29 ) and vascular ( or = 1.24 , 95% ci 1.121.38 ) surgeries . , there do not appear be significant differences in variables that contribute to morbidity , when comparing models for diabetic subgroups of general and vascular surgical patients . as expected , ventilator dependence , copd , current dialysis , weight loss , sepsis , emergent cases , increased asa and any wound classification increase the risk of any morbidity in diabetic patients . our objective was to understand the postoperative risk for morbidity and mortality in diabetics versus non - diabetic patients undergoing general or vascular surgery . we found that , with regard to mortality , the presence of diabetes is not predictive of mortality ; however , vascular surgery itself is predictive of mortality . there are no significant differences in variables that contribute to mortality , when comparing diabetic and non - diabetic subgroups for both general and vascular surgery . in our morbidity analysis , insulin dependent diabetics have an increased risk for any morbidity compared to non - diabetics or non - insulin ( oral ) dependent diabetics in the general and vascular surgery subgroups . our multivariate analyses confirm findings in the literature of risk factors in general surgery that contribute to morbidity and mortality , such as ventilator dependence , copd , cardiac risk factors , dialysis , steroid use , significant weight loss , sepsis , emergent cases , and increasing asa class . interestingly , notable differences were detected when analyzing separate groups of diabetics and non - diabetics undergoing general surgery . for instance , male non - diabetics undergoing general surgery have an increased risk for any morbidity as compared male diabetics . it is possible that higher risk male diabetics were more likely denied elective surgical intervention , as opposed to higher risk non - diabetic males , resulting in our finding . as in the mortality analysis , there do not appear be significant differences in variables that contribute to morbidity for diabetics and non - diabetics specifically undergoing vascular surgery . this is the first study in the literature of overall post - operative outcomes ( morbidity and mortality ) of diabetics undergoing general surgery with such a large population of patients . examined 55,000 diabetic patients in the veterans heath administration national surgical quality improvement program database and analyzed the effect of different levels of preoperative hyperglycemia on the rate of wound infection . however , in their cohort , there was no comparison of diabetics to a non - diabetic control group , as that was not the purpose of their study . within their study , they showed that higher preoperative serum glucose levels correlated with worse wound - related outcomes ; however , preoperative hba1c ( long - term glucose control in diabetics ) had no association with infection rates . in a study of preoperative hba1c levels , o'sullivan found that , after multivariate analysis , elevated hba1c levels were predictive of morbidity in non - diabetic patients , but not in diabetics , undergoing vascular surgery . the o'sullivan study does highlight the disturbing problem of undiagnosed diabetics undergoing surgery without proper control of their glycemia . the lack of correlation between diabetic diagnosis and mortality in general surgery in our study is not unique in the literature , where a clear consensus on the issue can not be found . . showed in a study of over 600 patients undergoing hepatic resection that multivariate analysis did not identify diabetes as an independent variable having an impact on mortality and that overall complications were equally frequent between diabetic and non - diabetic patients . diabetes was also shown not to be a statistically significant predictor of overall mortality or cardiac morbidity in patients undergoing open aortic aneurysm repair . in a matched cohort retrospective review , diabetes did not increase the mortality rates of cardiac surgery , but increased the risk for renal and neurological complications , blood transfusion , reoperation , and length of icu stay . however , a smaller study than ours from finland , with a long 7-year followup , showed quite convincingly that short - term and long - term mortality was higher in diabetics versus non - diabetics undergoing similar noncardiac surgeries . described a high mortality , due mainly to cardiovascular complications , in cohort of diabetics undergoing noncardiac surgery ; however , there was no non - diabetic control group in the study . our study revealed that the presence of cardiac risk predicted overall morbidity in diabetic and non - diabetics undergoing general and vascular surgery equally . however , cardiac risk predicted mortality only in non - diabetic general and vascular surgery patients . conversely , hollenberg et al . showed that diabetes was one of five risk factors predicted of cardiac events in patients undergoing non - cardiac surgery . one difference that may account for the results is that the latter study began from the premise of a population of patients that were already high risk for a cardiac event occurring and underwent non - cardiac surgery . in contrast , our study evaluated all patients from the perspective of being diabetic or not and assessing factors predictive of morbidity and mortality in diabetic versus non - diabetic , that is , a difference of study design to test a different hypothesis . most studies regarding surgical outcomes of diabetics involve studying the presence of perioperative hyperglycemia in diabetics or non - diabetics and its effect on morbidity and mortality . currently , the literature regarding optimal target glucose ranges for the perioperative patient is unclear . the ada recommends screening for diabetes preoperatively in select populations , especially in obese patients . the united states preventive services task force ( uspstf ) recommends screening only hypertensive adults for diabetes . some of the first studies to evaluate the benefits of tight glucose control were the leuven study in 2001 and 2006 , which sparked widespread interest in intensive insulin therapy in critically ill patients [ 33 , 34 ] . some researchers relate the benefits identified in this study to our current understanding of the bodies stress response . in the leuven study , the majority of the critically ill patients studied had spent more than five days in the intensive care unit . the effects of an anabolic steroid , such as insulin , may be more beneficial as the response to the acute injury resolves . however , during the acute onset of stressful stimulus such as surgery or possibly traumatic events the effect of treatment with these hormones has not been fully explained and may be harmful . despite the fact , improvements in surgical outcome have been seen in patients with tight intraoperative glucose control . as previously mentioned , a growing body of evidence suggests that even small changes in glycemic ranges are associated with impaired outcomes . in one study , in patients undergoing cardiac surgery , a 20 mg / dl increase in mean intraoperative glucose was associated with increased risk of more than 30% of adverse outcome . however , it is important to make the distinction that these findings can not be generalized to general surgical patients . the broad array of patient demographics and options for surgical care encompass an extensive variety of settings , and as many operations performed by the modern general surgeon are now performed in the ambulatory setting , the assessment and management of the dysglycemia patient continue to be a looming challenge . the msqc is an excellent source of data for studying general and specific 30-day morbidity for various surgical procedures on a larger scale . the administrative database provides risk and reliability adjusted reports for over 52 participating hospitals in the state of michigan . the other option to answer our question would have been to use the national surgical quality improvement program ( nsqip ) database or the national inpatient sample ( nis ) hospital discharge data . the nis system , although widely available and currently the largest all - payer database of hospital discharge records , poses a number of limitations . first , inconsistencies across states and providers in the data element reporting may compromise data quality . second , other data elements that would be needed to analyze risk factors for certain preoperative patient populations , such as race / ethnicity , detailed test results , whether a condition was present on admission , functional status , severity of illness , and behavioral risk factors , would be absent in many cases . first , collaboration in the msqc is purely voluntary , and these hospitals may have different characteristics from those who have chosen not to participate . therefore , our results may not be generalizable to the entire cohort of statewide hospitals . moreover , michigan hospital practices may not be representative of us hospitals , although this database includes academic and community as well as various sizes and geographic locations within the state . secondly , data in the msqc is collected retrospectively and analyzed by the end - user retrospectively and therefore will have the biases inherent to such a study design . third , the present analysis does not address procedure specific outcomes for diabetes . for example , although a cholecystectomy may not have the level of morbidity or mortality as a pancreatic operation for a diabetic , the volume of cholecystectomies in the database outweighs that of complex pancreatic operations . although cases are easily distinguished between vascular and nonvascular surgeries , we did not separate out or group the 3000 cpt codes to identify procedure specific risk . in summary , we found that a diagnosis of diabetes ( insulin or oral dependent ) did not affect outcomes in general surgical patients . however , the presence of insulin - dependent diabetes was associated with increased morbidity in the vascular and general surgery subgroups greater than non - diabetics or non - insulin dependent diabetics . however , the reassuring fact that the presence of diabetes did not have an overt effect on mortality , along with morbidity risk of insulin - dependent diabetes , may greatly impact the surgeon - patient preoperative dialogue and our ability to appropriately risk - stratify and risk - optimize these patients .

purposetransforming growth factor-induced epithelial mesenchymal transition ( emt ) is one of the main causes of posterior capsular opacification ( pco ) or secondary cataract ; however , the signaling events involved in tgf-induced pco have not been fully characterized . here , we focus on examining the role of -catenin / cyclic amp response element binding protein ( creb)-binding protein ( cbp ) and -catenin / t - cell factor ( tcf)-dependent signaling in regulating cytoskeletal dynamics during tgf-induced emt in lens epithelial explants.methodsrat lens epithelial explants were cultured in medium m199 in the absence of serum . explants were treated with tgf-2 in the presence or absence of the -catenin / cbp interaction inhibitor , icg-001 , or the -catenin / tcf interaction inhibitor , pnu-74654 . western blot and immunofluorescence experiments were carried out and analyzed.resultsan increase in the expression of fascin , an actin - bundling protein , was observed in the lens explants upon stimulation with tgf- , and colocalized with f - actin filaments . inhibition of -catenin / cbp interactions , but not -catenin / tcf interactions , led to a decrease in tgf-induced fascin and stress fiber formation , as well as a decrease in the expression of known markers of emt , -smooth muscle actin ( -sma ) and matrix metalloproteinase 9 ( mmp9 ) . in addition , inhibition of -catenin / cbp dependent signaling also prevented tgf-induced downregulation of epithelial cadherin ( e - cadherin ) in lens explants.conclusionswe show that -catenin / cbp dependent signaling regulates fascin , mmp9 , and -sma expression during tgf-induced emt . we demonstrate that -catenin / cbp dependent signaling is crucial for tgf-induced emt in the lens .

recombinant human tgf-2 was obtained from r&d systems ( minneapolis , mn , usa ) . ( st . louis , mo , usa ) and calbiochem - emd millipore ( temecula , ca , usa ) , respectively . as to primary antibodies , fascin was from millipore ( temecula , ca , usa ) , e - cadherin from bd transduction laboratories ( lexington , ky , usa ) , active -catenin ( clone 8e7 ) from upstate ( lake placid , ny , usa ) , mmp9 and gapdh from abcam ( cambridge , ma , usa ) , -sma fluorescein isothiocyanate ( fitc ) conjugated and unconjugated from sigma - aldrich corp . , pan actin from abcam . all secondary antibodies for immunofluorescence staining were purchased from molecular probes ( invitrogen , eugene , or , usa ) ; secondary antibodies for western blots were obtained from li - cor biosciences ( lincoln , ne , usa ) . rats for this research were obtained and cared for in accordance with the institutional animal care and use committee and canadian council of animal care rules . all animal procedures were carried out in accordance with the arvo statement for the use of animals in ophthalmic and vision research and were approved by the ethics committee on animal research of mcmaster university ( hamilton , ontario , canada ) . lens epithelial explants were obtained from 17- to 19-day - old wistar rats ( charles river laboratories , montreal , canada ) as previously described . briefly , rat lenses were isolated and placed in 35-mm culture dishes containing prewarmed , serum - free medium m199 , supplemented with antibiotics ( all from invitrogen ) . the posterior suture of the lens was located and a small incision was made to remove the posterior capsule . this revealed the fiber mass , which was then gently peeled off the anterior epithelium . once separated , the epithelium was then pinned to the culture dish with a blunt tool exposing lecs to culture medium . twenty - four hours after explanting , confluent epithelial explants were left untreated in serum - free m199 or treated with tgf-2 at 6 ng / ml for 48 hours . -catenin inhibitors icg-001 and pnu-74654 were dissolved in dimethyl sulphoxide ( dmso ) at a final concentration of 17.6 and 10 mm , respectively . the working concentration for icg-001 and pnu-74654 was 10 m ( 1.14 l/2 ml ) and 20 m ( 4 l/2 ml ) , respectively . the working concentration of icg-001 and pnu-74654 was determined based on dose response experiments performed on lens epithelial explants . for -catenin inhibitor studies , lens explants were pretreated with icg-001 ( 5 m ) or pnu-74654 ( 20 m ) for 1 hour before coincubating with tgf-2 ( 6 ng / ml ) for 48 hours . lens explants were fixed with 10% neutral buffered formalin for 15 minutes and washed with phosphate - buffered saline ( pbs ; invitrogen , carlsbad , ca , usa ) . explants were then detached from the culture dish and permeabilized at room temperature for 1 hour in 0.1% triton x-100 , 0.5% sodium dodecyl sulphate ( sds ) , and 5% donkey serum , followed by incubating overnight at 4c with primary antibody at a dilution of 1:200 in pbs . following further pbs washes , explants were incubated with corresponding secondary antibody , alexa fluor 488 or alexa fluor 568 ( invitrogen ) , at a dilution of 1:200 at room temperature for 1 hour , and washed three times with pbs ( 10 minutes for each wash ) . the explants were then mounted in prolong gold antifade reagent with 4,6-diamidino-2-phenylindole ( dapi , invitrogen ) to visualize nuclei . immunostained explants were analyzed using a zeiss 510 meta confocal microscope ( carl zeiss canada , toronto , canada ) or zeiss apotome inverted fluorescence microscope ( carl zeiss canada ) or leica dmra2 fluorescence microscope ( leica microsystems canada , richmond hill , canada ) equipped with a q - imaging retiga 1300i fast digital camera ( q - imaging , surrey , bc , canada ) . minor adjustments were made to the captured images using photoshop cs3 ( adobe systems , mountain view , ca , usa ) . lens explants ( 57 per sample ) were lysed in triton lysis buffer ( 50 mm tris , ph 8.0 , 150 mm nacl , 1% triton x-100 ) with complete mini , edta - free protease / phosphatase inhibitor cocktail ( roche , laval , qc , canada ) . equal amounts of total protein ( 5 g ) were loaded on a gel , as determined by dc protein assay ( bio - rad , mississauga , on , canada ) , and sds - page was performed . resolved bands were transferred onto a nitrocellulose membrane ( pall life sciences , pensacola , fl , usa ) . membranes were blocked with odyssey blocking buffer ( li - cor biosciences ) for 1 hour and incubated with primary antibody overnight at 4c . primary antibodies were diluted in tris - buffered saline with 0.1% tween-20 ( tbst ) at a dilution of 1:500 for fascin ( abcam ) , 1:1000 for e - cadherin ( millipore ) and mmp9 ( abcam ) , and 1:10,000 for -sma ( sigma - aldrich corp . ) and actin and gapdh ( abcam ) . blots were washed in tbst and probed with corresponding li - cor near - infrared respective secondary antibodies ( either 680- or 800-nm wavelengths ) at a dilution of 1:10,000 for 1 hour . the blots were then washed with tbst and scanned using odyssey near - infrared scanning system ( li - cor biosciences ) . densitometry on visualized bands was performed using fiji image processing software ( imagej , http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . the peaks from the densitometric scans ( imagej ) were used to calculate the average density and the ratio between protein of interest and loading control . the average band density was further used to make graphs and calculate standard deviation using graphpad prism software ( la jolla , ca , usa ) . unpaired tukey 's multiple comparison test was performed on the averages to calculate the p values using graphpad prism software . recombinant human tgf-2 was obtained from r&d systems ( minneapolis , mn , usa ) . ( st . louis , mo , usa ) and calbiochem - emd millipore ( temecula , ca , usa ) , respectively . as to primary antibodies , fascin was from millipore ( temecula , ca , usa ) , e - cadherin from bd transduction laboratories ( lexington , ky , usa ) , active -catenin ( clone 8e7 ) from upstate ( lake placid , ny , usa ) , mmp9 and gapdh from abcam ( cambridge , ma , usa ) , -sma fluorescein isothiocyanate ( fitc ) conjugated and unconjugated from sigma - aldrich corp . , pan actin from abcam . all secondary antibodies for immunofluorescence staining were purchased from molecular probes ( invitrogen , eugene , or , usa ) ; secondary antibodies for western blots were obtained from li - cor biosciences ( lincoln , ne , usa ) . rats for this research were obtained and cared for in accordance with the institutional animal care and use committee and canadian council of animal care rules . all animal procedures were carried out in accordance with the arvo statement for the use of animals in ophthalmic and vision research and were approved by the ethics committee on animal research of mcmaster university ( hamilton , ontario , canada ) . lens epithelial explants were obtained from 17- to 19-day - old wistar rats ( charles river laboratories , montreal , canada ) as previously described . briefly , rat lenses were isolated and placed in 35-mm culture dishes containing prewarmed , serum - free medium m199 , supplemented with antibiotics ( all from invitrogen ) . the posterior suture of the lens was located and a small incision was made to remove the posterior capsule . this revealed the fiber mass , which was then gently peeled off the anterior epithelium . once separated , the epithelium was then pinned to the culture dish with a blunt tool exposing lecs to culture medium . twenty - four hours after explanting , confluent epithelial explants were left untreated in serum - free m199 or treated with tgf-2 at 6 ng / ml for 48 hours . -catenin inhibitors icg-001 and pnu-74654 were dissolved in dimethyl sulphoxide ( dmso ) at a final concentration of 17.6 and 10 mm , respectively . the working concentration for icg-001 and pnu-74654 was 10 m ( 1.14 l/2 ml ) and 20 m ( 4 l/2 ml ) , respectively . the working concentration of icg-001 and pnu-74654 was determined based on dose response experiments performed on lens epithelial explants . for -catenin inhibitor studies , lens explants were pretreated with icg-001 ( 5 m ) or pnu-74654 ( 20 m ) for 1 hour before coincubating with tgf-2 ( 6 ng / ml ) for 48 hours . lens explants were fixed with 10% neutral buffered formalin for 15 minutes and washed with phosphate - buffered saline ( pbs ; invitrogen , carlsbad , ca , usa ) . explants were then detached from the culture dish and permeabilized at room temperature for 1 hour in 0.1% triton x-100 , 0.5% sodium dodecyl sulphate ( sds ) , and 5% donkey serum , followed by incubating overnight at 4c with primary antibody at a dilution of 1:200 in pbs . following further pbs washes , explants were incubated with corresponding secondary antibody , alexa fluor 488 or alexa fluor 568 ( invitrogen ) , at a dilution of 1:200 at room temperature for 1 hour , and washed three times with pbs ( 10 minutes for each wash ) . the explants were then mounted in prolong gold antifade reagent with 4,6-diamidino-2-phenylindole ( dapi , invitrogen ) to visualize nuclei . immunostained explants were analyzed using a zeiss 510 meta confocal microscope ( carl zeiss canada , toronto , canada ) or zeiss apotome inverted fluorescence microscope ( carl zeiss canada ) or leica dmra2 fluorescence microscope ( leica microsystems canada , richmond hill , canada ) equipped with a q - imaging retiga 1300i fast digital camera ( q - imaging , surrey , bc , canada ) . minor adjustments were made to the captured images using photoshop cs3 ( adobe systems , mountain view , ca , usa ) . lens explants ( 57 per sample ) were lysed in triton lysis buffer ( 50 mm tris , ph 8.0 , 150 mm nacl , 1% triton x-100 ) with complete mini , edta - free protease / phosphatase inhibitor cocktail ( roche , laval , qc , canada ) . equal amounts of total protein ( 5 g ) were loaded on a gel , as determined by dc protein assay ( bio - rad , mississauga , on , canada ) , and sds - page was performed . resolved bands were transferred onto a nitrocellulose membrane ( pall life sciences , pensacola , fl , usa ) . membranes were blocked with odyssey blocking buffer ( li - cor biosciences ) for 1 hour and incubated with primary antibody overnight at 4c . primary antibodies were diluted in tris - buffered saline with 0.1% tween-20 ( tbst ) at a dilution of 1:500 for fascin ( abcam ) , 1:1000 for e - cadherin ( millipore ) and mmp9 ( abcam ) , and 1:10,000 for -sma ( sigma - aldrich corp . ) and actin and gapdh ( abcam ) . blots were washed in tbst and probed with corresponding li - cor near - infrared respective secondary antibodies ( either 680- or 800-nm wavelengths ) at a dilution of 1:10,000 for 1 hour . the blots were then washed with tbst and scanned using odyssey near - infrared scanning system ( li - cor biosciences ) . densitometry on visualized bands was performed using fiji image processing software ( imagej , http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . the peaks from the densitometric scans ( imagej ) were used to calculate the average density and the ratio between protein of interest and loading control . the average band density was further used to make graphs and calculate standard deviation using graphpad prism software ( la jolla , ca , usa ) . unpaired tukey 's multiple comparison test was performed on the averages to calculate the p values using graphpad prism software . additionally , increased expression of fascin and its actin binding activity have also been demonstrated to play a key role in cell adhesion and migration during emt in cancer cells . to investigate the expression pattern of fascin during tgf-induced emt in our lens system , primary rat lens epithelial explants were treated with tgf-2 , a known emt inducer , followed by immunostaining for fascin . immunostaining revealed a low - level , basal expression of fascin in untreated lens explants ( fig . 1a ) , along with an absence of f - actin staining , the latter of which was determined by rhodamine phalloidin . in comparison , a significant increase in expression of fascin was observed in lens explants following treatment with tgf-2 for 48 hours , and this was correlated with the appearance of stress fibers ( f - actin filaments ) ( fig . importantly , total actin protein levels remained similar between untreated and tgf-2treated lens explants ( supplementary fig . interestingly , we observed a significant increase ( 13% , * p < 0.007 , n 1b ) in tgf-2treated explants , and this was specific to the filaments of fascin ( arrowheads , fig . finally , to quantify the increased expression of fascin upon tgf-2 stimulation , we performed western blot analyses on lysates from untreated and tgf-2treated explants . a significant increase in the expression of fascin protein was observed in tgf-2treated explants when compared to the untreated controls ( fig . stained slides were imaged using 63 water lens of zeiss lsm510 confocal microscope and analyzed using zeiss lsm software . ( b ) a graph for colocalization of fascin with actin was made by scoring at least six different areas of the untreated and tgf-2treated lens explants ( ( c ) western blot analysis for fascin and gapdh was carried out using protein lysates extracted from untreated lens explants and tgf-2treated lens explants . densitometric quantification ( lower ) shows fold increase in fascin normalized to untreated lens explants ( n = 4 , * wnt/catenin signaling has been shown to play a critical role during tgf-induced emt . in lens explants , -catenin translocates to the nucleus from the cell margin following treatment with tgf-. to corroborate our results with previous findings , we initially examined the status of -catenin following tgf-2 stimulation in the lens explants . immunofluorescence staining showed membranous localization of -catenin in untreated explants , whereas delocalization of -catenin from the cell membrane was observed in the lens explants at 20 hours post tgf-2 treatment ( inset , panel 2 , fig . additionally , an increase in nuclear accumulation of -catenin was observed in the explants incubated with tgf-2 for 20 hours ( inset , panel 2 , fig . a complete delocalization of -catenin from the cell membrane and concomitant increase in nuclear localization of -catenin were observed upon incubation of explants with tgf-2 at the later time point of 48 hours ( inset , panel 3 , fig . untreated lens explant shows membranous localization of -catenin ( inset , merge image , upper ) that is disrupted upon stimulation with tgf-2 starting at 20 hours post treatment . tgf-2 induces nuclear localization of -catenin ( inset , merge image , panels 2 , 3 ) . during tgf-induced emt , it has been proposed that -sma expression is regulated by the interaction between -catenin and smad3 , which is further dependent on the transcriptional coactivator cbp . inhibition of the -catenin / cbp complex by the inhibitor icg-001 prevents -catenin and smad3 interaction and has been shown to inhibit tgf-induced emt in other systems . we therefore utilized icg-001 to determine whether disruption in -catenin / cbp dependent signaling in tgf-2treated explants can prevent fascin expression and emt . lens explants were treated with tgf-2 in the presence or absence of icg-001 ( 10 m ) for 48 hours . upon stimulation with tgf-2 , lecs showed actin cytoskeleton remodeling , as demonstrated by a significant increase in both fascin and -sma expression ( inset , fig . interestingly , tgf-2induced expression of fascin was prevented by cotreatment with icg-001 , and this suppression in fascin expression was further confirmed by western blot analysis ( fig . 3b ) . a significant decrease in tgf-2induced expression of -sma was also observed upon disruption of -catenin / cbp dependent signaling by cotreatment with icg-001 when compared to tgf--treated lens explants ( inset , fig . 3a ; figs . untreated and icg-001treated explants did not exhibit any induction in either fascin or -sma expression . a previous report had shown that inhibition of wnt/catenin signaling by icg-001 induces cytotoxicity in multiple myeloma cells . however , we did not observe any significant increase in cell death in the lens explants treated with icg-001 alone or in combination with tgf-2 . effect of inhibition of -catenin / cbp dependent signaling by icg-001 on tgf-induced emt of lens explants . ( a ) untreated , icg - only , tgf-2only , and tgf-2 and icg-001-treated lens explants were immunostained for fascin and -sma . slides were imaged using 40 lens of zeiss apotome microscope and analyzed using zeiss zen software . ( b ) western blot analysis for fascin , -sma , and gapdh was carried out using protein lysates extracted from untreated , icg - treated , tgf-2treated , and tgf-2 and icg-001-treated lens explants . ( c ) densitometric quantification of -sma indicating fold reduction in -sma expression normalized to untreated lens explants ( n = 3 , * * p < 0.005 , * * * p < 0.0005 ) . since we had shown that fascin colocalizes with f - actin in tgf-2treated explants ( fig . 1 ) , we further examined f - actin expression following treatment with tgf-2 in the presence and absence of icg-001 . an increase in actin polymerization was observed in tgf-2treated explants , whereas coincubation with icg-001 completely inhibited tgf-2induced f - actin polymerization ( fig . 4a ) . inhibition of -catenin / cbp dependent signaling by icg-001 prevents stress fiber formation and tgf-induced e - cadherin delocalization . lecs incubated with icg only , tgf-2 only , and tgf-2 and icg-001 immunostained for actin and e - cadherin . stained slides were imaged using 40 lens of zeiss apotome microscope and analyzed using zeiss zen software . ( a ) actin staining reveals a decrease in tgf-2induced stress fiber formation ( panel 3 ) with inhibition of -catenin / cbp interaction by icg-001 ( panel 4 ) . ( b ) the tgf-2induced loss of membranous e - cadherin ( panel 3 ) was restored by icg-001 ( panel 4 ) . our lab and others have shown that during tgf-induced emt of lecs , e - cadherin is degraded , and its localization at the cell membrane is lost . loss of e - cadherin leads to a dissociation of the e - cadherin/catenin complex that results in translocation of -catenin to the nucleus and activation of its downstream targets . thus , we examined the effects of tgf-2 on e - cadherin expression in the presence of icg-001 . untreated lens explants showed hexagonal , cobblestone arrangement with e - cadherin exhibiting membranous localization ( inset , fig . 4b ) , which was also observed in cells treated with icg-001 alone . in comparison , explants treated with tgf-2 exhibited a substantial decrease in the localization of e - cadherin at the cell membrane . in comparison , coincubation with tgf-2 and icg-001 was able to partially rescue the tgf-induced e - cadherin delocalization ( fig . these results suggest the involvement of -catenin / cbp dependent signaling in e - cadherin localization downstream of tgf- signaling in lens explants . previous studies have speculated that -catenin / tcf dependent signaling may regulate tgf-induced cataract formation and emt of lens epithelial explants . to investigate this further , we inhibited the -catenin / tcf dependent signaling pathway utilizing pnu-74654 ( pnu ) , an inhibitor that selectively inhibits the interaction between nuclear -catenin and tcf4 . we did not observe any change in the cellular morphology in the lens explants cotreated with tgf-2 and pnu when compared to the cells of the explant incubated with tgf-2 . we first examined the change in expression of -sma following cotreatment of lens explants with tgf and pnu as compared to cells treated with tgf alone . we did not observe any change in expression of -sma positive stress fibers ( fig . these findings revealed that while untreated explants showed membranous localization of e - cadherin ( arrowhead , fig . 6a ) , explants treated with tgf-2 exhibited a decrease in the localization of e - cadherin at the cell membrane ( arrowhead , fig . as expected , we did not observe any change in the localization of e - cadherin in lens explants cotreated with tgf-2 and pnu when compared to explants treated with tgf-2 alone . these results were confirmed by western blot analyses showing that cotreatment with pnu did not prevent the tgf-induced decrease in e - cadherin expression ( figs . c ) . inhibition of -catenin / tcf dependent signaling fails to protect tgf-induced -sma expression and f - actin polymerization . ( a ) lecs incubated with tgf-2 and tgf-2 and pnu for 48 hours immunostained for -sma , and with rhodamine phalloidin for f - actin , and imaged using 40 lens of leica dmra2 fluorescent microscope . scale bar : 100 m ( b ) representative western blot of -sma protein from untreated , tgf-2treated , pnu - treated , and tgf-2 and pnu - treated lecs ( n = 3 ) . ( a ) e - cadherin stained untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs imaged using zeiss lsm 510 confocal microscope . ( b ) total protein from untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs was subjected to western blot analysis for e - cadherin and gapdh ( n = 3 ) . ( c ) graph showing average relative density sd of e - cadherin normalized to untreated lecs ( n = 3 , * p < 0.05 , * * p < 0.005 ) . previous studies have shown that fascin gene expression is regulated by the wnt/catenin signaling pathway , which promotes cancer cell migration and invasion . more specifically , the fascin promoter has been shown to contain binding sites for tcf / lef , transcription factors that are activated specifically by -catenin . to investigate the role of -catenin / tcf interaction on fascin expression , rat lens epithelial explants immunofluorescence results revealed no change in the expression of fascin in lens explants cotreated with tgf-2 and pnu when compared to explants treated with tgf-2 alone ( fig . this finding was further confirmed using western blot analysis , where the protein extracts from lens explants cotreated with tgf-2 and pnu revealed no change in the levels of fascin protein when compared to explants treated with tgf-2 alone ( figs . ( a ) untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs were stained for fascin and imaged using 40 lens of leica dmra2 fluorescent microscope ( n = 3 ) . ( b ) western blot analysis for fascin and gapdh was carried out using protein lysates from untreated , tgf-2treated , pnu - treated , and tgf-2 and pnu - cotreated lecs ( n = 3 ) . ( c ) graph representing the relative density sd of fascin normalized to untreated lecs ( n = 3 , * p < 0.05 ) . during emt , cells undergoing transformation express mesenchymal markers , such as the matrix metalloproteinases ( mmps ) , as they lean toward a mesenchymal phenotype . previous reports from our lab show an increase in mmp9 protein levels in the supernatants from tgf-2treated whole rat lenses . first , to show the mesenchymal phenotype of rat lens explants induced by tgf-2 and to investigate whether -catenin / cbp dependent signaling has a role in the regulation of mmp9 expression , as observed in other systems , we examined mmp9 expression in tgf-2treated lens explants in the presence and absence of icg-001 . western blot analyses revealed a significant 2.7 0.8-fold reduction ( p < 0.02 ) in tgf-2induced mmp9 protein levels upon inhibition of -catenin / cbp dependent signaling by icg-001 compared to explants treated with tgf-2 alone ( figs . next , we investigated the ability of the pnu inhibitor to block tgf-2induced mmp9 expression and found that it was ineffective ( data not shown ) . these results suggest a role for -catenin / cbp dependent signaling in regulating mmp9 expression during tgf-induced emt in lens epithelial explants . ( a ) untreated , icg-001treated , tgf-2treated , and tgf-2 and icg-001cotreated lens explants were subjected to western blot analysis for mmp9 and gapdh ( n = 3 ) . ( b ) graph representing the relative density sd of mmp9 normalized to untreated lecs ( n = 3 , * p < 0.02 across all samples ) . additionally , increased expression of fascin and its actin binding activity have also been demonstrated to play a key role in cell adhesion and migration during emt in cancer cells . to investigate the expression pattern of fascin during tgf-induced emt in our lens system , primary rat lens epithelial explants were treated with tgf-2 , a known emt inducer , followed by immunostaining for fascin . immunostaining revealed a low - level , basal expression of fascin in untreated lens explants ( fig . 1a ) , along with an absence of f - actin staining , the latter of which was determined by rhodamine phalloidin . in comparison , a significant increase in expression of fascin was observed in lens explants following treatment with tgf-2 for 48 hours , and this was correlated with the appearance of stress fibers ( f - actin filaments ) ( fig . importantly , total actin protein levels remained similar between untreated and tgf-2treated lens explants ( supplementary fig . interestingly , we observed a significant increase ( 13% , * p < 0.007 , n 1b ) in tgf-2treated explants , and this was specific to the filaments of fascin ( arrowheads , fig . finally , to quantify the increased expression of fascin upon tgf-2 stimulation , we performed western blot analyses on lysates from untreated and tgf-2treated explants . a significant increase in the expression of fascin protein was observed in tgf-2treated explants when compared to the untreated controls ( fig . stained slides were imaged using 63 water lens of zeiss lsm510 confocal microscope and analyzed using zeiss lsm software . ( b ) a graph for colocalization of fascin with actin was made by scoring at least six different areas of the untreated and tgf-2treated lens explants ( ( c ) western blot analysis for fascin and gapdh was carried out using protein lysates extracted from untreated lens explants and tgf-2treated lens explants . densitometric quantification ( lower ) shows fold increase in fascin normalized to untreated lens explants ( n = 4 , * wnt/catenin signaling has been shown to play a critical role during tgf-induced emt . in lens explants , -catenin translocates to the nucleus from the cell margin following treatment with tgf-. to corroborate our results with previous findings , we initially examined the status of -catenin following tgf-2 stimulation in the lens explants . immunofluorescence staining showed membranous localization of -catenin in untreated explants , whereas delocalization of -catenin from the cell membrane was observed in the lens explants at 20 hours post tgf-2 treatment ( inset , panel 2 , fig . additionally , an increase in nuclear accumulation of -catenin was observed in the explants incubated with tgf-2 for 20 hours ( inset , panel 2 , fig . 2 ) . a complete delocalization of -catenin from the cell membrane and concomitant increase in nuclear localization of -catenin were observed upon incubation of explants with tgf-2 at the later time point of 48 hours ( inset , panel 3 , fig . untreated lens explant shows membranous localization of -catenin ( inset , merge image , upper ) that is disrupted upon stimulation with tgf-2 starting at 20 hours post treatment . tgf-2 induces nuclear localization of -catenin ( inset , merge image , panels 2 , 3 ) . during tgf-induced emt , it has been proposed that -sma expression is regulated by the interaction between -catenin and smad3 , which is further dependent on the transcriptional coactivator cbp . inhibition of the -catenin / cbp complex by the inhibitor icg-001 prevents -catenin and smad3 interaction and has been shown to inhibit tgf-induced emt in other systems . we therefore utilized icg-001 to determine whether disruption in -catenin / cbp dependent signaling in tgf-2treated explants can prevent fascin expression and emt . lens explants were treated with tgf-2 in the presence or absence of icg-001 ( 10 m ) for 48 hours . upon stimulation with tgf-2 , lecs showed actin cytoskeleton remodeling , as demonstrated by a significant increase in both fascin and -sma expression ( inset , fig . interestingly , tgf-2induced expression of fascin was prevented by cotreatment with icg-001 , and this suppression in fascin expression was further confirmed by western blot analysis ( fig . 3b ) . a significant decrease in tgf-2induced expression of -sma was also observed upon disruption of -catenin / cbp dependent signaling by cotreatment with icg-001 when compared to tgf--treated lens explants ( inset , fig . untreated and icg-001treated explants did not exhibit any induction in either fascin or -sma expression . a previous report had shown that inhibition of wnt/catenin signaling by icg-001 induces cytotoxicity in multiple myeloma cells . however , we did not observe any significant increase in cell death in the lens explants treated with icg-001 alone or in combination with tgf-2 . effect of inhibition of -catenin / cbp dependent signaling by icg-001 on tgf-induced emt of lens explants . ( a ) untreated , icg - only , tgf-2only , and tgf-2 and icg-001-treated lens explants were immunostained for fascin and -sma . slides were imaged using 40 lens of zeiss apotome microscope and analyzed using zeiss zen software . ( b ) western blot analysis for fascin , -sma , and gapdh was carried out using protein lysates extracted from untreated , icg - treated , tgf-2treated , and tgf-2 and icg-001-treated lens explants . ( c ) densitometric quantification of -sma indicating fold reduction in -sma expression normalized to untreated lens explants ( n = 3 , * * p < 0.005 , * * * p < 0.0005 ) . since we had shown that fascin colocalizes with f - actin in tgf-2treated explants ( fig . 1 ) , we further examined f - actin expression following treatment with tgf-2 in the presence and absence of icg-001 . an increase in actin polymerization was observed in tgf-2treated explants , whereas coincubation with icg-001 completely inhibited tgf-2induced f - actin polymerization ( fig . 4a ) . inhibition of -catenin / cbp dependent signaling by icg-001 prevents stress fiber formation and tgf-induced e - cadherin delocalization . lecs incubated with icg only , tgf-2 only , and tgf-2 and icg-001 immunostained for actin and e - cadherin . stained slides were imaged using 40 lens of zeiss apotome microscope and analyzed using zeiss zen software . ( a ) actin staining reveals a decrease in tgf-2induced stress fiber formation ( panel 3 ) with inhibition of -catenin / cbp interaction by icg-001 ( panel 4 ) . ( b ) the tgf-2induced loss of membranous e - cadherin ( panel 3 ) was restored by icg-001 ( panel 4 ) . our lab and others have shown that during tgf-induced emt of lecs , e - cadherin is degraded , and its localization at the cell membrane is lost . loss of e - cadherin leads to a dissociation of the e - cadherin/catenin complex that results in translocation of -catenin to the nucleus and activation of its downstream targets . thus , we examined the effects of tgf-2 on e - cadherin expression in the presence of icg-001 . untreated lens explants showed hexagonal , cobblestone arrangement with e - cadherin exhibiting membranous localization ( inset , fig . 4b ) , which was also observed in cells treated with icg-001 alone . in comparison , explants treated with tgf-2 exhibited a substantial decrease in the localization of e - cadherin at the cell membrane . in comparison , coincubation with tgf-2 and icg-001 was able to partially rescue the tgf-induced e - cadherin delocalization ( fig . these results suggest the involvement of -catenin / cbp dependent signaling in e - cadherin localization downstream of tgf- signaling in lens explants . previous studies have speculated that -catenin / tcf dependent signaling may regulate tgf-induced cataract formation and emt of lens epithelial explants . to investigate this further , we inhibited the -catenin / tcf dependent signaling pathway utilizing pnu-74654 ( pnu ) , an inhibitor that selectively inhibits the interaction between nuclear -catenin and tcf4 . we did not observe any change in the cellular morphology in the lens explants cotreated with tgf-2 and pnu when compared to the cells of the explant incubated with tgf-2 . we first examined the change in expression of -sma following cotreatment of lens explants with tgf and pnu as compared to cells treated with tgf alone . we did not observe any change in expression of -sma positive stress fibers ( fig . these findings revealed that while untreated explants showed membranous localization of e - cadherin ( arrowhead , fig . 6a ) , explants treated with tgf-2 exhibited a decrease in the localization of e - cadherin at the cell membrane ( arrowhead , fig . as expected , we did not observe any change in the localization of e - cadherin in lens explants cotreated with tgf-2 and pnu when compared to explants treated with tgf-2 alone . these results were confirmed by western blot analyses showing that cotreatment with pnu did not prevent the tgf-induced decrease in e - cadherin expression ( figs . c ) . inhibition of -catenin / tcf dependent signaling fails to protect tgf-induced -sma expression and f - actin polymerization . ( a ) lecs incubated with tgf-2 and tgf-2 and pnu for 48 hours immunostained for -sma , and with rhodamine phalloidin for f - actin , and imaged using 40 lens of leica dmra2 fluorescent microscope . scale bar : 100 m ( b ) representative western blot of -sma protein from untreated , tgf-2treated , pnu - treated , and tgf-2 and pnu - treated lecs ( n = 3 ) . effect on e - cadherin by inhibition of -catenin / tcf interaction . ( a ) e - cadherin stained untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs imaged using zeiss lsm 510 confocal microscope . ( b ) total protein from untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs was subjected to western blot analysis for e - cadherin and gapdh ( n = 3 ) . ( c ) graph showing average relative density sd of e - cadherin normalized to untreated lecs ( n = 3 , * p < 0.05 , * * p < 0.005 ) . previous studies have shown that fascin gene expression is regulated by the wnt/catenin signaling pathway , which promotes cancer cell migration and invasion . more specifically , the fascin promoter has been shown to contain binding sites for tcf / lef , transcription factors that are activated specifically by -catenin . to investigate the role of -catenin / tcf interaction on fascin expression , rat lens epithelial explants immunofluorescence results revealed no change in the expression of fascin in lens explants cotreated with tgf-2 and pnu when compared to explants treated with tgf-2 alone ( fig . this finding was further confirmed using western blot analysis , where the protein extracts from lens explants cotreated with tgf-2 and pnu revealed no change in the levels of fascin protein when compared to explants treated with tgf-2 alone ( figs . ( a ) untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs were stained for fascin and imaged using 40 lens of leica dmra2 fluorescent microscope ( n = 3 ) . ( b ) western blot analysis for fascin and gapdh was carried out using protein lysates from untreated , tgf-2treated , pnu - treated , and tgf-2 and pnu - cotreated lecs ( n = 3 ) . ( c ) graph representing the relative density sd of fascin normalized to untreated lecs ( n = 3 , * p < 0.05 ) . during emt , cells undergoing transformation express mesenchymal markers , such as the matrix metalloproteinases ( mmps ) , as they lean toward a mesenchymal phenotype . previous reports from our lab show an increase in mmp9 protein levels in the supernatants from tgf-2treated whole rat lenses . first , to show the mesenchymal phenotype of rat lens explants induced by tgf-2 and to investigate whether -catenin / cbp dependent signaling has a role in the regulation of mmp9 expression , as observed in other systems , we examined mmp9 expression in tgf-2treated lens explants in the presence and absence of icg-001 . western blot analyses revealed a significant 2.7 0.8-fold reduction ( p < 0.02 ) in tgf-2induced mmp9 protein levels upon inhibition of -catenin / cbp dependent signaling by icg-001 compared to explants treated with tgf-2 alone ( figs . next , we investigated the ability of the pnu inhibitor to block tgf-2induced mmp9 expression and found that it was ineffective ( data not shown ) . these results suggest a role for -catenin / cbp dependent signaling in regulating mmp9 expression during tgf-induced emt in lens epithelial explants . ( a ) untreated , icg-001treated , tgf-2treated , and tgf-2 and icg-001cotreated lens explants were subjected to western blot analysis for mmp9 and gapdh ( n = 3 ) . ( b ) graph representing the relative density sd of mmp9 normalized to untreated lecs ( n = 3 , * p < 0.02 across all samples ) . understanding the mechanisms through which tgf- induces emt in the lens is important for developing novel therapeutic strategies for the prevention of cataract . in this study , we show that tgf-induced -catenin / cbp dependent signaling is critical during tgf-induced emt in the lens . in particular , we show that tgf-induced -catenin / cbp dependent signaling is responsible for the upregulation of fascin , an actin - bundling protein known to be involved in emt and migration and invasion in a number of different cancer cells . icg-001 , a specific inhibitor of -catenin / cbp interactions , prevented tgf-induced fascin , -sma , and mmp9 induction , demonstrating the requirement of cbp - dependent -catenin signaling in the regulation of proteins that facilitate tgf-induced emt in the lens . these data add to the growing list of mechanisms regulated during tgf-induced ocular fibrosis , and identify novel signaling events and molecular markers that contribute to tgf-induced emt , which could offer potential new therapeutic approaches to control fibrosis of the lens . mesenchymal transition plays a major role in the pathogenesis of numerous fibrotic diseases including fibrotic cataract . transforming growth factor- is the growth factor involved in emt of lecs following cataract surgery that results in the development of secondary cataract or pco . in addition , emt and the formation of anterior subcapsular plaques containing -sma expressing , spindle - shaped cells can be induced by tgf-. transforming growth factor-induced emt in lec explants involves a loss of epithelial characteristics with downregulation / degradation of epithelial markers , such as e - cadherin , and a transition to a mesenchymal / fibroblast phenotype characterized by an upregulation of myofibroblast markers , namely , -sma incorporated into f - actin stress fibers . formation of stress fibers requires the coordinated interaction between monomeric actin and its accessory proteins , such as fascin . fascin has been shown to facilitate formation of peripheral actin - rich protrusions through its actin - bundling activity that results in increased cell migration . recent reports have demonstrated more specific roles for fascin in the formation and turnover of adhesive structures of the cell . for example , fascin promotes cell migration by restricting the number and thickness of newly polymerized actin filaments into tight noncontractile bundles at the focal adhesions ; depletion of fascin results in thicker and more contractile actin filaments that generate higher tensile forces and slow turnover of focal adhesions , ultimately causing decreased cell migration . indeed , fascin along with other actin - modulating proteins helps cancer cells undergoing emt to change their cellular morphology , aiding in tumor cell migration and invasion . overexpression of fascin has also been shown to modulate emt during progression of breast cancer and pancreatic tumors ; however , its fate during tgf-induced emt in the lens had yet to be examined . in the current study , we demonstrate that stimulation of rat lens epithelial explants with active tgf- led to a significant increase in the expression of fascin ( figs . 1a , 1c ) . in accordance with the role of fascin discussed above , this increase in expression of fascin in our lens explant model system following tgf- treatment may contribute to the migratory phenotype of lecs often observed during pco . since our lens explant model often consists of a confluent culture of cells , migration is reserved only to the edges of the explant . thus , future studies examining the role of fascin in the cellular protrusions of migratory lens explant cells will be important . it is well known that tgf- signaling in the lens is mediated through a canonical , smad - dependent pathway ; however , smad - independent pathways are also involved in regulating emt . our group has demonstrated that overexpression of tgf- in the lens leads to the formation of asc in the absence of smad3 . this finding indicates that smad3 signaling alone is not required for tgf- to induce a lens fibrotic response . transforming growth factor-induced smad signaling has also been shown to cross - talk with other pathways , such as wnt/-catenin . in other epithelial cell systems , -catenin has been shown to form a complex with smad3 that then translocates to the nucleus , and cooperatively promotes the transcription of emt target genes . the translocation of -catenin from cell margins to the nucleus in tgf-treated lens epithelial explants as shown in this study ( fig . 2 ) , and as reported previously , suggests the role of -catenin dependent signaling during tgf-induced emt in our lens explant model system . it is possible that tgf- may be initiating -catenin signaling through upregulation of wnts and their receptors , frizzled ( fz ) , in our lens explant model system , as has been observed previously by others using a tgf- transgenic mouse model . the formation of -catenin / smad3 complex is further dependent on the -catenin transcriptional coactivator , cbp , which differentially regulates a subset of target genes . , we demonstrate that in the presence of icg-001 , tgf-induced -sma expression ( fig . 4a ) were completely inhibited in lens epithelial explants , indicating the prevention of a contractile , myofibroblast phenotype . further , the drastic decrease in tgf-induced expression of fascin in the presence of icg-001 ( figs . 3a c ) indicates a novel role of -catenin / cbp dependent signaling in regulation of cofactors that are essential for cell migration and invasion . these observations show a complete inhibition of the tgf-induced expression of emt - related proteins such as -sma by icg-001 in lecs . the morphology of lens explant cells cotreated with tgf-2 and icg-001 showed a cobblestone arrangement of cells resembling that of untreated control explants , indicative of a prevention of tgf-induced transformation of lecs ( supplementary fig . as well , the known membrane delocalization of e - cadherin induced by tgf- signaling was partially restored in the presence of icg-001 ( fig . however , since the inhibition was not complete , it is possible that tgf-induced loss of e - cadherin localization is also regulated by -catenin independent repressive activity of transcription factors such as slug on e - cadherin . nonetheless , our findings suggest the importance of -catenin / cbp dependent signaling during tgf-induced emt in the lens . dependent signaling has also been implicated in emt in other model systems . in the lens , it is speculated that -catenin / tcf signaling plays a role in regulating emt / fibrosis . to further understand the role of -catenin / tcf signaling during tgf-induced emt in lecs , a specific inhibitor of -catenin / tcf interaction , pnu-74654 , was used . the morphology of lens explant cells cotreated with tgf-2 and pnu resembles that of tgf-2treated lecs , indicating that pnu was unable to prevent tgf-induced transformation of lecs ( supplementary fig . the observed appearance of contractile , -sma expressing fibers and presence of polymerized f - actin / stress fibers in our lens explant cells in the presence of tgf- and pnu indicates that -catenin / tcf dependent signaling is not required in the regulation of tgf-induced -sma expression and stress fiber formation ( fig . these observations are in line with those reported in other emt model systems , in which -catenin / cbp dependent signaling as compared to -catenin / tcf dependent signaling is more critical during tgf-induced emt . the icg-001 inhibitor has , however , in some cases been shown to interfere with -catenin / tcf dependent signaling . hence , we can not rule out the possibility that -catenin / tcf dependent signaling may have partially contributed to tgf-induced emt in lecs . however , -catenin / cbp dependent signaling , as opposed to -catenin / tcf dependent signaling , has been shown to be required for -sma expression and , importantly , the cis elements of the -sma promoter do not possess -catenin / tcf binding sites , but instead are regulated by transcription factors that are controlled by -catenin / cbp / smad3 signaling . interestingly , inhibition of -catenin / cbp dependent signaling by icg-001 , and not inhibition -catenin / tcf dependent signaling by pnu , partially inhibited tgf-induced nuclear translocation of -catenin ( supplementary fig . neither icg-001 nor pnu treatment alone resulted in the decrease in -catenin expression ( supplementary fig . / tcf complex , has been shown to coincide with -sma expression during tgf-induced emt in other epithelial cells , such as tubular cells . indeed , further investigation is needed to elucidate the role of the interaction between -catenin / cbp and smad3 in the regulation of -sma expression during tgf-induced emt in lecs . the presence of fascin in the lens explants cells incubated with both tgf- and pnu ( fig . ahr signaling , as opposed to -catenin / tcf dependent signaling , is responsible for the regulation of fascin . furthermore , incubation of lens explants with pnu did not prevent tgf-induced downregulation of e - cadherin ( figs . c ) , a key component of adherens junction , which is in agreement with the previous report showing tgf-induced smad - dependent signaling , but not -catenin / tcf dependent signaling , as the key mechanism involved in downregulation of e - cadherin during tgf-induced emt . overall , the presence of -sma , fascin , and e - cadherin shows that inhibition of -catenin / tcf dependent signaling by pnu was unable to prevent the tgf-induced contractile , myofibroblast phenotype and emt in lecs . during tgf-induced emt , epithelial cells begin to express mesenchymal markers such as mmps as they lean toward a more mesenchymal phenotype . previously , we have shown that in the absence of mmp9 expression , tgf-induced emt - like changes , such as induction of -sma in lecs , are prevented . in tubular epithelial cells , it has been demonstrated that the inhibition of -catenin / cbp interactions by icg-001 decreases active levels of mmp9 , as assessed by gelatin zymography of supernatants , thereby completely preventing the mesenchymal transition . here , we found that inhibition of -catenin / tcf interactions by pnu was not able to prevent tgf-induced mmp9 expression , suggesting that -catenin / tcf dependent signaling may not be required in the regulation of mmp9 during tgf-induced emt in lecs ( data not shown ) . interestingly , inhibition of -catenin / cbp dependent signaling by icg-001 showed a significant decrease in tgf-induced mmp9 expression along with suppression of the mesenchymal transition of lecs ( fig . a decrease in tgf-induced mmp9 expression upon inhibition of -catenin / cbp interactions also shows the novel role of -catenin / cbp dependent signaling in regulation of mmp9 during tgf-induced emt . taken together , our findings have revealed a -catenin mediated mechanism underlying the regulation of fascin , -sma , and mmp9 expression during tgf-induced emt in the lens . we propose that tgf-mediated downregulation of e - cadherin results in disruption of e - cadherin/catenin complex that leads to increased cytosolic -catenin , which interacts and forms a complex with cbp ( fig . we demonstrate for the first time that -catenin / cbp dependent signaling is indispensable in tgf-mediated emt in the lens . -catenin / cbp complex may further interact with other regulators of emt and regulate tgf-induced emt of lecs . the interaction between -catenin and smad3 is known to promote transcription activity of myocardin - related transcription factor ( mrtf ) , the master regulator of cytoskeletal genes , during tgf-induced emt in tubular epithelial cells . interestingly , our previous finding suggests translocation of mrtf to the nucleus during tgf-induced emt in lecs . hence , it is possible that the interplay between -catenin , smad3 , and mrtf may be playing a crucial role in tgf-mediated emt in lecs . indeed , a detailed molecular investigation is needed to dissect out the role of -catenin , smad3 , and mrtf interactions in tgf-induced emt in the lens . in conclusion , the inhibition of tgf-induced emt by icg-001 may represent a new therapeutic approach to target ocular fibrosis of the lens . tgf-induced downregulation of e - cadherin leads to disruption of e - cadherin/-catenin complex that results in interaction of -catenin with cbp . free -catenin may interact with either smad in a cbp - dependent manner or tcf , and regulate tgf-induced -sma , fascin , and mmp9 expression . inhibition of interaction between -catenin and tcf fails to inhibit tgf-induced emt - like changes in lecs . however , inhibition of interaction between -catenin and cbp prevents tgf-induced emt in lens explants .

calcified coronary lesions are challenging to deal with , as they require optimal lesion preparation . direct stenting in this scenario is associated with risk of stent - underexpansion , which is related to in - stent restenosis , target lesion revascularization and stent - thrombosis . we report on the interventional management of an underexpanded bare - metal stent not amenable to high - pressure balloon dilation and cutting - balloon . by using rotablation we could abrade the underexpanded stent struts and the calcification with subsequent implantation of a drug - eluting stent . follow - up of 6 months revealed good results without evidence of significant restenosis . our clinical experience and case reports in the literature suggest that this strategy might be an option for underexpanded stents not amenable to conventional techniques .

direct coronary stent implantation is an elegant technique for coronary artery revascularization.1 however , calcified coronary lesions , often seen in older patients suffering from diabetes mellitus , renal failure and hypertension , are challenging to deal with , as they require optimal lesion preparation prior stenting for avoiding stent underexpansion which is related to in - stent restenosis , target lesion revascularization and subsequent stent thrombosis.2 several strategies and technologies have been developed to address the problem of heavily calcified coronary lesions . these include simple dilatation using standard non - compliant balloon , cutting balloon and plaque modification using rotational atherectomy . we report on the management of an underexpanded bare - metal stent in a patient with heavily calcified lesion not amenable to high - pressure balloon - dilatation . a 72-year old man suffering from progressive angina over the past 8 weeks presented to our chest pain unit . he had previously documented insulin - dependent diabetes , alimentary obesity , hyperlipidemia and arterial hypertension . an ambulatory performed myocardial perfusion scintigraphy revealed a reduced tracer - uptake in the apex , left posterior and antero - lateral wall during physical examination ( 100 watt - cycling ) . coronary angiography , which was performed via right radial access using a 5f sheath , revealed a 50% stenosis of the left anterior descending artery ( lad ) and ramus posterolateralis sinistra ( rpls ) while the right coronary artery ( rca ) had a critical 90% stenosis ( fig . due to the patients symptoms and the angiographic findings we decided to perform a percutaneous coronary intervention ( pci ) . the patient received 600 mg clopidogrel , 500 mg aspirin and 5000u heparin followed by primary pci and direct stenting of a bare - metal stent ( bms ) ( coroflex blue 3.5 mm/8 mm , b. braun , melsungen , germany ) with 18 atm for 30 sec ( fig . post - pci angiography revealed a 75% stenosis in the mid - portion of the stent ( fig . 1c ) . a subsequent dilatation with a semi - compliant balloon ( pantera 3,5/10 mm with [ biotronik , berlin , germany ] 18 atm over 30 sec ) , a non - compliant balloon ( quantum 3,5/8 mm [ boston scientific , natick , usa ] with 20 atm over 30 sec ) and a cutting - balloon ( 3,0/10 mm [ boston scientific , natick , usa ] with 18 atm over 30 sec ) could not expand the stent further ; pointing out the heavily calcified nature of this lesion . due to the fact that an underexpanded stent is a predictor for worse clinical outcome we decided on rotablation . additionally we introduced a 5f sheath in right femoral vein and inserted a transient pacemaker lead . after passing across the stenosis with the 0.009 rotawire we ablade the heavily calcified stenosis as well as the stent struts ( stentablation ) ( fig . all ablations were performed with a 1.75 mm burr with at least 150,000 rpm and ablation times < 30 sec without a decrease in rotational speed of > 5,000 rpm . the procedure was free of complications and we continued with dilatation with a non - compliant balloon ( quantum 3,5/8 mm with 20 atm over 30 sec ) and a cutting - balloon ( 3,0/10 mm with 16 atm over 30 sec ) . with complete expansion of the balloons the procedure was continued with implantation of a drug - eluting stent ( taxus libert 4.0/12 mm [ boston scientific , natick , usa ] with 16 atm over 30 sec ) ( rotastenting ) ( fig . finally , there was timi 3 without evidence of dissection or residual stenosis ( fig . 2c ) . following uneventful hospital stay without evidence of myocardial necrosis the patient was discharged after 3 days on 100 mg aspirin , 75 mg clopidogrel , 5 mg bisoprolol , 5 mg of ramipril and 40 mg simvastatin with a recommendation for dual antiplatelet therapy of 1 year without any change in his extra - cardiovascular medications . a routine coronary angiography performed 6 months after index - pci revealed a good result with a mild ( 25% ) restenosis ( fig . 2d ) . direct stenting is the implanation of stents in coronary lesions without predilatation.1 from animal restenosis models , direct stenting without the need for predilatation appears to reduce vessel trauma , in particular as a result of less endothelial denudation , resulting in less neointimal hyperplasia subsequently.1 pci of calcified and complex lesions has been associated with lower success rates , an increased frequency of acute complications , and higher restenosis rates than pci of simple lesions.2 as seen in our case , delivering the stent may be difficult and stent expansion may be inadequate in heavily calcified lesions , resulting in smaller acute gain compared to non - calcified lesions.2 it is widely accepted that achieving postprocedural residual stenosis is a major determinant of restenosis during follow - up and optimal stent expansion is a crucial factor in minimizing the risk of stent thrombosis pointed out by the fact that only 22% of patients that experienced subacute stent thrombosis have an acceptable pci result as assessed by ivus.2,3 a variety of strategies and technologies have been developed to address the problem of an underexpanded stent . the postit trial revealed that in case of using only the stent delivery balloon over 70% of patients did not achieve optimal stent deployment.4 use of non - compliant balloon to achieve full distension in resitant lesions is a reasonable first - step . however , focal points of resistance within a lesion result in non - uniform balloon expansion and characteristic dog - boning with overexpansion in the more compliant segments . in this non - uniform expansion may cause vessel dissection and rupture acutely as well as restenosis due to deep - wall injury in the follow - up . cutting - balloon , designed to score the vessel longitudinally rather than causing uncontrolled plaque disruption , have been used successfully in the treatment of undilatable lesions.5 in our case , none of these techniques were successful in reducing the underexpansion , demonstrating the nature of the heavily calcification , which was not assumed on initial fluoroscopy . thus , despite the existence of limited data,6 we decided to rotablade the remaining calcification and the underexpanded stent struts to avoid aforementioned complications . high - speed rotational atherectomy preferentially cuts hard plaque , increases plaque compliance and thereby renders the lesion more amenable to balloon dilatation.7 the rotablator is able to ablate inelastic tissue selectively while maintaining the integrity of elastic tissue due to the principle of differential cutting . these particles are small enough to pass through the coronary microcirculation and ultimately undergo phagocytosis in the liver , spleen , and lung.7 the procedure performed in our case was uneventful with no dissection , slow - flow , heamodynamic compromise or myocardial necrosis . we had applied a transient pacemaker via the right femoral vein to overcome possible conduction disturbances when handling in the right coronary artery . several observational studies have confirmed that rotational atherectomy prior to stent deployment in severely calcified lesions does facilitate stent delivery and expansion , but incidence of restenosis remains unsatisfactory ( 23% ) when bms are used.8 there is limited information about rotational atherectomy followed by des implantation , but initial results seem promising.9 a comparison of bms ( n = 84 ) and des ( n = 213 ) after rotablation with cardiac death and recurrent myocardial infarction being defined as primary endpoint and binary restenosis as secondary endpoint revealed lower rates for primary endpoint in des group ( 2.3% versus 7.1% ; p = 0.04 ) during a follow - up of 1300 days.10 despite our procedural success and good midterm result , there are no data on long - term follow - up after stentablation and rotastening . thus , it should be emphasized that a better lesion preparation is needed to avoid stent underexpansion in undilatable lesions .

aim of databasethe aim of the danish quality database for prehospital emergency medical services ( qems ) is to assess , monitor , and improve the quality of prehospital emergency medical service care in the entire prehospital patient pathway . the aim of this review is to describe the design and the implementation of qems.study populationthe study population consists of all 112 patient contacts defined as emergency patients , where the entrance to health care is a 112 call forwarded to one of the five regional emergency medical coordination centers in denmark since january 1 , 2014 . estimated annual number of included 112 patients is 300,000350,000.main variableswe defined nine quality indicators and the following variables : time stamps for emergency calls received at one of the five regional emergency medical coordination centers , dispatch of prehospital unit(s ) , arrival of first prehospital unit , arrival of first supplemental prehospital unit , and mission completion . finally , professional level and type of the prehospital resource dispatched to an incident and end - of - mission status ( mission completed by phone , on scene , or admission to hospital ) are registered.descriptive datadescriptive data included age , region , and danish index for emergency care including urgency level.conclusionqems is a new database under establishment and is expected to provide the basis for quality improvement in the prehospital setting and in the entire patient care pathway , for example , by providing prehospital data for research and other quality databases .

the emergency care system in denmark has been reorganized during recent years , and a danish quality database on emergency care has been established to assess , monitor , and improve quality . the danish quality database for prehospital emergency medical services ( qems ) is one of the three national sub - databases . the aim of the qems is to assess , monitor , and improve the quality of prehospital emergency medical service ( ems ) care in the entire patient pathway and acts as basis for clinical and health - service research . provision of prehospital emergency care is subject to variations in organization among ems providers , with domestic and regional as well as international differences . there are few uniform quality indicators and standards based on high - level evidence , and most standards are based on expert panel opinions.14 some specific diagnostic groups benefit from quality indicators and standards , such as cardiac arrest,5,6 myocardial infarction ( mi),7,8 stroke,9,10 and severe injury . the major challenge and difficulty in emergency care , especially in the prehospital setting , is that the vast majority of patients present with symptoms and not diagnoses . thus , ems care has to be initiated and delivered on the basis of the symptoms with limited knowledge about the patient s objective condition and medical history . in 2013 , a working group was established by the danish regions with medical and administrative representatives from all five health regions , with the task of formulating recommendations on indicators and standards of ems quality . the working group consisted of medical directors and administrative professionals from prehospital organizations from each of the five regions . the selection of quality indicators was a consensus process , based on literature and the actual availability of prehospital data in our system and national registries on specific emergency conditions , cardiac arrest , mi , and stroke . the danish clinical registries a national improvement program was established in 2014 with medical representatives from the five public regional ems organizations , a professor in prehospital and emergency medicine and representatives from the danish society of cardiology , the danish society of neurology , and a clinical epidemiologist and a statistician from the danish clinical registries . an initial pilot test of indicators is currently underway in 2015 . due to lack of high evidence , we could not define all standards a priori . a process of defining the remaining standards will follow evaluation of the pilot test . ultimately , aggregated data at a regional level will be available monthly , and national and open - source annually , and provide stimulus for ems improvement . the aim of this review is to describe the design and the implementation of qems , a new danish clinical quality database . in denmark , access to health care is a tax - financed service , and access to ems and emergency hospital care is free of charge . during the last 7 years five regional health care regions were established and were responsible for the pre hospital ems in their own region . these regional ems organizations are responsible for the entire prehospital patient care pathway , from the 112 call made to the recently established emergency medical coordination centers ( emcc ) , through the care given on scene and during transport , and until the patient arrives at hospital , or is taken care of without being brought to hospital . at the emcc , the national emergency number 112 covers both ems , fire and police , and is answered by the police , except in the capital region . here all decisions regarding medical prehospital responses are made at the emcc , based upon a focused , structured interview of the person calling 112 . assessment is performed according to a criteria - based dispatch protocol , the danish index for emergency care ( danish index).11 the danish ems is a two- or three - tier system , with ambulances as the basic - level response , an intermediate level with paramedic- or nurse - manned cars , and an advanced level with dedicated prehospital doctors ( consultant anesthesiologists ) in cars ( mobile emergency care unit ) or helicopter ( helicopter emergency medical service [ hems ] ) in a rendezvous model . the danish hems has only recently been added to the system , established as national hems in 2014 . ambulances are run by municipal or private companies and staffed with emergency medical technicians or paramedics , educated and trained to national minimum standards according to national regulations.12 the regional ems organizations contract with ambulance providers after european union - regulated tenders . the regional ems is responsible for the quality of care delivered in the entire ems pathway , including the care delivered by private ambulance contractors . all ems providers nationwide are to use the same electronic prehospital patient record system , under implementation in 2015 . the study population consists of all 112 patient contacts defined as patients in need of emergency help , where access to the health care system is a 112 call forwarded to the emcc . . the corresponding number of 112 patient contacts will be lower , estimated to be 300,000350,000 as there might be more 112 calls on the same patient . the database includes data acquired from each of the five regional emcc and when fully implemented data from the prehospital electronic patient record . data from the danish database for acute hospital contacts will also be included in the future . emcc call - takers assess the level of emergency and respond in accordance with the danish index for emergency care , which consists of 37 main symptoms . the index defines five levels of urgencies : a : life- threatening or potentially life - threatening condition , immediate response required ; b : urgent , but not life - threatening condition ; c : nonurgent condition that needs an ambulance ; d : nonurgent supine patient transport ; and e : other service or advice / instruction including taxi transportation ( no ambulances dispatched ) . the emcc call - taker assigns the letter appropriate for the level of urgency coupling it with a specific number to the case according to the danish index criteria code that corresponds to the main symptom and specific subgroup symptom . the indicators aim at describing quality at the regional ems organizational level and the quality of the medical care . as the prehospital electronic patient record has not yet been fully implemented , only some of the indicators are monitored in the initial pilot test phase . indicator numbers 13 describe quality at an ems organizational level ; these include only responses of level of urgency a ( table 1 ) . time from arrival until the patient is handed over to the hospital personnel ( indicator 3 ) awaits the electronic patient record . concerning the three medical emergencies , cardiac arrest , st - elevation mi , and stroke , all these await full implementation in the electronic patient record . in the meantime , data will be obtained from the national research database , the danish out - of - hospital cardiac arrest registry , and regional and national databases.5 indicator number 4 , the proportion of 112 calls coded according to danish index , was included to assure registration of the initial symptom as this is the only patient information available at time of the decision on level of urgency . indicator number 5 was defined in order to monitor the proportion of patients calling 112 classified as having no need for an ambulance and/or no need for hospital care , who died or called 112 again within 24 hours . in denmark , each citizen is assigned a unique ten - digit civil registration ( cpr ) number , universally used in all patient records and danish registries , and enables unambiguous linkage between these registries . thus , the registration of this number , as indicator 9 , is essential as a key to patient care and for follow - up . table 3 describes the variables included in the primary data set . apart from being an identifier for an event and person , the variables comprise mainly date and time stamps identifying when the 112 call was received at the emcc , when the dispatcher sent a prehospital resource , and when the first resource arrived . the danish index , including urgency level , is also included in the data set . it is , however , a fact that the cpr number is missing in a substantial proportion of patients because it is not always possible to extract this information from the caller . patients with a valid danish cpr number are followed from the date of 112 call and until death for up to 2 days after the task has been completed using the danish civil registration system.13 the database is under establishment , and no studies have been performed yet , but there are previous studies using some of similar data from three regions comparing the danish index with diagnosis and outcome after hospitalization.11,14,15 data will be available to researchers after application to the steering committee , provided that permissions from the danish data protection agency and/or ethical committee were obtained . the qems is an approved clinical quality database ( the danish data protection agency record no 2013 - 58 - 0026 , the central denmark region record no 1 - 16 - 02 - 508 - 14 , and the state serum institute record no 14/18767 ) funded by the danish regions and receives administrative , epidemiological , and biostatistical support from the danish clinical registries ( danish regions ) . aggregated data at regional level will be reported annually in a published report , and data will be provided monthly to the danish regions for use in each regions information system . aggregated data at a regional level will be available monthly , and national and open - source annually , providing incentive for improvement of patient care pathways in the prehospital setting .

complex diseases such as allergy change gene expression in several cell types and tissues . benson and colleagues have now shown , in a paper in bmc systems biology , that this complexity can be studied effectively using an integrated experimental and computational modular analysis . their strategy revealed a core of allergy - associated genes of potential therapeutic value .

recent advances in array - based and sequence - based instrumentation have opened up new ways to monitor the inner molecular world of the cells and tissues that might be relevant to human diseases . yet it is far from evident how these large datasets should be analyzed and how they can be integrated with other sources of data in order to become informative . conversely , the medical community expects nothing less than a list of predictive biomarkers reflecting the risk of disease or its progression and an understanding of the cellular mechanisms involved in disease . however , comparing microarray samples from healthy and diseased individuals using a differential gene expression protocol generates a list of thousands of genes , and it is not clear which genes are important for what . a key idea , originating from engineering science in general and computer science in particular , is the notion of ' divide and conquer ' , which refers to first breaking down a problem into smaller sub - problems that are simple enough to allow an analysis and then combining the solutions to the subproblems , which gives the solution to the original problem . modular analysis of genomic data implements this strategy by dividing the original genomic data into smaller number of modules and then conquering the reduced complexity by using these modules for prioritization to give a shorter list of disease - associated genes . such genes could either be causal drivers of disease or secondary reactions to disease that could potentially be useful biomarkers . benson and colleagues , in a recent paper in bmc systems biology , have used a modular approach to study allergic asthma . they managed to divide the complexity and arrive at the gene encoding the interleukin-7 receptor ( il7r ) as a putative key regulator in allergic asthma . here , i put their analysis in the context of other modular approaches and discuss the possible use of this methodology for finding and prioritizing useful candidates for therapeutics . not surprisingly , there are several different ideas on how to divide and conquer high - throughput functional genomics data . i will restrict my discussion here to gene expression data , although similar remarks could be made for sequence data . one is : given a module of disease - associated genes , how can we compute and/or experimentally predict which genes are good candidates for therapeutics ? before discussing this problem i will first give an overview of different approaches to the other problem : identifying a module of genes . a module is a group of genes that are related in some way to each other and therefore a module is effectively a measure of similarity . for example , if similarity is defined as the distance in a network , then a graph theoretical calculation will be used . however , if gene functional associations are used , then gene similarity will be measured in terms of gene ontology ( go ) or correlation in gene expression values . therefore , different algorithms are used for dividing the genes into modules , a fact that could be confusing for the clinical researcher . the need to reduce the complexity of the original high - throughput gene expression data applying established engineering concepts , such as principal component analysis ( pca ) and singular value decomposition ( svd ) , reduced the dimensionality of the data . instead of analyzing scattered points ( the samples ) in a high - dimensional space equaling the number of genes , the data could thereby be projected into a two- to four - dimensional space . however , it turned out to be difficult to make a biological interpretation of the resulting linear combinations of large numbers of genes . this problem forced the development of different strategies in which the available knowledge on a limited number of genes could be used to predict the functions of as - yet uncharacterized genes . the use of hierarchical clustering in the classic compendium study on yeast data by rosetta inpharmatics grouped genes ( shown as rows ) by their similarity of expression across several experimental conditions ( columns ) . novel gene function was then predicted by inspecting genes in the same cluster as genes with known functions . subsequent work by eran segal and colleagues developed more statistically sound procedures for identifying robust modules using a bayesian formalism applied to microarray data generated from cancer samples . it became clear , however , that a similarity measure based only on correlations was insufficient , because the clusters ( modules ) or bayesian modules did not have an internal network structure that could be used for a more refined analysis . as a consequence , a large number of studies addressing this problem appeared in the literature at the beginning of 2004 . the idea was that if we could identify the wiring within cellular networks , various different algorithms could be applied to find ' connected groups ' in such networks . now , how can such networks be found using only a small number of experimental samples with a large number of genes ? this is an impossible problem from the point of view of engineering system identification , because the number of possible networks consistent with the data is prohibitively large . the key simplifying insight came from ideker and lauffenburger and was later developed by nicolas luscombe and colleagues in a pioneering paper . here , the edges ( or connections ) in the network were simply defined by transcription factor binding experiments , and gene expression data were used to select the subsets of edges that were active under different conditions . this idea of defining edges in a network using a static scaffold has since been reused using various data types ( protein - protein interaction data , pathways from a database , text mining and dna variants ) . the network of interest is then defined by combining the gene expression data with the scaffold , leaving only the active edges . by searching through such an active network using graph algorithms it is then possible to define ' more ' connected parts in a well defined manner , thereby providing modules with an intrinsic network structure . all the above approaches basically begin with a large , complex dataset , which is then simplified by dividing the data into smaller modules . they instead began with four well characterized breast cancer genes and , by using these ideas , constructed a module in which the genes were ' close ' as defined by expression and proteomic data in several species . benson and colleagues have now contributed to a disease - oriented modular analysis by combining several of the above ideas in a novel manner , as summarized in the flow chart in figure 1 . first , because allergic disease involves multiple cells in different tissues and because no prior characterization of key genes was available , they turned to several different sets of gene expression microarray data in order to find a reference disease - associated gene around which they could construct a module . using the idea that disease - associated genes tend to interact , they could search for other disease - associated genes that were ' close ' . for this purpose , the authors used a graph algorithm that identified a connected clique of 103 disease - associated genes from the microarray data . integration of several public gene expression datasets revealed a group of shared ( blue ) and closely connected clique ( red and black ) disease - associated genes . a subset of these genes were found to share the t - cell receptor signalling pathway , an observation that was then validated by independent experimentation . to identify a transcription factor ( gata3 ) regulating one of this subset , the itk gene , the final module of 37 disease - associated genes consisted of genes listed in public databases as having relevant expression patterns and interacting with gata3 . the t - cell receptor signaling pathway turned out to be a pathway shared by these 103 genes , as detected by the ingenuity pathway analysis tool , which identifies physical , transcriptional and enzymatic interactions from the literature . experimental analysis of this pathway in patient - derived cells revealed strong activation of the itk gene , which is also known to be located in the genomic susceptibility region for allergy . combining a promoter analysis of the itk gene with expression data revealed that the transcription factor gata3 regulated itk . the expression data were used to filter out 10 inactive genes , thus leaving a final module of 37 disease - associated genes around the gata3 transcription factor . the construction of this module was accompanied by several experimental tests at various stages , providing confidence to the analysis . the problem of selecting therapeutic targets within a module has not received much attention in studies that have used a modular approach for reducing complexity . there are various ideas from graph theory on how to compute mathematically defined properties , such as clustering and connectivity in large networks , which then could suggest which nodes are essential . however , essentiality is not necessarily equivalent to disease association . experimental investigators have instead performed target selection using the full dataset in combination with extensive experimental testing . the analysis by benson and colleagues is important because it highlights the difficulty of selecting a disease - associated target from a module of 37 genes despite the elegant prior reduction of complexity . they resorted to using a connectivity criterion , selecting the il7r gene because it had the largest number of connections , and they were also able to demonstrate that perturbing the il7r gene affected other genes and the t - cell phenotype . there are probably several other disease - associated genes in their module that warrant further experimental investigation . benson and colleagues have introduced a useful procedure for defining a module of disease - associated genes . as with most complex diseases , the study of allergy is complicated by the fact that the disease affects several cell types and tissues . the process of identifying such modules therefore requires the kind of stringent experimental validation as was performed by the benson team . despite their careful analysis , because there are other transcription factors for the itk gene that are active in the expression datasets there is a significant risk that several disease - associated genes remain that were not captured in their module . the second step of selecting a gene for therapeutics from a module is even more problematic because we are currently lacking systematic tools for this selection problem . furthermore , it is not unlikely that an efficient therapy could require targeting of several disease - associated genes simultaneously . however , the number of combinations of three genes that can be chosen from a small ten - gene module , for example , quickly exceeds what is experimentally feasible to study . in conclusion , benson and colleagues have devised an interesting method for finding disease - associated genes , but it needs to be evaluated on other complex diseases . their study also makes clear that the problem of prioritizing disease - associated genes within a module for therapeutic studies in the clinic is still unsolved .

anesthesiologists frequently see asymptomatic patients with diastolic dysfunction or heart failure for various surgeries . these patients typically show normal systolic function but abnormal diastolic parameters in their preoperative echocardiographic evaluations . the symptoms that are sometimes seen are similar to those of chronic obstructive pulmonary disease . patients with diastolic dysfunction , and even with diastolic heart failure , have the potential to develop a hypertensive crisis or pulmonary congestion . thus , in addition to conventional perioperative risk quantification , it may be important to consider the results of diastolic assessment for predicting the postoperative outcome and making better decisions . if anesthesiologists see female patients older than 70 years of age who have hypertension , diabetes , chronic renal disease , recent weight gain , or exercise intolerance , they should focus on the patient 's diastologic echocardiography indicators such as left atrial enlargement or left ventricular hypertrophy . in addition , there is a need for perioperative strategies to mitigate diastolic dysfunction - related morbidity . specifically , hypertension should be controlled , keeping pulse pressure below diastolic blood pressure , maintaining a sinus rhythm and normovolemia , and avoiding tachycardia and myocardial ischemia . there is no need to classify these diastolic dysfunction , but it is important to manage this condition to avoid worsening outcomes .

the efficiency and function of the left ventricle ( lv ) as a pump are represented as systole and diastole . the systolic function is energy dependent ; the diastolic function is an important part of cardiac output ( as preload ) and also includes active processes such as the suction effect , which is also energy dependent and can be weakened by ischemia . thus , diastolic dysfunction ( ddf ) is an early marker of ischemia , because it is sensitive to impaired perfusion . most physicians pay attention to the lv ejection fraction ( ef ) for systolic function in the preoperative echocardiographic evaluation of patients about to undergo surgery ; however , lv ddf is often overlooked in such cardiac risk evaluation . lv ddf , and even diastolic heart failure ( dhf ) , are usually not significant problems in the daily life of patients who do not suffer from exercise intolerance , dyspnea , or pulmonary edema . however , when they reach the ward after an operation , they often end up in the intensive care unit ( icu ) with unstable vital signs in combination with pulmonary edema , which are not responsive to the normal use of intravenous ( iv ) epinephrine or norepinephrine . this can lead to poor outcomes , caused by lv ddf regardless of lv systolic dysfunction ( sdf ) , with hemodynamic instability developing due to surgery - induced adrenergic stimulation and anesthesia - induced sympathetic attenuation , resulting in a lengthy stay in the icu or even death . in american heart association - american college of cardiology ( aha - acc ) guidelines , lv ddf is still underestimated in preoperative risk assessment and few clinical data supporting its importance are available . in particular , such patients need more appropriate anesthetic management to prevent postoperative complications and to aid in recovery from anesthesia . because overlooking patients with lv ddf or lv dhf during the perioperative period , regardless of lv systolic function , can be very dangerous and cause poor outcomes , it is important that anesthesiologists are familiar with the pathophysiology , management , and surgical outcomes and are able to notice their phenotypic characteristics , and diagnostic parameters . regardless of lv systolic function and the patient 's symptoms , ddf indicates impaired filling of the ventricles at low pressure and abnormalities in diastolic distensibility , filling , or relaxation , which are usually associated with hypertension , diabetes , or ischemia . the diagnosis of ddf can come from cardiac catheterization and doppler patterns of lv diastolic filling . ddf shows increased lv diastolic pressure ( > 16 mmhg ) with preserved systolic function and lv volume . ddf itself is a physiological or preclinical state that can be compensated for with an increase in left atrial pressure , so there is essentially an asymptomatic period . in addition to echocardiographic or angiographic evidence , when accompanied by symptoms and signs that can be confused with chronic obstructive pulmonary disease such as exercise intolerance , exertional dyspnea , and pulmonary edema with normal ef , it could be considered dhf . dhf is also characterized by a stiff and thickened ventricle , with a small cavity and low cardiac output . dhf can also show a normal ef ; this is called heart failure with a normal ejection fraction ( hfnef ) or preserved ef ( hfpreef ) in preclinical ddf , hypertension , hyperlipidemia , coronary artery disease , and renal dysfunction are prevalent . in one study , the 2-year cumulative probability of developing any symptom was as high as 31% and the probability of cardiac hospitalization was 21% . asymptomatic ddf is generally common in patients without heart failure , increases with age , and is particularly seen in elderly women with hypertension and hypertrophy of the ventricle . ddf alone can predict worse outcomes in patients and their prognosis worsens according to its increasing degree . moreover , more than half of all patients with heart failure have a normal ef . compared to sdf , the incidence and prevalence of ddf are increasing , and in geriatric patients in particular , it can cause hospitalization , increasing hospital costs , and even mortality . a study of two cohorts of patients with decompensated heart failure showed that 35% had preserved lv ef and that they were elderly and mainly women . phillip et al . reported that ~60% of elderly patients with normal ef presenting for geriatric cardiac and non - cardiac surgery had lv - filling abnormalities . moreover , ~15% of these patients had moderate to severe ddf in the absence of symptoms . in patients undergoing high - risk vascular surgery , dhf increased proportionally with age , up to 70% in those aged > 70 years . there are also age - related changes in the cardiovascular system including coronary artery disease , systemic hypertension , and hypertrophic or infiltrative cardiomyopathy . the effects of these conditions can also increase filling pressure due to abnormalities in relaxation and decreased compliance of the lv . diastole can be divided into four stages : isovolumetric ventricular relaxation , rapid passive ventricular filling , diastasis , and atrial contraction . these can also be subdivided into early and late phases . in the isovolumetric relaxation period , after mitral valve closure , calcium is sequestered back into the sarcoplasmic reticulum ; this is an active process for the suction effect , which needs adenosine triphosphate ( atp ) . in ddf , the breakdown of this process causes the ventricle to fail to relax and become stiff / less compliant . this dysfunction can be caused by impaired lv compliance , a passive process , or by changes in lv active relaxation . anatomical diastole occurs from aortic valve closing to mitral valve closing , but at the molecular level , relaxation of the lv starts during the early systolic period , because it starts with the dissociation of actin - myosin cross bridges . because lv segmental and atrio - ventricular synchronization are indispensable in guaranteeing effective relaxation , loss of the atrial kick in atrial fibrillation not only changes lv filling but also slows myocardial relaxation . before doppler - derived variables , the gold - standard measurement of lv diastolic function was the invasive measurement of lv pressures using cardiac catheterization . this can show pressure / volume loops , lv pressure decline , and the time constant of isovolumetric relaxation ( ) . , lv ddf shows a diastolic loop ( compliance curve ) shifted to the left and upwards . in such cases , lv end - diastolic volume is normal but end - diastolic pressure is increased , resulting in pulmonary congestion . however , sdf causes the end - systolic slope to shift to the right and downwards ( fig . 1 ) . in active diastole with isovolumetric relaxation , which is energy dependent , abnormalities in relaxation can be caused by myocardial ischemia , hypertension , aortic stenosis , and hypertrophic cardiomyopathy . in contrast , passive diastole can be impaired by increased stiffness of the lv chamber , caused by an infiltrative disorder ( amyloidosis ) , myocardial fibrosis , and progression from impaired relaxation . age - related changes in the cardiovascular system can be condensed into a reduction of cardiovascular reserve , which includes increased arterial and myocardial stiffness , impaired -adrenergic responsiveness and lv relaxation , and reduced sinus nodal function and baroreceptor responsiveness . aging is related to ventricular and arterial stiffness , which increase and exacerbate myocardial oxygen consumption for cardiac output . apoptotic myocytes are not replaced , but decrease in number with an increased amount of connective tissue matrix . the afterload causes increased arterial stiffness that affects lv relaxation , resulting in ventricular hypertrophy to preserve systolic function . age - related arterial stiffening has two important consequences : decreased aortic distensibility and increased pulse wave velocity , which result in higher pulse pressure ( pp ) and prolonged systolic contraction , respectively . increased pp is an established cardiovascular risk factor and the early reflected wave , because of prolonged systolic contraction , impinges on early diastole , which can cause greater myocardial stroke work , wall strain , and oxygen consumption such as a mistimed intra - aortic balloon pump . beyond this , impaired calcium homeostasis and reduced -receptor responsiveness also cause lv ddf . at the cellular level , reduced sarcoplasmic / endoplasmic reticulum calcium atpase 2 ( serca2 ) activity and increased phospholamban activity contribute to impaired lv diastolic function . the signal dampening of -receptors may be due to receptor down - regulation or diminished coupling of -receptors with intracellular adenylate cyclase activity . consequently , despite increased circulating catecholamines , there is a limited increase in heart rate and contractility in response to endogenous and exogenous catecholamines , so there is impaired baroreflex control of blood pressure ( bp ) . apoptosis , fibrosis , fatty infiltration , and calcification of pacemaker and his - bundle cells can result in conduction block , sick sinus syndrome , and arterial fibrillation , with loss of the atrial kick for preload in the end - diastole stage . arrhythmias ( e.g. , atrial fibrillation ) and myocardial ischemia affecting diastolic timing can decompensate pre - existing ddf further . treatment with blockers or calcium channel blockers has been recommended to control tachycardia or arrhythmias to improve lv filling . patients with ddf in the operating room have some common phenotypic characteristics such as being elderly women and having peripheral edema , hypertension , and obesity . they also tend to have limitations in functional capacity caused by reduced chronotropic , vasodilator , and cardiac reserve . in addition to a functional capacity evaluation , risk factors and phenotypes for heart failure should be checked in the preoperative assessment for ddf because a heart failure history alone is associated with increased morbidity and mortality after non - cardiac surgery ( table 2 ) . biomarkers of ddf are important as prognostic parameters in patients with myocardial ischemia , atrial fibrillation , aortic stenosis , and general heart failure . cardiac catheterization can show increased ventricular diastolic pressure ( > 16 mmhg ) with preserved systolic function and normal ventricular volumes in ddf . beyond the analysis of pressure / volume curves from cardiac catheterization , echocardiographic assessment of diastolic function becomes very important in perioperative risk stratification , surgical care , and anesthetic management of patients undergoing operations . however , before considering echocardiographic findings , the different application of variables in transthoracic echocardiography ( tte ) and transesophageal echocardiography ( tee ) in the outpatient clinic from intraoperative tee should be acknowledged because of differences in the postures of patients and their ventilation status , as well as the effects of anesthetic drugs . the classification and assessment of ddf comes from the initial , transmitral flow ( tmf ) examination with a pulsed - wave doppler ( pwd)-based evaluation of la and lv filling and its abnormalities , according to current guidelines . however , because of the known limitations of the rigid application of conventional doppler indices , the most recent guidelines no longer recommend determining e and a waves on tmf with pwd as the initial step in the decision for grading ddf with normal lv ef . new guidelines based on tte have been simplified and focus on the increase in left atrial pressure ( lap ) rather than grading diastolic dysfunction . in patients with normal lv ef , more than half of the following should be positive to determine the presence of ddf : average e / e > 14 , lateral e < 10 cm / s , tricuspid regurgitation ( tr ) velocity > 2.8 m / s , and la volume index > 34 ml / m . in patients with depressed lv ef and with normal lv ef but myocardial disease , the mitral inflow ratio and the criteria above are used for evaluating lap and grading ddf ( table 3 ) . some doppler variables are valuable for the assessment of myocardial diastolic function including tmf with pwd ( e / a wave , deceleration time [ dt ] , a duration ) , pulmonary venous inflow with pwd ( s / d , atrial reversal [ ar ] ) , tissue doppler image ( tdi ) with pwd ( e/a wave ) , and transmitral color m - mode ( tmf propagation velocity [ vp ] ) , usually on midesophageal ( me ) four - chamber and me long - axis views . conventionally , the lv filling pattern of tmf ( e and a wave , e / a ratio ) and dt are most commonly used in echocardiographic examinations regarding ddf , but these , along with pulmonary venous flow patterns , are dependent on preload variation and heart rate and rhythm disturbances . in this regard , the patient 's preload can be reduced using nitroglycerin ( ntg ) or with the use of the valsalva maneuver in an awake state ( or reverse trendelenburg position or a prolonged lung recruit maneuver in a patient under general anesthesia ) potentially unmasking an e , a pattern and impaired relaxation to discriminate normal from a pseudo - normal state . in contrast , tissue doppler imaging of the mitral annulus is considered a more sensitive tool in the assessment of ddf , in which measurements of e indicate the myocardial velocity , and not blood flow velocity . e may be influenced by preload variation in the normal heart , but becomes preload - independent in a heart with ddf . the abnormal range of e values is also influenced by age : an e < 10 cm / s in those < 50 years and e < 8 in those > 50 . the value of e is higher at the lateral annulus than the septal annulus , because the motion of the lateral annulus is freer than that of the septal annulus due to the surrounding structures . it is convenient to use the lateral e in intraoperative tee and the septal e in tte . measurements of e represent the early diastolic active relaxation phase , but do not represent lv compliance for which the e / e ratio ( the ratio of the transmitral e wave velocity to the mitral annular velocity ) normalizes early transmitral lv filling to mitral annular motion . this is used to estimate mean left atrial pressure , with values > 15 representing elevated filling pressures and < 8 reflecting normal filling pressures . with a ratio between 8 and 15 , this measurement has been considered accurate and relatively independent of lv systolic function , rhythm abnormalities , lv hypertrophy , and functional mitral regurgitation . the prediction of lv filling pressure using doppler modalities is important in patients with ddf . elevated lv filling pressures may be the most important factor in poor outcomes , rather than merely the presence of delayed relaxation . after cardiac surgery , it can also predict a prolonged icu / hospital stay and mortality independently of systolic function . when evaluating diastolic function with echocardiographic tools , measurements should be performed multiple times , considering multiple doppler variables ( table 4 ) . according to these echocardiographic variables , based on tee , it might be suggested that predictors for increased perioperative cardiovascular risk would include e / a of 12 , dt of 150200 ms , e < 8 cm / s , e / e > 15 , vp < 40 cm / s , an enlarged la , and lv hypertrophy in tte . in fact , tdi is of limited use in analyzing regional wall motion abnormalities ; however , there are some recent advances in color tdi , such as strain , strain rate , speckle tracking , and velocity vector imaging that may allow a better appreciation of lv filling dynamics and a more accurate classification of diastolic function . brain natriuretic peptide ( bnp ) and n - terminal probnp ( nt probnp ) are recognized as specific markers of heart failure ( hf ) with acute dyspnea and are elevated in both hfpreef and hf with reduced hf ( hfref ) . these markers tend to be higher in hfref than hfpreef , but do not distinguish hfpreef from hfref effectively . suggested levels for a diagnosis of acute hf are bnp > 100 pg / ml or nt probnp > 300 pg / ml , which are independent predictors of adverse cardiovascular events in hfpreef . other reasons for elevated bnp factors include renal failure and pulmonary hypertension , caused by lung disease , and obesity , which decrease bnp levels . as mentioned above , it is important not to overlook patients with asymptomatic lv ddf in the preoperative evaluations , and risk factors for heart failure should also be considered . although not specific to geriatric patients , the latest acc / aha guidelines should be used for the perioperative care of cardiac patients undergoing general surgery , and if needed , preoperative cardiovascular risk factors should be evaluated and treated by cardiologists . in ischemic heart disease - related procedures , bare - metal stenting can be performed with dual anti - platelet therapy for 46 weeks . statins should not be discontinued before surgery . because the renin - angiotensin - aldosterone system plays an important role in the development of dhf and particularly in myocardial remodeling and fluid retention , angiotensin - converting enzyme inhibitors , angiotensin receptor antagonists , and aldosterone antagonists as aforementioned mentioned , drugs for the treatment of pre - existing heart failure , arrhythmias , hypertension , and ischemic heart diseases ( e.g. , diuretics , -blockers , calcium channel blockers , antiplatelet agents ) should be maintained during the perioperative period . the principles of anesthetic management for patients with ddf must be addressed on a case - by - case basis . in addition to standard monitoring tools , monitoring the volume status in these patients is important , because they are very sensitive to volume changes due to surgical bleeding and sympathetic tone changes due to anesthesia in the perioperative period . thus , instituting an arterial cannula is necessary for invasive arterial monitoring , indirect assessments of preload by stroke volume variance , and frequent blood sampling ; the timing of each should be based on experience and local practice . in major or vascular surgery , central venous catheterization , pulmonary artery catheterization , and/or tee may be needed , which can contribute to evaluating intravascular volume during anesthesia . during anesthesia , patients with ddf or dhf are commonly elderly and may have hemodynamic instability because of general and regional anesthesia disturbing the baseline sympathetic tone , thereby leaving the patient with a higher resting sympathetic tone and altered -receptor sensitivity . in addition , the frequent use of diuretics due to pre - existing diseases , a reduction in the thirst response in the elderly , and age - related renal function changes can aggravate the reduction of preload , increasing the sensitivity to volume status . moreover , direct effects of anesthetics on cardiac inotropy and lusitropy as well as arterial and venous vasodilation can not be ignored with regard to unstable hemodynamics either . the choice of regional versus general anesthesia is still debated and there are no definitive recommendations either way . however , epidural anesthesia is preferable to spinal anesthesia because of slower hemodynamic changes from the slow onset time and the smooth removal of sympathetic tone . in addition , for general anesthesia , iv induction and maintenance with balanced anesthesia of volatile agents and opioids are recommended . sevoflurane during spontaneous ventilation preserves diastolic relaxation better than propofol , but there are no differences between them regarding diastolic function during positive pressure ventilation through balanced anesthesia . the common volatile agents ( sevoflurane , desflurane , isoflurane ) , iv anesthetics ( propofol , midazolam ) , and opioids ( morphine , remifentanil ) have no significant effects on diastolic performance in healthy people , unlike some animal models . however , in high - risk dhf patients , anesthesiologists should avoid acute changes in hemodynamic load conditions , heart rate , and myocardial oxygen balance . to achieve this , a 3050% reduced dose of induction agents should be used , followed by waiting for their delayed effects due to the reduced cardiac reserve and slower circulation time , as well as titrating the anesthetics . intraoperative bp control is important , in that systolic bp must be within 1020% of the baseline value and pp should be kept below the diastolic bp . an increased pp increases ventricular wall stress , alters ventricular contraction time , and impairs early diastolic filling , increasing the cardiac workload . the rule of 70s is useful for controlling bp : age > 70 , diastolic bp > 70 , hr = 70 , and pp < 70 . given the effects on cardiac work by controlling pp , the combined use of low - dose ntg ( 0.54 g / kg / min ) and phenylephrine titration ( from 0.25 g / kg / min ) is applicable and helpful , but either agent alone can worsen hemodynamics . this protocol preserves vascular distensibility , and prevents reductions in preload and coronary perfusion pressure , thereby maintaining stroke volume with minimal cardiac work . in the postoperative period in patients with known ddf , hypoxemia and/or atrial fibrillation are the most common complications , because restored vascular sympathetic tone due to emergence and resolution from general or regional anesthesia causes a volume shift to central blood volume . the subsequent volume overload can result in pulmonary edema and/or atrial fibrillation due to decompensation . because these events can happen acutely in a patient with an initially stable appearance , continued and careful monitoring are essential . maintenance of ntg at a low dose ( 25 g / min ) is also useful for the pulmonary vasculature , and it is important to avoid the risk factors that predispose patients to hf ( table 5 ) . among these factors , a hypertensive crisis can be managed with sound anesthetic practices , planning for appropriate postoperative analgesia , preventing shivering by control of body temperature during the operation , and using iv calcium channel blockers ( nicardipine , diltiazem ) and high doses of iv ntg . specific drugs with positive lusitropic effects are often invaluable in circulatory failure related to ddf and include phosphodiesterase iii inhibitors ( milrinone ) and calcium channel sensitizers ( levosimendan ) , both of which are inodilators . milrinone has inotropic , vasodilatory , and lusitropic ( particularly in a heart failure state ) effects with minimal chronotropy , by increasing calcium ion uptake in the sarcoplasmic reticulum . it can be used at a bolus dose of 50 g / kg over 10 min and an infusion of 0.3750.75 g / kg / min . levosimendan sensitizes the contractile elements to calcium and has positive inotropic effects , modulating the interaction between troponin and calcium . it also has vasodilatory effects by opening atp - sensitive potassium channels and improving both systolic and diastolic functions . ddf of the lv and dhf with preserved ef are not rare pathologies , but are frequently underestimated . in the preoperative evaluation of asymptomatic patients undergoing high - risk surgery , echocardiographic assessment of lv diastolic function should be included in cardiovascular assessments , as should consideration of the risk factors for dhf . early detection of and not overlooking preclinical lv diastolic dysfunction can be useful for the choice of appropriate surgery , appropriate bp control , timely monitoring tools , and proper volume management to maintain normovolemia on a case - by - case basis . although anesthesiologists may not appreciate the stages of ddf , those who are involved in the perioperative care of these patients with cardiovascular disorders need to be able to manage them appropriately . because there is not yet an evidence - based treatment of choice regarding acute decompensated dhf , its management should focus on resolving known precipitating factors such as myocardial ischemia , hypertensive crisis , arrhythmias , and pulmonary congestion .

introduction : motion artifacts are a common problem in pediatric radiographic studies and are a common indication for pediatric procedural sedation . this study aimed to compare the combination of oral midazolam and ketamine ( omk ) with oral midazolam alone ( om ) as procedural sedatives among children undergoing computed tomography ( ct ) imaging . methods : the study population was comprised of six - month to six - year old patients with medium - risk minor head trauma , who were scheduled to undergo brain ct imaging . patients were randomly allocated to two groups : one group received 0.5 mg / kg midazolam ( om group ; n = 33 ) orally and the other one received 0.2 mg / kg midazolam and 5 mg / kg ketamine orally ( omk group ; n=33 ) . the vital signs were monitored and recorded at regular intervals . the primary outcome measure was the success rate of each drug in achieving adequate sedation . secondary outcome measures were the time to achieve adequate sedation , time to discharge from radiology department , and the incidence of adverse events . results : adequate sedation was achieved in five patients ( 15.2% ) in om group and 15 patients ( 45.5% ) in omk group , which showed a statistically significant difference between the groups ( p = 0.015 ) . no significant difference was noted between om and omk groups with respect to the time of achieving adequate sedation ( 33.80 7.56 and 32.87 10.18 minutes , respectively ; p = 0.854 ) and the time of discharging from radiology department ( 89.60 30.22 and 105.27 21.98 minutes , respectively ; p=0.223 ) . the complications were minor and similar among patients of both groups . conclusion : this study demonstrated that in comparison with om , omk was more effective in producing a satisfactory level of sedation in children undergoing ct examinations without additional complications ; however , none of these two regimens fulfilled clinical needs for procedural sedation .

the results of imaging studies are negatively affected by patients movements ; hence , patient s cooperation is required during imaging . children s movements and lack of cooperation are common indications for pediatric procedural sedation during imaging studies such as computed tomography ( ct ) scans ( 1 ) . different rates of success have been achieved by various sedation regimens such as diphenhydramine , propofol , ketamine , midazolam ( 2 ) , chloral hydrate ( 3 ) , pentobarbital ( 4 ) , and dexmedetomidine ( 5 ) ; therefore , the efforts to find an ideal regimen are continued . the selected sedative should have a rapid onset of action , few adverse effects , short and sufficient duration of action , self - maintenance of a patent airway , minimal effects on respiration or hemodynamics , and rapid recovery ( 6 ) . over the last few years , researchers have shown a special interest in finding effective , nonparenteral , sedative agents that do not have injection problems ( 3 ) . ketamine is a noncompetitive antagonist of the n - methyl - d - aspartate receptor ( nmdar ) , which is used for premedication , sedation , and induction as well as maintenance of general anesthesia . quick onset , short duration of action , and maintenance of laryngeal reflexes have made it a popular sedative choice for pediatric patients in the emergency department ( 7 ) . although ketamine is known as a parenteral agent , some researchers have successfully used it as an oral sedative drug ( 8 , 9 ) . midazolam is a potent benzodiazepine with rapid onset and offset effects as well as anxiolytic and amnestic properties , which can be administered through different routes ( i.e. oral , intravenous , intramuscular , rectal , sublingual , and intranasal ) . oral midazolam ( om ) is a safe and effective choice for sedation in children ( 10 ) . in previous studies , the authors have used a combination of oral midazolam and ketamine ( omk ) as premedication and sedative regimens ; besides , some studies reported that combination therapy has higher efficacy without any additional adverse effects in comparison to the methods using each drug alone ( 11 , 12 ) . to the best of our knowledge , limited number of studies have reported the use of om during imaging , which had reported lower effectiveness rate in comparison with chloral hydrate ( 13 , 14 ) ; however , omk has not been used for this purpose , yet . therefore , this study was conducted to compare the effects of omk with om as procedural sedatives in pediatric patients undergoing ct imaging . study design and setting this randomized , double - blinded , clinical trial was conducted from november 2012 to november 2013 in two teaching hospitals ( ayatollah kashani hospital and alzahara hospital ) affiliated to isfahan university of medical sciences , isfahan , iran . the study protocol was approved by ethics committee of the isfahan university of medical sciences . all parents or guardians were informed of the study 's protocol , risks , and benefits and were asked to sign an informed written consent . we included six - month to six - year old children with medium - risk minor head trauma who were scheduled to undergo brain ct scan ( 15 ) . medium - risk minor head trauma was defined as initial glasgow coma scale score of 15 with any history of brief loss of consciousness , posttraumatic amnesia , vomiting , headache , or intoxication . all of these patients were classified as status i or ii according to american society of anesthesiologists ( asa ) physical status classification . children with neurological disorders , anomalies of the cardiovascular system , known allergy to midazolam , gastritis , any serious systemic diseases , those on long - term treatment with hepatic enzyme - inducing drugs or those receiving erythromycin concurrently ( due to drug interaction ) , and patients who had received medication within the preceding 48 hours were excluded . patients were randomly allocated to two groups ; om group received 0.5 mg / kg midazolam ( produced by ; tehran shimi , tehran , iran ) orally and omk group received 0.2 mg / kg midazolam and 5 mg / kg ketamine ( produced by ; rotexmedica , trittau , germany ) orally . demographic and basic characteristics such as age , height , gender , weight , body mass index ( bmi ) , and history of any medical condition were recorded before administration of study drugs . then a nurse , who was blinded to the study , mixed both of the medications with 5 ml of sugar syrup to make it palatable . pulse rate and oxygen saturation ( sao2 ) were continuously monitored by a portable pulse oximeter . blood pressure , heart rate , respiratory rate , and sao2 were recorded at baseline ( just before drug administration ) and every 30 minutes until the patient was discharged from the radiology department . the level of sedation of patients after drug administration was assessed using ramsay sedation scale ( rss ) ( 16 ) . patients who did not show satisfactory response to the sedative drugs within 40 minutes and those who were awakened or moved during the imaging were excluded from further analysis . the interval between administration of sedative drugs and achieving rss of four was considered as the time to achieve adequate sedation . once the patients achieved adequate sedation , they were transferred to a scanner room and the imaging was performed according to the protocol . after the scan , the patients were transferred to another room in the radiology department to monitor and observe their conditions . the time to discharge from radiology department was defined as the interval between the start of sedative administration and return to the baseline alertness and spontaneous breathing . statistical analyses all statistical analyses were performed using the spss 19.0 ( spss inc . , two - way anova was used to compare changes of vital signs during 180 minutes after drug administration between two groups . consort flow diagram of study inter - group differences of vital signs at baseline and at various time points after administration of midazolam and combination of midazolam - ketamine ( df : 18 , p > 0.05 for all comparisons ) . demographic characteristics of the participants a , data are presented as mean standard deviation . the outcomes of patients in midazolam and midazolam - ketamine groups om : oral midazolam ; omk : combination of oral midazolam - ketamine ; rd : radiology department . the mean age of participants was 2.8 1.6 years ( range : 6 months to 6 years ) and 54.5% of the participants were male . table 1 shows the basic characteristics of patients . there was no significant difference between the two groups in age , height , weight , and male to female ratio ( table 1 ) . adequate sedation ( rss of 4 ) was achieved in five patients ( 15.2% ) in the om group and 15 patients ( 45.5% ) in the omk group , which showed a statistically significant difference ( p = 0.015 ) . no significant difference was found between the groups in time of achieving adequate sedation ( p = 0.854 ) and the time of discharging from radiology department ( p = 0.223 ) . adverse effects observed in two patients ( one with nausea and another with vomiting ) in the om group and three patients ( two with nausea and one patient with vomiting ) in the omk group . complications were minor and transient and did not differ between study groups ( p > 0.05 ) . systolic blood pressure , sao2 level , pulse rate , and respiratory rate showed no significant inter - group differences at baseline and at various time points after administration of the drugs ( for all analysis : df = 18 , p > 0.05 , figure 2 ) . our investigation showed that in comparison to om , omk successfully induced sedation in 45% of the patients . om failed to sedate the patient in 85% of the cases and led to administration of additional medication and longer time to obtain the scan . ketamine is used for premedication , sedation , and induction as well as maintenance of general anesthesia with minimal effect on respiration and tendency to preserve autonomic reflexes ( 7 ) . it has been reported that 10 mg / kg of ketamine provides effective sedation and analgesia in young children undergoing wound repair processes ( 9 ) . moreover , 6 mg / kg of ok was reported to be effective in sedation for outpatient pediatric dental surgeries ( 10 ) . om is the most commonly used premedication in the united states ( 18 ) , which has been used as a safe and effective sedative in pediatrics , mainly in pediatric dentistry ( 10 , 17 ) ; however , intravenous midazolam has been found to induce more sedation ( 18 ) . intravenous or nasal midazolam has gained widespread popularity as a sedative for children undergoing radiographic studies such as ct scan ( - ) . the omk and om alone are safe , effective , and practical approach to manage children for minor dental procedures ( 22 , 23 ) . the efficacy of om and ketamine , alone or in combination has been studied in pediatric dentistry sedation and premedication ( - ) . reported better sedative effects during suturing lacerations with ok ( 10 mg / kg ) in comparison to om ( 0.7 mg / kg ) ( 27 ) . although our study was consistent with the previous studies regarding the higher efficacy of omk in comparison to om , most of the previous studies reported success rates of 60% to 90% for om ( 11 , 17 , 28 ) and 46% to 95% for omk ( 11 , 22 ) . showed that the combination of oral midazolam ( 0.5 mg / kg ) and ketamine ( 5 mg / kg ) led to deeper sedation in comparison with om ( 0.5 mg / kg ) alone in children requiring laceration repair . in addition , 27% of patients in the om group and 6% in the omk group needed further intravenous sedation ( 11 ) . ( 12 ) compared the efficacy of om ( 1 mg / kg ) and the omk ( midazolam , 0.5 mg / kg ; and ketamine , 3 mg / kg ) for guiding the behavior of children undergoing dental treatment and reported higher efficacy of omk . on the other hand , there are several studies reporting lower success rate , which are similar to our findings . ( 6 ) compared the sedative effects of intravenous midazolam with pentobarbital during brain ct imaging in children aged six months to six years . they administered pentobarbital to 29 patients ( 53% ) and midazolam ( mean dose , 0.2 0.03 mg / kg ) to 26 patients ( 47% ) . in the midazolam group , only five patients ( 19% ) were successfully scanned with midazolam alone and the remaining 21 patients ( 81% ) required additional medication and took a longer time to scan . another investigation reported a failure rate of 60% in administrating intranasal midazolam ( 0.2 mg / kg ) for sedation of children undergoing ct scan ( 3 ) . ( 29 ) used 0.4 mg / kg of om 20 minutes before the induction of general anesthesia and reported a mild or no sedative effect in 76% to 84% of patients . in this study , the onset time of adequate sedation was 33 minutes in om group and 32 minutes in omk group . ( 27 ) reported an onset time of 20 minutes for ok and 43 minutes for om . other studies reported that the time to reach optimal sedation level would be 15 minutes for om ( 10 , 17 ) . studies , which used om ( 0.5 - 1 mg / kg ) as premedication reported that the best time for optimal preoperative sedation would be 30 to 45 minutes before scan ( 30 ) . barkan observed that the needed time to achieve adequate sedation was 17 minutes for om and 14 minutes for omk . the patients ' blood pressure , respiratory rate , pulse rate , and sao2 levels did not show any significant changes during the sedation . although ketamine and midazolam affect the respiratory and hemodynamics responses ( 11 ) , our results were in line with the findings of previous studies that reported minimal effects of low - dose om and ok on these parameters ( 17 , 31 ) . although further studies are needed to confirm or refute our results and attention to the pharmacological aspects is necessary , lower efficacy of om in this study could be explained as follows : first , we used these drugs for ct imaging that needs a deeper level of sedation in comparison with some other studies that used those drugs in other settings as premedication . second , in the previous studies , intravenous forms of these drugs were used , which might bring about unexpected results ; the same drugs in different studies should be compared with caution . third , the ethnic differences in response to these sedative agents might affect the results . a major cause of variations in drug responses is hepatic cytochrome p450 oxidase ( cyp450)-mediated drug metabolism ( 29 ) . midazolam is almost exclusively metabolized by cyp450 3a ( cyp3a ) isoenzymes ( 32 ) . although previous studies have shown that these parenteral forms can be used orally , the oral form of drugs might bring about results that are more precise . second , the number of participants who were included in the final analysis ( as sedated patients ) was small . this study demonstrated that in comparison with om , omk was more effective in producing a satisfactory level of sedation in children undergoing ct examinations without additional complications ; however , none of these two regimens fulfilled clinical needs for procedural sedation . all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors .

we present an extremely rare case of noninfiltrating angiolipoma ( al ) of the buccal mucosa in a 90-year - old patient reaching a size of 14 cm 10 cm . al is rare in the soft tissues of oral cavity , and till date , only 22 cases have been reported , including our case . if size be taken into consideration , our case can be considered to be the first in literature to reach this massive size . surgical excision of the tumor mass was performed . the histopathological findings confirmed the diagnosis of noninfiltrating al . follow - up for 1 year revealed no signs of recurrence .

the common occurrence of these tumors is prevalent among males in their second or third decades of life with site predilection toward trunk and extremities , especially in the forearm . however , the incidence of al is extremely rare in the intraoral region , with only 21 reported cases in english literature . a 90-year - old woman with no significant medical history was referred to our department for evaluation of painless mass arising from oral cavity . the tumor was noticed by the patient 20 years ago as a small nodule on the right buccal mucosa and since then it gradually increased in its size . the growth reached a size , which could not accommodate in mouth , and due to constant irritation from the tumor , the patient coughed the growth for 5 years . since then on extraoral examination , a solitary , smooth , nontender and nonpulsatile pedunculated growth of size 14 cm 10 cm with variable consistency was evident with intraoral origin [ figure 1 ] . clinical image shows preoperative tumor on intraoral examination , single , smooth , firm and nontender stalk - like pedicle of diameter 2 cm was seen originating from the right buccal mucosa . based on the history and clinical examination , we provisionally diagnosed it as a benign growth . surgical excision of the tumor was done under general anesthesia [ figures 2 and 3 ] . clinical image shows excised tumor mass clinical image shows postoperative frontal view histopathology confirmed it as noninfiltrating al [ figures 46 ] . one - year follow - up of the patient revealed no evidence of recurrence . ethical approval from the institutional review board was obtained for the publication , and patient release form was signed by the patient . clinical image shows histopathology section ( 4 magnifications ) clinical image shows histopathology section ( 10 magnifications ) clinical image shows histopathology section ( 40 magnifications ) lipomas are the most frequently found soft - tissue benign tumors , but their occurrence in head and neck region is rare . al , in the oral cavity , was first documented by davis et al . as a growth on the hard palate . history of trauma , lipomatous differentiation by hormones during puberty , vascular proliferation of a congenital lipoma and fatty degeneration of a central hemangioma have been implicated as possible etiological factors . the mean diameter at the largest portions of all the 21 documented als occurring in oral region was 3 cm , and the mean onset age of the patients was about 29 years old . although diagnostic modalities such as magnetic resonance imaging , computed tomography , ultrasonography and aspiration biopsy have been used to differentiate between hemangioma , lipomas and al , the diagnosis of these tumors is confirmed only by histopathology . the standard treatment of choice is surgical excision for the noninfiltrating als and complete surgical excision with a clear surgical margin for poorly encapsulated infiltrating als to avoid recurrence . the case we have presented showed the typical clinical and histological findings of a noninfiltrating al . its most striking and unique features were its occurrence in a 90-year - old female with a 20-year long history and pedunculated mass of 14 cm 10 cm . with regard to the size and its typical pedunculated appearance , our present case can be considered to be the first of its kind in literature .

highlights22q11ds offers a compelling model to understand the neural substrates of attentional dysfunction.first study directly comparing neural function in 22q11ds vs. adhd patients22q11ds and adhd patients show a shared deficit in ri - related activation.adhd patients showed greater activity in the middle frontal gyrus than 22q11ds during ri.neural activity is inversely correlated with self - reported cognitive impulsivity in 22q11ds .

attention deficit hyperactivity disorder ( adhd ) is a behaviorally defined disorder characterized by a persistent pattern of inattention and/or hyperactivity impulsivity that is more frequent and severe than typically observed in individuals at a comparable level of development ( american psychiatric association , 2013 ) . substantial heterogeneity at both the genetic and phenotypic level has significantly impeded our understanding of the underlying biological mechanisms of adhd . as the past two decades of research on causal genetic and neurobiological risk factors for this illness have been met with limited success ( for reviews , see elia and devoto , 2007 ; franke et al . , 2009 ) , one promising strategy for investigating the pathogenesis of adhd involves the investigation of a disorder with known genetic etiology that shares similar phenotypic characteristics an approach that is supported by findings that large , rare chromosomal deletions and duplications ( copy number variants ; cnvs ) are implicated in adhd etiology ( williams et al . , 22q11.2 deletion syndrome ( 22q11ds ; velocardiofacial syndrome ) is a particularly compelling model , as it is a relatively common cnv that is highly penetrant for adhd ; approximately 40% of 22q11ds patients meet clinical criteria for the disorder ( green et al . , 2009 ) . however , it is not yet known whether the neural substrates of attentional dysfunction in 22q11ds are shared with idiopathic adhd . impaired response inhibition ( ri ) has been consistently implicated as a hallmark of idiopathic adhd ( dickstein et al . , 2006 ; durston , 2003 ; epstein et al . , 2007 ; schulz et al . , 2004 ; tamm et al . , 2004 ; van rooij et al . , 2015 ) . most commonly measured with either go / no - go or stop - signal task paradigms , successful inhibition of a prepotent response has been shown to rely upon engagement of a right - lateralized fronto - striatal network in healthy individuals ( aron and poldrack , 2006 ; aron et al . studies on the neural underpinnings of ri in adults with adhd have yielded inconsistent results , with some studies reporting less ri - related activation in adhd patients than controls ( e.g. , mulligan et al . , 2011 ; sebastian et al . , 2012 ; van rooij et al . , 2015 ) and , 2012 ; congdon et al . , 2010 ) or greater activity for adhd patients as compared to controls ( dillo et al . , 2010 ; karch et al . , however , the bulk of the evidence suggests a disturbed pattern of ri - related neural activation in adults with adhd . discrepancies in previous findings may be attributable to several factors , including differences in task parameters , medication status , symptom severity , and small sample size . consistent with findings of impaired performance on ri - related behavioral tasks in 22q11ds ( shapiro et al . , 2013 ) , using functional magnetic resonance imaging ( fmri ) we previously found that young adults with 22q11ds show significantly less activation in ri - related regions during successful ri relative to healthy controls ( montojo et al . , 2015 ) . further , we found that ri - related activity within the left middle frontal gyrus and basal ganglia was associated with the severity of self - reported cognitive impulsivity , suggesting that reduced engagement of ri - related regions in 22q11ds may be relevant to the behavioral manifestations of the disorder . despite symptomatic overlap between 22q11ds and idiopathic adhd , it is not yet known whether disturbances in the neural circuitry underlying inhibitory control difficulties are shared between these clinical populations . using fmri , we examined neural activity in patients with 22q11ds , idiopathic adhd , and demographically comparable healthy controls during performance of a stop - signal task . we hypothesized that , relative to controls , individuals with 22q11ds and those with idiopathic adhd would show reduced recruitment of fronto - striatal regions involved in inhibitory control . in addition , we explored the relationship of neural activity during task performance to dimensional measures of cognitive and behavioral impulsivity , to determine whether brain behavior associations were similar across groups . based on prior literature in 22q11ds and adhd ( montojo et al . , 2015 ; van rooij et al . , 2015 ) we hypothesized that , while 22q11ds and idiopathic adhd patients would share similar dysfunction within ri - related neural systems , this shared dysfunction would show different relationships to behavior . specifically , we hypothesized that 22q11ds patients would show an inverse relationship between neural activity during ri and cognitive impulsivity ( montojo et al . , 2015 ) . given the phenotypic and genetic heterogeneity known to exist within the adhd population , we hypothesized that we would not observe a significant relationship between neural activity and cognitive or behavioral impulsivity within this group . seventy - two participants ( 19 patients with 22q11ds , 23 with an adhd diagnosis , and 30 healthy adults ) were included in the study . 22q11ds participants include individuals with a molecularly confirmed diagnosis of 22q11.2 deletion syndrome recruited from an ongoing longitudinal study at the university of california los angeles ( ucla ) . individuals with adhd were recruited from the los angeles area as part of the consortium for neuropsychiatric phenomics ( cnp ) at ucla . the current study includes a partially overlapping subset of patients with 22q11ds and adhd that were included in previous publications ( congdon et al . , 2014 ; montojo et al . , four 22q11ds participants whose data were collected for the stop - signal task were excluded from analysis for the following reasons : low signal - to - noise ratio ( n = 1 ) , too much translational movement ( n = 1 ) , and poor registration between functional and anatomical scans ( n = 2 ) . thus , our final sample consisted of 15 patients with 22q11ds , 23 with adhd , and 30 healthy adults . all diagnoses were determined using the structured clinical interview for the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) ( scid ; first et al . , 2004 ) by trained raters who participated in an ongoing quality assurance program ( ventura et al . , adhd diagnoses in the cnp sample were confirmed with the adult adhd interview ( a structured interview derived from the kiddie schedule for affective disorders and schizophrenia , present and lifetime version ( ksads - pl ) ( kaufman et al . , 1997 ) ) , in order to provide a more detailed characterization of lifetime history of adhd in adults . prior to conducting diagnostic interviews , clinicians had to achieve good to excellent reliability with a set of gold standard ratings ( kappa coefficients of .90 or greater for categorical diagnoses and intra - class correlation coefficients ( iccs ) ranging from 0.85 to 1.00 for quantitative symptom ratings ) . diagnostic formulations for all 22q11ds cases were additionally reviewed in consensus diagnosis meetings led by the study director , a board - certified psychologist ( ceb ) . training , reliability and ongoing quality assurance procedures for psychiatric diagnostic assessments are detailed in a prior publication ( jalbrzikowski et al . , 2013 ) . exclusion criteria for all study participants included the following : neurological or medical condition that might affect performance , insufficient fluency in english , substance or alcohol abuse and/or dependence with the past 6 months , any contraindications to scanning , and left - handedness ( further details are provided in the supplementary material ) . the scid was used to ensure that healthy controls did not meet criteria for any current major mental disorder ( see the supplementary material and thakkar et al . , 2014 for additional details of inclusion / exclusion criteria ) . stable medications were permitted in 22q11ds and adhd participants , as it was not ethically permissible to ask participants to stop taking physician - prescribed medication in order to participate in the study . the ucla institutional review board ( irb ) approved all study procedures and informed consent documents . in both studies , after screening and neuropsychological assessment , participants took part in a behavioral testing session immediately prior to a 1-hour scan . in the behavioral testing session , participants received training on the stop - signal task in the form of one initial demonstration and trial run before completing one experimental run while inside the scanner . stimulus presentation and timing of all stimuli and response events were achieved using matlab ( mathworks ) and the psychtoolbox ( http://www.psychtoolbox.org , brainard , 1997 ) on an apple powerbook . for the experiment block administered in the scanner , each participant viewed the task through mri - compatible goggles and responded with his or her right hand on an mr - compatible button box in the scanner . participants completed a tracking version of the stop - signal task ( logan , 1994 ) enabled isolation of neural activation associated with the inhibition of an already - initiated motor response , and calculation of an individualized measure of inhibitory control ( stop - signal reaction time , ssrt ) . participants were instructed to respond quickly when a go stimulus was presented on the computer screen ( which consisted of left- or right - ward pointing arrows ) , except on the subset of trials where the go stop signal ( a 500 hz tone presented through headphones ) , in which case participants were instructed to withhold their response . the onset of the stop - signal , or stop - signal delay ( ssd ) , was adjusted according to the participant 's performance such that the ssd increased following a successful inhibition ( making the next trial more difficult ) and decreased following a failed inhibition ( making the next trial easier ) which ensured that subjects successfully inhibited on approximately 50% of stop trials . a complete description of the task and fmri acquisition parameters is presented in the supplementary material . the barratt impulsiveness scale ( bis ) version 11 ( bis-11 ; patton et al . , 1995 ) is a 30-item questionnaire assessing aspects of impulsivity which has been applied to various clinical groups , including those with adhd ( crunelle et al . in addition to calculating a total bis score , we used the revised , two - factor scoring method of the bis , which has been proposed as an alternative multidimensional structural representation of impulsivity ( reise et al . , 2013 ) . the revised method of scoring the bis results into two correlated factors : ( 1 ) cognitive impulsivity , reflecting difficulties in attentional control , concentration , careful and deliberate thinking , and planning ( e.g. , not a steady thinker , no self - control / concentration and not planful ) , and ( 2 ) behavioral impulsivity ( with some cognitive elements ) , which reflects acting impulsively , changing jobs , moving residences relatively often , and a scattered quick - paced cognitive tempo ( e.g. , extraneous racing thoughts , acts impulsively , and changes , moves around ) . we note that while the bis-11 measures an individual 's subjective view of his / her own behavior , there is a strong correlation between scores on this measure and clinical diagnoses of adhd ( malloy - diniz et al . , 2007 ) . for both scales , higher scores reflect higher levels of trait impulsivity . supervised clinical psychology doctoral students or phd staff administered a comprehensive neurocognitive battery . iq data were acquired for all participants using the wechsler abbreviated scale of intelligence ( wasi ; wechsler , 1999 ) or the wechsler adult intelligence scale ( wais - iv ; wechsler , 2008 ) . stop - signal task data were analyzed as has been previously described ( congdon et al . , 2010 , 2012 ) and as detailed in the supplementary material . briefly , ssrt was estimated using the quantile method ( band et al . , 2003 ) , with longer ssrt values reflecting poorer inhibitory control . additional performance measures included mean and standard deviation of reaction time ( rt ) on go trials , percent inhibition on stop trials ( i.e. , successful inhibition ) , and percent correct on go trials . to examine demographic differences between the three groups ( 22q11ds vs. adhd vs. controls ) we conducted univariate anovas for continuous variables or chi - square tests for categorical variables . pearson correlations were conducted to assess the relationship between demographics and stop - signal task performance . to examine group differences with regard to stop - signal task performance and bis-11 scores as iq and years of education showed significant differences between groups , ancovas were also conducted with years of education included as a covariate , in order to verify anova results ( iq was omitted given the high correlation between years of education and iq : r(68 ) = 0.577 , p < 0.01 ) . age was also included as a covariate given that this variable showed associations with task performance ( see results section ) . analyses were performed using tools from the fmrib software library ( http://www.fmrib.ox.ac.uk/fsl ) , version 5.0 ( s.m . smith et al . , 2004 ) , and preprocessing steps are outlined in the supplementary material . for each subject , stop successful null , and stop unsuccessful stop successful contrasts were computed , and the output from the subject - specific analyses was then analyzed using a mixed - effects model with flame for between - group comparisons . to examine between - group differences , group - level statistics images were thresholded with a cluster - forming threshold of z > 2.3 and a cluster probability of p < 0.05 , corrected for whole - brain multiple comparisons using the gaussian random field theory . as our primary goal was to examine between - group differences in ri - related activation , and as patterns associated with ri have been previously reported in each group alone ( congdon et al . 2015 ) , we present below only the results from stop successful go between - group comparisons . the whole - brain , between - group contrasts included the following : controls > 22q11ds , controls > adhd , 22q11ds > adhd , and adhd > 22q11ds . results for additional task contrasts are presented in the supplementary material . in order to identify differences in neural activity during successful ri that were common to both clinical groups relative to controls , we used activity maps for the contrasts of controls > 22q11ds and controls > adhd and performed a conjunction analysis to define a set of significantly overlapping regions between these two contrasts . we then extracted percent signal change values from this overlapping set of regions in order to visualize the patterns of group differences in neural activation for these regions of overlap ( for details , see methods for visualizing group differences below ) . in order to investigate correlates of ri - associated neural activity in these clinical groups , we characterized relationships between neural activity and bis-11 scores using whole - brain regressions , within 22q11ds and adhd patients only , given the limited range of bis-11 scores within control participants . first , to test for differences in the relationship between ri - related activation and bis-11 scores between the 22q11ds and adhd groups ( i.e. , an interaction effect ) , whole - brain regressions were defined for the stop successful go contrast with either bis cognitive or behavioral impulsivity subscale scores and group membership as covariates of interest , including age and sex as covariates of no interest . second , to further explore the pattern of association between ri - related activation and bis-11 scores within the 22q11ds and adhd patient groups separately without regard to group differences in these relationships we defined whole - brain regressions for the stop successful go contrast with either bis cognitive or behavioral impulsivity subscale scores as covariates of interest , including age and sex as covariates of no interest . each whole - brain regression was thresholded with a cluster - forming threshold of z > 2.3 and a cluster probability of p < 0.05 , corrected for whole - brain multiple comparisons using the gaussian random field theory . this approach allowed us to : 1 ) test whether there were differences in the association between neural activity during task performance and bis scores between groups ( 22q11ds > adhd ; adhd > 22q11ds ) ; and 2 ) subsequently investigate the associations between neural activity and bis scores within each group alone , in order to understand the directionality of any significant interaction effects . in order to visualize : 1 ) the common regions of reduced ri - related neural activity across both patient groups relative to controls ; and 2 ) the relationship between ri - related neural activity and bis-11 scores within each group , we extracted percent signal change values corresponding to a 1-s stimulus convolved with a double - gamma hemodynamic response function from each contrast of interest ( following mumford and poldrack , 2007 ) following inspection of group contrasts . for the conjunction analysis , after inspecting clusters of activation that significantly differed between both patient groups as compared with controls , we intersected the group conjunction map with an anatomical atlas ( the fsl harvard oxford probabilistic atlas thresholded at 25% ) to extract percent signal change from anatomically - constrained regions that contained the clusters of activation identified in the conjunction map . the anatomically constrained regions that contained these clusters of activation included the anterior cingulate cortex ( acc ) , cerebellum , inferior parietal lobe , middle frontal gyrus , middle temporal gyrus ( posterior ) , paracingulate gyrus , superior frontal gyrus , and left and right thalamus . by intersecting our group conjunction map with an anatomical atlas , we were able to extract percent signal change from multiple clusters within anatomically - defined regions . in order to visualize the relationship between stopping - related activation and self - reported impulsivity within each group , percent signal change was extracted from significant clusters identified in each whole brain regression of stop successful go activity on impulsivity scores . follow - up analyses were conducted to investigate : 1 ) the effects of psychostimulant use and iq on fmri results , and 2 ) the effects of group differences in performance on go trials on ri - related neural activity ( see the supplementary material ) . the total sample consisted of 68 participants ( 15 22q11ds , 23 adhd , and 30 healthy adults , age 1838 years old ) . as shown in table 1 , 22q11ds , adhd , and control groups were matched on all demographic factors except for years of education and iq . adhd patients and controls had significantly higher iq and more years of education than 22q11ds patients . there was no significant difference in years of education or iq between adhd patients and controls . 33% ( 5 out of 15 ) had a clinical diagnosis of adhd , and 20% ( 3 out of 15 ) were taking psychostimulants . in adhd patients , 40% ( 8 out of 23 ) reported currently taking psychostimulants . as shown in table 2 , there were no significant differences in ssrt between the three groups and given the tracking nature of the task design percent inhibition on stop trials was close to 50% in each group . however , there were significant differences in percent correct on go trials and median reaction time ( rt ) on go trials , with adhd patients and controls showing higher accuracy and faster response times on go trials as compared to 22q11ds patients . there were no significant differences between patients with adhd and controls in accuracy or median rt on go trials . these results remained unchanged when controlling for age and years of education . across the three groups , there was a significant relationship between percent correct on go trials with age ( r = 0.365 , p < 0.01 ) and years of education ( r = 0.575 , p < 0.01 ) . participants who were older or had more years of education performed with higher accuracy on go trials relative to younger or less educated participants . there was no association between any of the other demographic or performance variables ( see the supplementary material ) . , there were significant group differences in cognitive impulsivity . specifically , 22q11ds and adhd patients showed significantly elevated cognitive impulsivity scores relative to controls , while there were no significant differences between adhd and 22q11ds patients . there were also significant group differences in behavioral impulsivity . specifically , adhd patients showed significantly elevated behavioral impulsivity scores relative to 22q11ds patients and controls , while there was no significant difference between 22q11ds patients and controls . these results remained unchanged when controlling for years of education ( see the supplementary material ) . as shown in fig . 1 and table 3 , a direct comparison between controls and 22q11ds patients during successful ri ( stop successful go contrast ) revealed significantly increased activation in controls relative to 22q11ds patients in the bilateral medial frontal cortex / acc / paracingulate gyrus , bilateral middle / inferior / superior frontal gyrus , bilateral occipital and parietal regions , right middle temporal gyrus , posterior cingulate cortex ( pcc ) , bilateral basal ganglia ( thalamus ) and cerebellum . a direct comparison between controls and adhd patients revealed a similar pattern of increased activation for controls relative to adhd patients in the medial frontal cortex / acc paracingulate , right middle frontal gyrus , bilateral inferior parietal cortex , bilateral middle temporal gyrus , bilateral occipital cortex , bilateral basal ganglia ( thalamus ) , and cerebellum , while there were no regions showing greater activation for adhd patients as compared to controls . as controls showed greater ri - related activation than both 22q11ds and adhd patients across multiple brain regions , we conducted a conjunction analysis in order to determine the regions common to both clinical groups ( price and friston , 1997 ) . as illustrated in fig . 1 , conjunction regions that showed overlap for 22q11ds patients and adhd patients compared to controls included the medial frontal cortex / anterior cingulate / paracingulate gyrus , right middle frontal gyrus , left inferior parietal cortex , cerebellum ( bilaterally ) , thalamus ( bilaterally ) , and right middle temporal gyrus . corresponding percent signal change plots are illustrated in fig . 2 , showing that controls show greater activity than both adhd and 22q11ds patients within ri - related regions , including the acc , bilateral cerebellum , left inferior parietal cortex , right middle frontal gyrus , right middle temporal gyrus , paracingulate gyrus , superior frontal gyrus , and bilateral thalamus . as shown in fig . 3 and table 3 , a direct comparison between adhd and 22q11ds patients revealed significantly increased activation that was specific to the bilateral middle frontal gyrus for patients with adhd relative to those with 22q11ds . there were no regions showing greater activation for 22q11ds patients as compared with adhd patients . since we observed a significant difference in performance for go trials , which may affect the neural activity findings for the stop successful go contrast ( see the supplementary material for details ) . to further explore differences in ri - related activation between clinical groups , we examined the relationship between ri - related activation and trait impulsivity as a function of group status ( 22q11ds vs. adhd ) . 4a and detailed in table 4 , a significant interaction was observed between group and cognitive impulsivity , such that 22q11ds patients showed an inverse correlation between activity in the medial frontal cortex and precuneus with cognitive impulsivity , while adhd patients showed a positive correlation in these same regions . follow - up regressions within 22q11ds patients alone confirmed a negative association between cognitive impulsivity and ri - related activation in the medial frontal gyrus , precuneus / posterior cingulate cortex , and inferior parietal cortex ( table 4 ) . no regions were positively associated with cognitive impulsivity in the 22q11ds patients , and no significant correlation with cognitive impulsivity was evident in the adhd group alone after correction for multiple comparisons . as illustrated in fig . 4b and detailed in table 4 , a significant interaction in the opposite direction was observed between patient group and behavioral impulsivity , 22q11ds patients showed a positive correlation between activity in the left motor cortex and behavioral impulsivity , while adhd patients showed an inverse correlation . follow - up regressions within 22q11ds patients alone confirmed a positive association between successful stopping - related activation in the left motor cortex and behavioral impulsivity , such that increased neural activity in this region during successful stopping was associated with more severe self - reported behavioral impulsivity . no brain regions were inversely correlated with behavioral impulsivity in the 22q11ds patients , and no significant correlation with behavioral impulsivity was evident in the adhd group alone after correction for multiple comparisons . in a follow - up analysis , we excluded one outlier that was evident within the 22q11ds sample for the behavioral impulsivity score . while the interaction was no longer significant , the 22q11ds within - group correlation between ri - related activity and behavioral impulsivity remained significant to our knowledge , this is the first study to directly compare the neural substrates of response inhibition ( ri ) across distinct clinical populations associated with attentional dysfunction ( 22q11ds and idiopathic adhd ) and healthy controls . we hypothesized that , while 22q11ds and idiopathic adhd patients would share similar dysfunction within ri - related neural systems , this shared dysfunction would show different relationships to behavior . specifically , we anticipated that 22q11ds patients would show an inverse relationship with cognitive impulsivity ( montojo et al . , 2015 ) , whereas we would not observe a significant relationship between neural activity and cognitive or behavioral impulsivity within the adhd cohort . supporting our hypotheses , our fmri conjunction findings indicate that , during performance of the stop - signal task , 22q11ds and adhd patients show reduced ri - related neural activity compared to healthy controls within multiple overlapping brain regions , specifically the acc , middle and superior frontal gyri , middle temporal gyrus , inferior parietal cortex , paracingulate gyrus , bilateral thalamus and cerebellum . in addition to this shared pattern of hypoactivation across both clinical groups , 22q11ds patients showed reduced ri - related neural activity relative to adhd patients within the bilateral middle frontal gyrus . also in agreement with our predictions , in 22q11ds patients ( but not adhd patients ) , reduced neural activity within the medial frontal gyrus , precuneus / posterior cingulate , and inferior parietal cortex was associated with greater self - reported cognitive impulsivity . this measure of cognitive impulsivity was elevated in 22q11ds patients , in agreement with prior findings regarding the nature of adhd symptomatology in this population ( antshel et al . , 2008 ; niklasson et al . , these findings suggest that 22q11ds and idiopathic adhd patient groups share a pattern of reduced neural engagement during ri , relative to healthy controls , but that 22q11ds and idiopathic adhd patient groups differ with regard to the relationship of ri - related neural activity to trait impulsivity . specifically , these findings suggest that neural dysfunction during ri in 22q11ds is associated with variability in cognitive impulsivity , and not behavioral impulsivity . this relationship was not observed in patients with adhd . in the current study , both 22q11ds and idiopathic adhd patients showed significantly less engagement of several ri - related regions that have been implicated in prior research ( aron et al . , 2007 ; aron and poldrack , 2006 ; whelan et al . , 2012 ) . the use of a tracking stop - signal task ensured that participants successfully inhibited responses 50% of the time , which results in an equal number of successful stop trials for comparison and controls for difficulty level across participants . as such , findings of reduced activation in these cortical regions and the thalamus during successful inhibition in both patient groups are consistent with models of ri suggesting that the right inferior frontal cortex , via connections with a prefrontal - basal ganglia network , implements inhibition of a prepotent motor response ( aron et al . , 2014 ) . furthermore , findings of reduced ri - related neural activation in these patient groups , both of which are characterized by deficits in inhibitory control , are consistent with prior research ( mulligan et al . , 2011 ; sebastian et al . , 2012 ; van rooij et al . , 2015 ) . with regard to behavioral performance , it is somewhat surprising that there were no differences in ssrt between groups , given that deficits in response inhibition are widely reported in adhd . along these lines , we observed non - significantly increased accuracy on go trials in our adhd sample as compared with controls . because this study included adult participants with adhd who were able to complete up to 2 days of testing and from whom we have complete , usable data , it is possible that these individuals are not characterized by pronounced deficits in behavioral indices of response inhibition , or that they have acquired compensatory mechanisms to overcome response inhibition deficits . from that standpoint , the findings of anomalous patterns of neural activity during ri in the adhd group we previously found that healthy controls showed significantly greater activation than 22q11ds patients within the frontal cortical and basal ganglia regions during successful ri ( montojo et al . , 2015 ) . one prior study on adolescent 22q11ds patients showed greater activation in the left parietal regions compared to healthy controls during performance of a go / no - go task ( gothelf et al . however , in addition to a difference in tasks used , the different findings may be accounted for by age differences , as differences in neural activity during this age range may reflect changes in neural trajectories during this developmentally sensitive period . in terms of ri - related activation in idiopathic adhd patients , although reports are mixed , the bulk of the evidence supports a pattern of reduced neural activation during ri as compared with controls ( cortese et al . , 2012 ; mulligan et al . , 2011 ; sebastian et al . , 2012 ; strong support comes from a recent large - scale study reporting a pattern of relative hypoactivity in 185 adolescents with adhd , as well as in their unaffected siblings , as compared with healthy controls . employing the stopsignal task , the authors found reduced activation during successful ri seen in the left inferior and superior frontal gyri in adhd patients as compared with unaffected siblings and controls , and reduced ri - related activation seen in the left supramarginal gyrus , right postcentral gyrus , and right temporal parietal junction in both adhd patients and their unaffected siblings as compared with controls ( van rooij et al . , 2015 ) . these findings suggest that ri - related deficits in activity within these regions may be useful endophenotypes that extend beyond the affected individuals of the family . while there are also reports of greater fronto - striatal activation ( dillo et al . , 2010 ; karch et al . , , 2012 ; congdon et al . , 2014 ) , during ri in adhd patients as compared with controls , a review of this heterogeneous literature suggests that differences in the cognitive paradigm employed ( e.g. , stop - signal task vs. go / no - go task ) , medication status , symptom severity , and small sample size may account for these discrepant findings . our findings of common deficits in neural activity during ri between idiopathic adhd and patients with a specific genetic mutation that is highly penetrant for the illness suggest that this pattern of hypoactivation during ri may be a valuable neural endophenotype underlying attentional dysfunction . the direct comparison in ri - related neural activity between 22q11ds and adhd patients was of particular interest to determine whether the clinical groups differed in ri - related neural circuitry . this analysis revealed that adhd patients showed greater activity in the bilateral middle frontal gyrus regions compared to 22q11ds patients . although the right inferior frontal cortex has been shown to be central to ri , it does so through a network of prefrontal basal ganglia regions ( aron et al . , 2014 ) and the middle frontal gyrus is often engaged during suppression of a prepotent response ( e.g. , dambacher et al . , neuroimaging studies have additionally implicated the middle frontal gyrus in the processing of higher order information , including functions of working memory ( leung et al . , 2002 ; mccarthy et al . , 1994 ) and episodic retrieval ( rajah et al . , 2011 ) . our findings indicate substantial overlap across clinical groups in terms of hypoactivation in ri - related neural circuitry relative to controls , but with additional dysfunction in the middle frontal gyrus in 22q11ds patients . impulsivity is a key symptom of adhd ( robbins et al . , 2012 ) and has been defined as a trait that leads to actions which are poorly conceived , prematurely expressed , unduly risky or inappropriate to the situation and that often result in undesirable consequences the complexity of defining impulsivity has led researchers to suggest that impulsivity may not be a unitary construct ( dalley et al . , 2011 ) . in line with this , a recent study proposed that a two - factor model including cognitive and behavioral impulsivity may best describe the multidimensional structural representation of self - reported trait impulsivity as measured by the bis-11 ( reise et al . , 2013 ) . in terms of trait impulsivity , both 22q11ds and adhd patients showed greater cognitive impulsivity compared to healthy controls , as measured by the bis-11 . the elevated cognitive impulsivity scores that we observed within the 22q11ds group are of interest given that 1 ) the cognitive impulsivity subscale specifically captures difficulties in attentional control ( reise et al . , 2013 ) , and 2 ) adhd diagnoses in 22q11ds are primarily of the inattentive subtype ( antshel et al . , 2008 ; niklasson et al , we observed that while adhd patients showed elevated behavioral impulsivity relative to both 22q11ds patients and controls , 22q11ds patients did not show increased behavioral impulsivity relative to controls . thus , these findings are in general agreement with previous literature and suggest that 22q11ds and adhd patients share elevated cognitive impulsivity , while adult adhd patients exhibit elevations in both cognitive and behavioral impulsivity . our findings further indicate that deficits in ri - related neural activation are associated with increased cognitive impulsivity in 22q11ds patients , but not behavioral impulsivity . specifically , as cognitive impulsivity increases , ri - related neural activity decreases within the medial frontal cortex , precuneus / posterior cingulate , and inferior parietal cortex . we have previously reported an inverse correlation between ri - related activation and cognitive impulsivity in 22q11ds patients ( montojo et al . , 2015 ) within a set of a priori ri - related regions of interest ( primarily frontal and basal ganglia regions ) . here , we extend those findings in a whole - brain regression analysis , to demonstrate an association between ri - related neural activity and cognitive impulsivity in a broader set of regions . together , these complementary analysis approaches provide convergent evidence that self - reported cognitive impulsivity is inversely associated with ri - related neural activation in multiple brain regions . in contrast , the finding of a positive association between behavioral impulsivity and ri - related neural activity in the left motor cortex in 22q11ds patients is novel . although this association may reflect a compensatory mechanism in patients required to achieve sufficient ri , the presence of an outlier driving the interaction effect leads us to exercise caution in interpreting these findings and suggests that replication is necessary . while pathophysiologic models of adhd have primarily focused on dysfunction within fronto - striatal neural circuitry ( arnsten , 2009 ; cortese et al . , 2012 ; precuneus interactions may constitute a new locus of dysfunction in adhd ( castellanos and proal , 2012 ; castellanos et al . , 2008 ) . specifically , a prior study reported reduced functional connectivity between the anterior cingulate cortex and precuneus / posterior cingulate cortex regions during resting state within adhd patients ( castellanos et al . , 2008 ) . we extend those findings here to patients with attentional dysfunction associated with homogeneous genetic etiology ( 22q11.2 deletions ) , by reporting an inverse correlation between ri - related neural activity and cognitive impulsivity symptoms within the precuneus / posterior cingulate . in addition , 22q11ds patients show decreased cortical volume and surface area within the precuneus ( jalbrzikowski et al . , 2013 ) . these findings are in agreement with the idea of this region as another locus of dysfunction , with variability in neural activity being related to the severity of trait impulsivity . with regard to the medial prefrontal cortex , both primate and human studies implicate this region in cognitive control functions ( ridderinkhof et al . , 2004 ) , including : the detection of unfavorable outcomes ( ito et al . , 2003 ; shidara and richmond , 2002 ; stuphorn et al . , 2000 ) , response errors ( gemba et al . , 1986 ; ito et al . , 2003 ) , response conflict ( botvinick et al . , 2001 ) , and decision uncertainty ( botvinick et al . , the inferior parietal cortex is activated during go / no - go tasks ( braveret al . , 2001 ; garavan et al . , 1999 ; kiehl et al . , 2000 ) and has been related to response conflict ( braver et al . , 2001 ; carter et al . , 2000 ) and , more generally , multiple aspects of sensory processing and sensorimotor integration ( caspers et al . , 2006 ; clower et al . , 2001 ) . we suggest that reduced engagement of neural circuitry within these regions has potential significance for the clinical presentation of 22q11ds . despite clear elevations in trait impulsivity in idiopathic adhd , the relationship between impulsivity and ri - related neural activation is still an active area of investigation . in a large - scale study of adhd patients , van rooij et al . ( 2015 ) reported an inverse correlation between adhd symptom count ( based on hyperactive / impulsive and inattentive symptoms according to dsm - iv criteria ) and neural activation in the inferior frontal gyrus during successful ri . here , our within - group analyses did not reveal an association between ri - related neural activity and cognitive or behavioral impulsivity for adhd patients that survived correction . this may be related to the relatively small sample of adhd patients included in our study . given the genetic and phenotypic heterogeneity within adhd patients , it may be necessary to include a larger sample to detect neural function behavior relationships . these findings also suggest that there may be more power to detect associations between dysfunctional ri - related neural activity and trait impulsivity within 22q11ds patients , given the more homogeneous genetic etiology . notably , the 22q11.2 locus includes the catechol - o - methyltransferase ( comt ) gene , which codes for an enzyme involved in prefrontal dopamine metabolism ( egan et al . , 2001 ; fallgatter and lesch , 2007 ; gothelf et al . , 2007 ; lachman et al . , 1996 ) which may be relevant to behavioral manifestations of inattention and impulsivity in 22q11ds ( cole et al . , 2013 ; cools et al . , 2007 ; gothelf et al . , 2007 ; shashi et al . , 2006 ; soeiro - de - souza et al . , 2013 ) . investigation of the relationships between impulsive behavior , response inhibition - related neural circuitry , and allelic variation and expression of comt and other genes involved in dopaminergic function is warranted in future , larger - scale studies of this syndrome . the findings reported here help to elucidate the neurobiological basis of attentional dysfunction in the following ways . first , we directly compared patients with adhd , a group that traditionally exhibits substantial heterogeneity at both genotypic and phenotypic levels , with 22q11ds patients , a group representing a relatively homogeneous genetic etiology with common downstream phenotypic characteristics . 22q11ds offers a compelling model for the investigation of the neurobiological substrates of attentional dysfunction in adhd . direct comparison of our adhd and 22q11ds patients allowed us to demonstrate a shared deficit in ri - related neural activation between groups , as compared with controls , but circumscribed differences in adhd patients as compared with 22q11ds patients . second , we investigated the relationship between ri - related neural activity and different aspects of trait impulsivity cognitive and behavioral . we used a dimensional approach to assess these aspects of impulsivity symptoms , which is in agreement with the rdoc initiative ( cuthbert and kozak , 2013 ; insel et al . , 2010 ) that aims to characterize psychopathology on dimensions of observable behaviors or neurobiological measures rather than traditional diagnostic categories . as such , our results provide initial evidence for how dimensional assessments of impulsivity may characterize distinct etiologies of downstream adhd diagnosis . finally , our ability to detect significant within - group correlations between impulsivity measures and ri - related neural activation in 22q11ds patients , but not adhd patients , has implications for future efforts , as there may be more power to detect associations between dysfunctional ri - related neural activity and trait impulsivity within 22q11ds patients , given the more homogeneous genetic etiology . taken together , these findings suggest a pattern of reduced neural engagement during ri in both idiopathic adhd and 22q11ds , seen in the acc , cerebellum , inferior parietal cortex , middle frontal gyrus , middle temporal gyrus , paracingulate gyrus , superior frontal gyrus , and bilateral thalamus . notably , 22q11ds patients differed relative to idiopathic adhd patients in that they showed hypoactivation of the bilateral frontal regions . our results further indicate that deficits in ri - related activation are associated with increased cognitive impulsivity in 22q11ds patients , but not increased behavioral impulsivity , consistent with the behavioral presentation of the disorder . these results offer initial evidence that similar underlying ri - related deficits in adhd and 22q11ds may manifest as different forms of real - world trait impulsivity between patient groups .

background : multiple approaches have been used to replace lost , damaged or diseased gingival tissues . the connective tissue graft ( ctg ) procedure is the golden standard method for root coverage . although multiple sites often need grafting , the palatal mucosa supplies only a limited area of grafting material . to overcome this limitation , expanded mesh graft provides a method whereby a graft can be stretched to cover a large area . the aim of this study was to evaluate the effectiveness and the predictability of expanded mesh ctg ( e - mctg ) in the treatment of adjacent multiple gingival recessions.materials and methods : sixteen patients aged 2050 years contributed to 55 sites , each site falling into at least three adjacent miller 's class 1 or class 2 gingival recession . the ctg obtained from the palatal mucosa was expanded to cover the recipient bed , which was 1.5 times larger than the graft . clinical measurements were recorded at baseline and 3 months , 12 months postoperatively.results:a mean coverage of 1.96 mm 0.66 mm and 2.22 mm 0.68 mm was obtained at the end of 3rd and 12th month , respectively . twelve months after surgery a statistically significant increase in cal ( 2.2 mm 0.68 mm , p < 0.001 ) and increasing wkt ( 1.75 0.78 , p < 0.001 ) were obtained . in 80% of the treated sites , 100% root coverage was achieved ( mean 93.5%).conclusions : the results of this study demonstrated that multiple adjacent recessions were treated by using e - mctg technique can be applied and highly predictable root coverage can be achieved .

gingival recession is defined as the partial denudation of the root surface due to the apical migration of the gingival margin . etiological factors include trauma from tooth brushing , malposition of teeth , ectopic insertion of frenum , and muscle attachments . the major therapeutic goals in mucogingival surgery are a correction of esthetic problems and management of hypersensitivity . numerous surgical procedures have been described to achieve soft tissue coverage of exposed root surfaces including coronally repositioned flaps , pedicle grafts , free gingival grafts , subepithelial connective tissue grafts ( ctg ) , and guided tissue generation ( gtr ) were commonly used procedures . the treatment of isolated or multiple buccal recessions with different surgical procedures depends on many factors such as defect size , presence or absence of keratinized tissue adjacent to the defect , and thickness of the gingiva . since the patients are concerned about their esthetic appearance , every effort should be made to achieve complete root coverage up to the cementoenamel junction ( cej ) . originally sullivan and atkins described a technique for coverage of exposed root surfaces using the free gingival autogenous graft . the graft survival over large expanses of avascular root surfaces was unpredictable , and complete root coverage was rarely achieved . demonstrates that the underlying connective tissue has a direct bearing on the type of epithelium that is superimposed upon it . edel showed that a significant increase in the volume of gingiva can be achieved by grafting gingival connective tissue alone . langer and langer described the ctg technique in root coverage on both single and multiple adjacent teeth . the advantage is the dual blood supply from the overlying flap and palatal connective tissue , which maximizes graft survival . when multiple adjacent teeth with gingival recessions are present in esthetic regions of the mouth , the preferred surgical technique should be such the one , which provides the possibility of achieving maximum root coverage . one of the problems with multiple root coverage grafting is the unavailability of the large blood supply of donor tissue . if connective tissue supply is limited , more than one surgical procedure may be needed . the purpose of the present study was to evaluate the effectiveness and the predictability of expanded mesh ctg ( e - mctg ) procedure for the treatment of multiple adjacent gingival recession defects . the study population 16 patients , ( age range 2055 years mean age 37 years ) with either dentin hypersensitivity or esthetic problems caused due to the recession defects were included in the study . prior to initiation of the study , ethical approval was obtained from institution ethical committee . all the patients agreed to the study protocol , and signed informed consent was obtained prior to inclusion in the study . the inclusion criteria are ( 1 ) the presence of at least three adjacent miller 's class i or class ii gingival recession on the buccal / facial aspect with recession depth ( rd ) of 2 mm , ( 2 ) probing depth ( pd ) of 3 mm , ( 3 ) a minimum width of keratinized gingival ( kg ) of at least 1 mm . nine subjects contributes three sites , and seven subjects contributed four sites [ figure 1 ] . preoperative facial view of gingival recession the exclusion criteria are ( 1 ) the presence of severe cervical abrasion / root caries , ( 2 ) the presence of abnormal frenal attachment , ( 3 ) current smokers , ( 4 ) medically compromised patients , ( 5 ) miller 's class iii and iv gingival recession . the patients initially completed a plaque control program , so as to achieve a full mouth plaque score ( fmps ) < 25% . the following clinical measurements were taken by a single examiner at baseline and 3 months , 12 months postoperatively . ( 1 ) rd measured from the cemento - enamel junction ( cej ) to the gingival margin , ( 2 ) recession width ( rw ) measured across the buccal surface at the cej level , ( 3 ) pd measured from the gingival margin to the bottom of the gingival sulcus , ( 4 ) width of keratinized tissue ( kt ) measured from the gingival margin into the mucogingival junction , ( 5 ) clinical attachment level ( cal ) measured from cej to the bottom of the gingival sulcus . all measurements were performed at the mid buccal level using a william 's periodontal probe ( hu - friedy ) and rounded to the nearest 0.5 mm . all surgical procedures were done by the same operator . following the induction of local anesthesia ( lignocaine hydrochloride with 1:100,000 adrenaline ) , an intra - crevicular incision was made through the bottom of the crevice and horizontal incision was placed at the level of cej extending 3 mm on either side of the involved tooth including their papilla . two vertical incisions were placed from the end point of the horizontal incision to the alveolar mucosa to establish a trapezoidal flap [ figure 2 ] . line diagram depicting the incisions a full thickness flap was elevated to 34 mm apical to the bone dehiscence followed by a split thickness flap and all muscle interferences were eliminated in order to facilitate its coronal advancement . the root surface was mechanically instrumented using gracey curettes followed by conditioning with 1 ml tetracycline hydrochloride solution for 3 min with subsequent rinsing with saline [ figure 3 ] . split thickness trapezoidal flap was elevated ctg was harvested from the molar- premolar area of the palate on one side [ figures 4 and 5 ] . the donor site was then sutured with 4 - 0 black silk to ensure primary intention healing . alternating incisions were then made on each edge of the harvested graft to expand it [ figure 6 ] so that it would cover the recipient bed completely , which was 1.5 times larger than the graft [ figure 7 ] . subsequently , the graft was positioned at the cej with interrupted 5 - 0 vicryl bioabsorbable sutures [ figure 8 ] . the mucogingival flap was coronally repositioned without tension to cover the e - mctg with 4 - 0 silk sutures [ figure 9 ] . the area was re - examined to ascertain that the graft was completely covered by the flap . a periodontal dressing ( coe - pak ) was placed over the recipient site and removed after a week . original size of the connective tissue graft was harvested from palatal mucosa original size of the connective tissue graft was positioned in the recipient area the expanded mesh connective tissue graft the expanded mesh connective tissue graft was positioned in the recipient area initial stabilization of the graft with 5 - 0 absorbable suture coronal repositioning of the flap with complete closure all patients were instructed to discontinue tooth brushing in the surgical site for 1-week so as to avoid trauma or pressure at the surgical site . a 0.12% chlorhexidine digluconate mouth rinse analgesics ( ibu profen and paracetamol tds for 5 days ) and antibiotics ( amoxycillin 500 mg tds for 5 days ) were prescribed . mechanical tooth cleaning of the treated areas using a soft toothbrush and a careful roll technique was resumed following the removal of periodontal dressing . the patients were recalled for oral prophylaxis after 2 , 4 weeks , and every 3 months for 1-year . the postoperative clinical measurements were taken at the end of 3 and 12 month [ figures 10 and 11 ] . postoperative facial view at the end of 3 month [ compare with figure 1 ] postoperative facial view at the end of 12 month [ compare with figure 1 ] the data were collected , and statistical analysis was performed using statistical package for social science version 16 ( spss , ibm corp . , chicago , usa ) . the following clinical measurements were taken by a single examiner at baseline and 3 months , 12 months postoperatively . ( 1 ) rd measured from the cemento - enamel junction ( cej ) to the gingival margin , ( 2 ) recession width ( rw ) measured across the buccal surface at the cej level , ( 3 ) pd measured from the gingival margin to the bottom of the gingival sulcus , ( 4 ) width of keratinized tissue ( kt ) measured from the gingival margin into the mucogingival junction , ( 5 ) clinical attachment level ( cal ) measured from cej to the bottom of the gingival sulcus . all measurements were performed at the mid buccal level using a william 's periodontal probe ( hu - friedy ) and rounded to the nearest 0.5 mm . all surgical procedures were done by the same operator . following the induction of local anesthesia ( lignocaine hydrochloride with 1:100,000 adrenaline ) , an intra - crevicular incision was made through the bottom of the crevice and horizontal incision was placed at the level of cej extending 3 mm on either side of the involved tooth including their papilla . two vertical incisions were placed from the end point of the horizontal incision to the alveolar mucosa to establish a trapezoidal flap [ figure 2 ] . line diagram depicting the incisions a full thickness flap was elevated to 34 mm apical to the bone dehiscence followed by a split thickness flap and all muscle interferences were eliminated in order to facilitate its coronal advancement . the root surface was mechanically instrumented using gracey curettes followed by conditioning with 1 ml tetracycline hydrochloride solution for 3 min with subsequent rinsing with saline [ figure 3 ] . split thickness trapezoidal flap was elevated ctg was harvested from the molar- premolar area of the palate on one side [ figures 4 and 5 ] . the donor site was then sutured with 4 - 0 black silk to ensure primary intention healing . alternating incisions were then made on each edge of the harvested graft to expand it [ figure 6 ] so that it would cover the recipient bed completely , which was 1.5 times larger than the graft [ figure 7 ] . subsequently , the graft was positioned at the cej with interrupted 5 - 0 vicryl bioabsorbable sutures [ figure 8 ] . the mucogingival flap was coronally repositioned without tension to cover the e - mctg with 4 - 0 silk sutures [ figure 9 ] . the area was re - examined to ascertain that the graft was completely covered by the flap . a periodontal dressing ( coe - pak ) was placed over the recipient site and removed after a week . original size of the connective tissue graft was harvested from palatal mucosa original size of the connective tissue graft was positioned in the recipient area the expanded mesh connective tissue graft the expanded mesh connective tissue graft was positioned in the recipient area initial stabilization of the graft with 5 - 0 absorbable suture coronal repositioning of the flap with complete closure all patients were instructed to discontinue tooth brushing in the surgical site for 1-week so as to avoid trauma or pressure at the surgical site . a 0.12% chlorhexidine digluconate mouth rinse analgesics ( ibu profen and paracetamol tds for 5 days ) and antibiotics ( amoxycillin 500 mg tds for 5 days ) were prescribed . mechanical tooth cleaning of the treated areas using a soft toothbrush and a careful roll technique was resumed following the removal of periodontal dressing . the patients were recalled for oral prophylaxis after 2 , 4 weeks , and every 3 months for 1-year . the postoperative clinical measurements were taken at the end of 3 and 12 month [ figures 10 and 11 ] . postoperative facial view at the end of 3 month [ compare with figure 1 ] postoperative facial view at the end of 12 month [ compare with figure 1 ] the data were collected , and statistical analysis was performed using statistical package for social science version 16 ( spss , ibm corp . , chicago , usa ) . at baseline , mean rd was 2.56 mm 0.62 mm , mean rw was 3.44 mm 0.44 mm , mean kt was 2.13 mm 0.73 mm , mean pd was 1.15 mm 0.23 mm , and a mean cal was 3.71 mm 0.61 mm . three months following surgical intervention , the mean rd reduced from 2.56 mm 0.62 mm to 0.61 mm 0.63 mm ( mean 1.96 mm 0.66 mm ) , the mean root coverage was 86% , the mean rw reduced from 3.44 mm 0.44 mm to 1.52 mm 1.40 mm ( mean 1.92 1.34 ) , the mean kt increased from 2.13 mm 0.73 mm to 3.55 mm 0.69 mm ( mean 1.42 0.62 ) , the pd from 1.15 mm 0.23 mm to 1.22 mm 0.25 mm ( 0.07 0.33 ) , and cal increased from 3.71 mm 0.61 mm to 1.83 mm 0.70 mm ( 1.88 0.69 ) . on statistical analysis , there was a significant reduction in rd and rw , and kt and clinical attachment gain at 3 months ( p < 0.001 ) compared to the baseline [ table 1 ] . mean and sd of baseline and end of 3 months postoperative parameters ( n=55 sites ) at the end of 12 months , the mean rd reduced from 2.56 mm 0.62 mm to 0.35 mm 0.54 mm ( mean 2.22 0.68 ) , the mean root coverage was 93.5% , the mean rw reduced from 3.44 mm 0.44 mm to 0.77 mm 1.16 mm ( 2.71 1.23 ) , the mean kt increased from 2.13 mm 0.73 mm to 3.87 mm 0.80 mm ( 1.75 0.78 ) , the pd from 1.15 mm 0.23 mm to 1.20 mm 0.25 mm ( 0.06 0.34 ) , and cal increased from 3.71 mm 0.61 mm to 1.51 mm 0.61 mm ( 2.20 0.68 ) . all parameters were statistically significant ( p < 0.001 ) compared to base line except pd ( p > 0.05 ) but there was no statistical significance improvement between 3 months to 12 months interval ( p > 0.05 ) [ table 2 ] . mean and sd of baseline and end of 12 months postoperative parameters ( n=55 sites ) at the end of 12 months postoperatively , favorable results were obtained using the e - mctg procedure . totally , 44 out of 55 sites , 44 ( 80% ) sites showed 100% root coverage 7 out of 55 sites , 7 ( 12.7% ) sites showed 90% root coverage 4 out of 55 sites , 4 ( 7.3% ) sites showed 65.5% root coverage at the end of 12 months [ figure 12 ] . gingival recession involves groups of adjacent teeth and is seldom localized to a single tooth . when multiple recession defects affecting adjacent teeth in esthetic areas of the mouth are present , they should all be treated at the same time to help ensure the best esthetic results . autogenous ctg have been extensively used for root coverage procedures in teeth and implants . whereas subepithelial ctg was extensively used for one or two adjacent gingival recession defects excellent result with color matching . harris 's study proposed that the use of acellular dermal matrix graft would improve the gingival color , reduce patient morbidity , provide a uniform thickness of material and eliminate the need for multiple surgeries because of unlimited availability . in the present study , a new approach of the ctg technique the most common problem for root coverage with ctg procedure is the amount that can be harvested . the rugae area is not suitable for graft material , and an extensive palatal wound will be uncomfortable for the patient . because of the high rate of complications and a limited amount of palatal mucosa available for grafting , it is advisable to refrain from covering large or multiple defects . formerly e - mctg was used for free gingival grafts and was generally applied to increase the width of keratinized tissue without root coverage . e - mctg provides more graft material since it can be expanded as much as 50% to cover a larger area . totally , 44 ( 80% ) sites showed 100% root coverage , 7 out of 55 sites , 7 ( 12.7% ) sites showed 90% root coverage , 4 out of 55 sites , and 4 ( 7.3% ) sites showed 65.5% root coverage at the end of 12 months regardless of the number of patients treated . , 94.68% ; romangno - genon 84.84% ; rosetti et al . , 95.6% ; and harris 97.7% . however , it has some limitations and complications such as color match , painful postoperative wound healing , and scar tissue formation in the donor area . it has been reported that using ctg to increase kt has a more rapid , maturation , and less traumatic healing of the graft in the recipient site . similar clinical observations were noticed in our study with the use of the e - mctg technique . as mentioned in previous studies , an increase in kt may be great when there is a narrow band of attached gingival ( 1 mm ) apical to the defects . in this study , a significant increase of kt was obtained after surgery and maintained over time for 12 months , and this was comparable to other studies . on the other hand , we did not achieve complete coverage of the graft in four cases where there were more than three adjacent gingival recession sites and the average amount of kt was 1.5 mm at baseline . it is suggested that attempting to cover completely the graft should be avoided when the initial height of keratinized tissue is poor . the mean gain in cal was 2.20 mm 0.68 mm at 12 months follow - up . histological studies have demonstrated that the blood supply from the periosteum and overlying flap results in a more rapid re - establishment of circulation . in the present study , we took care to place the graft with the periosteal side facing the root surface . borghetti and gardella are documented that creeping attachment may continue for 1-year postoperatively , when thick grafts were used . in this study , there was no statistically significant creeping attachment between the 3 and 12 months interval . however , five patients did show a 1 mm improvement in attachment from the 3 to 12 month . recently , an acellular dermal matrix has been shown to be effective in root coverage procedures as a substitute for ctg . tat et al . and wei et al . have shown that acellular dermal matrix was not as successful as the autogenous free graft and connective tissue free graft in increasing the kt , and a histologic report suggested that placing an acellular dermal matrix does not increase kt . ctg is the gold standard for treatment of gingival recession , but the disadvantages are the inadequate graft availability . the results of the present study demonstrated that the e - mctg procedure was an effective and predictable treatment modality for the management of multiple adjacent gingival recessions in terms of root coverage in the treatment of multiple adjacent gingival recessions .

we quantified chicken calpain 2 ( capn2 ) expression in two chinese chicken breeds ( mountainous black - bone chicken breed [ mb ] and a commercial meat type chicken breed [ s01 ] ) to discern the tissue and ontogenic expression pattern and its effect on muscle metabolism . real - time quantitative pcr assay was developed for accurate measurement of the capn2 mrna expression in various tissues from chickens of different ages ( 0 , 2 , 4 , 6 , 8 , 10 , and 12 weeks ) . results showed that the breast muscle and leg muscle tissues had the highest expression of capn2 compared to the other tissues from the same individual ( p < .05 ) . overall , the capn2 mrna level exhibited a rise developmental change in all tissues . the s01 chicken had a higher expression of the capn2 mrna in all tissues than the mb chicken . our results suggest that chicken capn2 expression may be related to chicken breeds and tissues .

nowadays a highlighted issue for an increasing number of consumers is the problem of meat tenderness , as the result of its physicochemical and biochemical mechanisms acting mainly on myofibrillar structures postmortem . because calpastatin inhibits both calpain1 and calpain2 , it remains unclear whether one or both calpains are active in postmortem muscle . most of the studies that have used appropriate methodology have found the same effect of postmortem storage on the activity of the components of the calpain system [ 24 ] . this conclusion seems to be based on the finding that calpain2 is not autolyzed during postmortem storage [ 5 , 6 ] . but some studies also have now suggested that both unautolyzed calpain1 and calpain2 are proteolytically active [ 7 , 8 ] . the lack of expression data of the calpain2 gene in chicken makes it difficult to verify the role of calpain2 in control of meat quality and carcass traits . in this study , we aimed to ( 1 ) develop a convenient approach to quantify the abundance of the calpain2 transcripts in chicken tissues and ( 2 ) determine tissue distribution and ontogenic expression of this gene , particularly in muscle tissues . thirty - six chickens at different ages from two breeds / populations , mountainous black - bone chicken ( mb ) and a commercial chicken ( s01 ) from sichuan province , were used in this study . tissue samples ( including heart , liver , breast muscle , leg muscle , brain , and abdominal fat ) from mb chickens were collected at 0 , 2 , 4 , 6 , 8 , and 10 w , respectively ; we slaughtered four chickens at each time point . we also collected tissue samples from six s01 chickens at 10 w and six mb chickens at 12 w. tissue samples were quick - frozen in liquid nitrogen and then stored at 80c for total rna extraction . total rna was isolated from the heart , liver , brain , breast muscle , and leg muscle tissues by using the trizol reagent ( invitrogen ) . the quality of rna was determined by the a260/280 absorbance ratio ( 1.61.8 ) and the integrity of the 18s and 28s rrna bands on 1% formaldehyde agarose gel . isolated rnas were treated with 8 l dnase ( fermentas ) for 20 minutes at 37c and stored at 80c . the cdna was synthesized using the improm - ii reverse transcription system ( takara ) according to the manufacturer 's instructions . the reaction was performed in a volume of 10 l containing 5primerscript buffer , 10 mm of each dntps , 40 u/l rnase inhibitor , and 2.5 m oligo - dt primer . the reverse transcription was maintained at 30c for 10 minutes , 45c for 25 minutes , 99c for 5 minutes , and ended with a 4c for 5 minutes , then stored at 20c . according to the chicken capn2 mrna sequence in genbank ( accession number nm_205080.1 ) , a pair of primers were designed by using oligo 6.0 ( table 1 ) . the expression levels of chicken capn2 gene were detected by using the sybr green i assay on an iq5 real - time pcr thermal cycle instrument ( bio - rad , german ) and were normalized to the expression of the -actin gene ( actb ; genbank accession no . relative transcript quantification was performed using standard curves generated for the actb and capn2 genes based on a 7-fold serial dilution of the pooled cdna product prepared from a subset of the gastrocnemius samples . the cycling conditions consisted of an initial single cycle of 95c for 3 minutes , followed by 35 cycles of 15 s at 94c and 1 minute at 60c . reactions were performed in a volume of 25 l and included 2.0 l cdna template , 1.0 l of each specific primer ( table 1 ) , 12.5 l sybr green pcr master mix ( takara , japan ) , 1.0 l calibration liquid ( bio - rad ) , 7.5 l pcr - grade water , and 1.0 l cdna . all real - time quantitative pcr amplifications were performed in triplicate for each sample and were analyzed using the 2 method previously described . expression data were described as ( least square mean standard error ) and were analyzed using the sas 8.0 for windows software ( sas institute inc . , the expression levels of the capn2 gene between the tissue and age - related samples of the same breed and those samples from the two breeds with the same age were analyzed by the one - way anova and student 's t - test , respectively . relative mrna quantification was performed using standard curves generated for the actb and capn2 genes based on a serial dilution of cdna . in the current assay , the amplification efficiency of the actb and capn2 genes were 91.9% and 92.60% , respectively , which were approximately within the expected theoretical values . quantitative pcr analysis showed that the capn2 gene was expressed in all six mb chicken tissues . the capn2 gene transcript had the highest expression level in breast muscle and leg muscle whereas it had the lowest expression level in liver tissue . the mrna levels of the capn2 gene in breast muscle and leg muscle were higher than those in other tissues from the same chicken ( p < .05 ) except at 10 w ( table 2 ) . we analyzed the developmental changes of the capn2 mrna expression for each tissue in mb chickens with different ages . as shown in figure 1 , the capn2 mrna in breast muscle had the highest expression at 6 w and the lowest expression at birth . in leg muscle tissue , the highest expression of the capn2 gene was at 12 w and the lowest expression at birth . overall , the capn2 mrna levels exhibited a rise developmental change in all tissue . to characterize whether the expression of the capn2 gene had a breed - specific feature figure 2 presents the expression pattern of the capn2 mrna in the mb and s01 chickens at 10 w. the meat - type s01 chicken had a higher expression of the capn2 gene than that of mb chicken in all tissues . although previous studies on calpain protease activity indicate that the 2 calpains , calpain1 and calpain2 , have an important role in the postmortem proteolysis that increases meat tenderness , it remains unclear how the calpains function in postmortem muscle . in birds , and in particular the chicken breast muscle , the role of postmortem proteolysis is poorly documented , and the few studies performed did not take into account the particularities of the calcium - dependent proteases in these species [ 912 ] . in this study , we quantified the chicken capn2 gene tissue distribution and ontogenic expression . the capn2 mrna was expressed in all six different tissues studied in the current study , with a dominant expression in breast muscle and leg muscle tissues . the overall pattern of the capn2 expression in breast muscle was different from that of leg muscle . it is well known that the breast muscle of chicken is made of white fast oxidative glycolytic fibers that whereas the leg muscle has slow oxidative red aerobic fibers . we also found the relative expression level of the capn2 mrna in the s01 chicken was higher than that in the mb chicken . investigation of the role of the calpain 2 and its muscle protein substrates in these two chicken breeds may further explain the observed variation in meat tenderness . these results are necessary for knowing the effects of capn2 on the regulation of muscle protein metabolism and for defining the biological significance of degradation of the myofibrillar proteins in chicken , as well as potential applications in marker - assisted selection in chicken breeding . in conclusion , we have developed a highly sensitive real - time pcr method to detect tissue distribution and ontogenic expression of the capn2 mrna in chicken . future studies will be essential to determine the biochemical character of the muscles from the two chicken breeds and to discern the factors that contribute to differences in their meat quality , as well as to define the role of the calpain 2 .

backgroundacupuncture is a promising treatment approach in patients with chronic low back pain ( clbp ) but little is known about the quality of acupuncture in randomized controlled trials ( rct ) of acupuncture clbp.objectiveto determine how international experts ( ies ) rate the quality of acupuncture in rcts of clbp ; independent international validation of the low back pain acupuncture score ( lbpas).methodologyfifteen experts from 9 different countries outside china were surveyed ( ies ) . they were asked to read anonymized excerpts of 24 rcts of clbp and answer a three - item questionnaire on how the method of acupuncture conformed to 1 ) chinese textbook standards , 2 ) the expert 's personally preferred style , and 3 ) how acupuncture is performed in the expert 's country . likert scale rating , calculation of the mode for each answer , and spearman 's rank correlation coefficient between all three answers and the lbpas were calculated.resultson comparison with chinese textbook standards ( question 1 ) , 6 rcts received a good rating , 8 trials a fair and 10 trials a poor or very poor rating . 5 of the 6 trials rated good , received at least a good rating also in question 2 or 3 . we found a high correlation of 0.85 ( p < 0.0001 ) between the ies and lbpas ratings for question 1 and question 2 , and a correlation of 0.66 ( p < 0.0001 ) for question 3.conclusionthe international expert survey ( ies ) revealed that only 6 out of 24 ( 25% ) rcts of acupuncture for clbp were rated " good " in respect to chinese textbook acupuncture standards . there were only small differences in how the acupuncture quality was rated in comparison to chinese textbook acupuncture , personally preferred and local styles of acupuncture . the rating showed a high correlation with the low back pain acupuncture score lbpas .

acupuncture is a promising treatment approach in patients with low back pain ( clbp ) with nearly the same or even better results compared to conventional therapy . since in fact various acupuncture strategies are used in clinical practice , research in order to define the best acupuncture technique is of clinical interest for many medical practitioners as well as for clinical researchers designing acupuncture trials . allready for chinese acupuncture , representing the most widely used style , the specific manner in which acupuncture is applied ( treatment regimens , point selection , needle techniques ) appears to vary among , regions and countries . the same holds true for randomized clinical trials ( rct ) on clbp , with point selection being fixed for all patients , or points being selected on an individual basis according to trigger points , chinese meridians , syndromes , various microsystems to name a few . in rcts needle stimulation may range from non - existent to strong , needle insertion may be deep or shallow , the number of acupuncture treatments may vary from 1 to 15 , and the stimulation of ahshi points may be mandatory or forbidden . despite the multiplicity of existing approaches to acupuncture , in a previous study we ascertained that there is a high degree of concurrence on certain aspects ( minimal criteria ) of chinese acupuncture for the treatment of clbp . a systematic review by yuan et al . arrived at a similar conclusion , while detecting differences between chinese expert opinions , textbooks and acupuncture treatments in rcts . on the basis of a broad , international delphi survey the aim of this study was to find out how leading acupuncture experts from non - chinese countries would rate the acupuncture methods used in existing rcts of acupuncture for clbp . furthermore we calculated the correlation between the results of this expert survey and with a low back pain acupuncture score ( lbpas ) , derived from our recently published acupuncture questionnaire for low back pain . acupuncture experts in a number of countries were selected on the basis of personal contact and scientific publications in the field of acupuncture . additional criteria were clinical experience , a history of teaching , and textbook authorship . all experts were invited to recommend other experts to take part in the survey . those who had already taken part in our previous acupuncture survey establishing minimal criteria for chinese acupuncture in clbp were excluded from selection . twenty - five papers on rcts of acupuncture for clbp published between 1976 and 2007 , were identified via a computerbased search of the cochrane complementary field trials register , the cochrane controlled trials register , medline , embase and reference lists of articles . the acupuncture method for one of the 24 trials was published in two different papers ( papers 2 and 10 ) . for the purpose of this study we did not aim at completeness but sought to ensure that all major trials included in the recent meta - analyses of rcts on clbp were included . from each trial , the paragraph describing the acupuncture method was extracted , and supplemented by a table summarizing all relevant acupuncture treatment data for that trial ; a pdf document was created presenting this information for each trial in an anonymous way , thus minimizing the possibility that the surveyed experts would be able to identify the authors of the reported studies , which could be considered as a relevant source of bias . the pdf document was sent to the selected experts , asking them to answer the following three questions about the acupuncture treatment method of each trial .... please specify how you rate the acupuncture treatment for chronic low back pain in this trial .... 1 . ... according to your personal knowledge of chinese acupuncture as you have studied it from textbooks on chinese acupuncture . 2 . ... according to your personal concept of individualized point selection ( e.g. dry needling , trigger point stimulation , individual usage of chinese point selection rules such as ahshi points , channels , syndromes , pathogenic factors ... ) 3 . ... according to the acupuncture commonly performed in your country ( local style , not necessarily chinese style ) . answers were to be given on a likert scale : 1 = very poor ; 2 = poor ; 3 = fair ; 4 = good ; 5 = very good if the experts felt that the excerpt for a particular trial did not contain adequate information about the acupuncture treatment , they were asked to mark : " the trial does not show a sufficient amount of information for any further judgment . can not say " . recently we published the ' acupuncture questionnaire for low back pain ' based on an international expert survey . this survey revealed that a broad consensus exists with regard to the fundamental aspects of chinese acupuncture for clbp across different categories of practitioners and different countries . a typical treatment regime would consist of 11 sessions of 25 minutes each , given twice weekly . the practitioner would insert 12 needles , select points according to channels and syndromes , identify syndromes such as kidney deficiency ( yin and yang ) , cold dampness , and qi and blood stagnation . preferred points besides ahshi points are bl 23 , bl 40 , gb 34 , bl 54 , bl 60 , gb 30 and huatuo , although there is a high degree of variation concerning additional local and distal points . needling depth is dependent on the point location or other individual patient factors , varying from 0.5 mm in very sensitive patients to 10 cm for points such as gb 30 or bl 54 . acupuncture should be combined with other techniques of chinese medicine , the favourites being electrical stimulation and moxibustion . based on the acupuncture questionnaire for low back pain , a score was developed in such a way that the conformity of the acupuncture treatment applied in a given trial with the criteria derived from the acupuncture questionnaire for low back pain could be expressed in a single number from 0 to 8 , from 0 = very poor to 8 = very good ( table 1 ) . previously published data from 18 experts from 10 different countries showed that a typical acupuncture treatment regimen or treatment protocol should at least meet the above criteria . these results were the basis for the development of the low back pain acupuncture score this number is referred to as the low back pain acupuncture score ( lbpas ) . for the present study , we ( 2author ) calculated the lbpas for all 25 publications and compared it to the ies score . for each trial , the frequencies of the ratings given by the 15 experts were counted . for each question , the mode was defined as the true rating , since it had been selected by the majority of the experts . for all 25 papers we then counted how often experts missed the mode for each question ( false rating ) . the number of false ratings per expert is presented with its measures of average and variability . we calculated the spearman 's rank correlation coefficient of the expert 's true rating for the three questions of the ies . the same statistical procedure was used to calculate the correlation between the ies and the lbpas . acupuncture experts in a number of countries were selected on the basis of personal contact and scientific publications in the field of acupuncture . additional criteria were clinical experience , a history of teaching , and textbook authorship . all experts were invited to recommend other experts to take part in the survey . those who had already taken part in our previous acupuncture survey establishing minimal criteria for chinese acupuncture in clbp were excluded from selection . twenty - five papers on rcts of acupuncture for clbp published between 1976 and 2007 , were identified via a computerbased search of the cochrane complementary field trials register , the cochrane controlled trials register , medline , embase and reference lists of articles . the acupuncture method for one of the 24 trials was published in two different papers ( papers 2 and 10 ) . for the purpose of this study we did not aim at completeness but sought to ensure that all major trials included in the recent meta - analyses of rcts on clbp were included . from each trial , the paragraph describing the acupuncture method was extracted , and supplemented by a table summarizing all relevant acupuncture treatment data for that trial ; a pdf document was created presenting this information for each trial in an anonymous way , thus minimizing the possibility that the surveyed experts would be able to identify the authors of the reported studies , which could be considered as a relevant source of bias . the pdf document was sent to the selected experts , asking them to answer the following three questions about the acupuncture treatment method of each trial .... please specify how you rate the acupuncture treatment for chronic low back pain in this trial .... 1 . ... according to your personal knowledge of chinese acupuncture as you have studied it from textbooks on chinese acupuncture . 2 . ... according to your personal concept of individualized point selection ( e.g. dry needling , trigger point stimulation , individual usage of chinese point selection rules such as ahshi points , channels , syndromes , pathogenic factors ... ) 3 . ... according to the acupuncture commonly performed in your country ( local style , not necessarily chinese style ) . answers were to be given on a likert scale : 1 = very poor ; 2 = poor ; 3 = fair ; 4 = good ; 5 = very good if the experts felt that the excerpt for a particular trial did not contain adequate information about the acupuncture treatment , they were asked to mark : " the trial does not show a sufficient amount of information for any further judgment . can not say " . recently we published the ' acupuncture questionnaire for low back pain ' based on an international expert survey . this survey revealed that a broad consensus exists with regard to the fundamental aspects of chinese acupuncture for clbp across different categories of practitioners and different countries . a typical treatment regime would consist of 11 sessions of 25 minutes each , given twice weekly . the practitioner would insert 12 needles , select points according to channels and syndromes , identify syndromes such as kidney deficiency ( yin and yang ) , cold dampness , and qi and blood stagnation . preferred points besides ahshi points are bl 23 , bl 40 , gb 34 , bl 54 , bl 60 , gb 30 and huatuo , although there is a high degree of variation concerning additional local and distal points . needling depth is dependent on the point location or other individual patient factors , varying from 0.5 mm in very sensitive patients to 10 cm for points such as gb 30 or bl 54 . acupuncture should be combined with other techniques of chinese medicine , the favourites being electrical stimulation and moxibustion . based on the acupuncture questionnaire for low back pain , a score was developed in such a way that the conformity of the acupuncture treatment applied in a given trial with the criteria derived from the acupuncture questionnaire for low back pain could be expressed in a single number from 0 to 8 , from 0 = very poor to 8 = very good ( table 1 ) . previously published data from 18 experts from 10 different countries showed that a typical acupuncture treatment regimen or treatment protocol should at least meet the above criteria . these results were the basis for the development of the low back pain acupuncture score this number is referred to as the low back pain acupuncture score ( lbpas ) . for the present study , we ( 2author ) calculated the lbpas for all 25 publications and compared it to the ies score . for each trial , the frequencies of the ratings given by the 15 experts were counted . for each question , the mode was defined as the true rating , since it had been selected by the majority of the experts . for all 25 papers we then counted how often experts missed the mode for each question ( false rating ) . the number of false ratings per expert is presented with its measures of average and variability . we calculated the spearman 's rank correlation coefficient of the expert 's true rating for the three questions of the ies . the same statistical procedure was used to calculate the correlation between the ies and the lbpas . twenty - five publications on 24 rcts were selected and evaluated by 15 out of 18 experts from the following countries : australia ( 2 ) , austria ( 1 ) , england ( 3 ) , france ( 1 ) , germany ( 2 ) , korea ( 1 ) , norway ( 1 ) , spain ( 2 ) , and us ( 2 ) . three declined to take part , two because lack of time ( france , china ) , the other ( china ) reasoning that acupuncture treatment is a holistic approach and therefore information would have to be presented about every single patient 's tcm diagnoses for all complaints , not merely clbp . since ultimately we could not include a chinese expert in our survey , we restrict our results to non - chinese experts and countries . for each paper conformity of the acupuncture method with chinese textbook standards ( question 1 ) was rated good ( rating 4 or 5 ) in 6 trials , fair ( rating 3 ) in 8 trials , and poor or very poor ( rating 1 or 2 ) for 10 trials . five of the 6 trials rated good [ on question 1 ] received at least a good rating also in questions 2 , asking the experts to grade the degree to which the method in the trial accorded with their personally preferred style , or - question 3 - the country - specific acupuncture style . here too 6 of 10 trials that were rated poor or very poor received the same rating for all three questions . the results for each trial , differentiated by question , are presented in table 2 . questions : " please specify how you rate the quality of acupuncture treatment in this trial for chronic low back pain according to question 1 ... chinese acupuncture as you have studied it from textbooks on chinese acupuncture ? answers were to be given on a five point likert scale : 1 = very poor ; 2 = poor ; .3 = fair ; 4 = good ; 5 = very good . lbpas = low back pain acupuncture score , from minimum of 0 to a maximum of 8 points . lbpas rating : 1 to 4 = poor ; 5 and 6 = fair ; 7 and 8 = good and very good . we also counted how often each expert rated differently from the mode , which was defined as a false rating . , the experts made 8 false ratings in question 1 and 9 false ratings in questions 2 and 3 . the minimum number of false ratings was 4 for question 2 , and 5 for questions 1 and 3 . the maximum number of false ratings was 12 for question 2 , and 13 for questions 1 and 3 . the variability , as shown by the coefficient of variation , was low for all three questions and almost identical at nearly 24% . regarding papers 2 and 10 , the mode was 4 for each of the three questions in both papers . there was a high correlation between the rating of question 1 ( conformity with chinese textbook acupuncture ) , question 2 ( conformity with individual point selection ) and question 3 ( conformity with acupuncture as performed in the expert 's country ) . for 8 trials ( nos . 2 , 3 , 8 , 9 , 10 , 14 , 15 and 22 ) , the same rating was chosen for questions one , two and three . for all other trials the difference between the ratings was one mark only ( table 2 ) . the spearmann 's ranking correlation coefficient is 0.81 for question 1 to question 2 , and 0.80 for question 1 to question 3 . for question 2 to question 3 it is 0.72 ( two - sided p value < 0.0001 for all correlations ) . with the lbpas , the conformity of a given acupuncture method with the criteria derived from the low back pain survey published earlier is expressed as a single number from 0 ( very poor ) to 8 ( very good ) . two trials had an lbpas of 8 ( 15 , 25 ) , and two had an lbpas of 7 ( 2/10 , 11 ) . the lowest lbpas was 1 , for two trials ( 8 , 12 ) ( table 2 ) . a high correlation was found between the ies rating and the lbpas for questions 1 and 2 ( 0.85 , p < 0.0001 ) , and a slightly lower correlation was found for question 3 ( 0.66 , p < 0.0001 ) . twenty - five publications on 24 rcts were selected and evaluated by 15 out of 18 experts from the following countries : australia ( 2 ) , austria ( 1 ) , england ( 3 ) , france ( 1 ) , germany ( 2 ) , korea ( 1 ) , norway ( 1 ) , spain ( 2 ) , and us ( 2 ) . three declined to take part , two because lack of time ( france , china ) , the other ( china ) reasoning that acupuncture treatment is a holistic approach and therefore information would have to be presented about every single patient 's tcm diagnoses for all complaints , not merely clbp . since ultimately we could not include a chinese expert in our survey , we restrict our results to non - chinese experts and countries . for each paper conformity of the acupuncture method with chinese textbook standards ( question 1 ) was rated good ( rating 4 or 5 ) in 6 trials , fair ( rating 3 ) in 8 trials , and poor or very poor ( rating 1 or 2 ) for 10 trials . five of the 6 trials rated good [ on question 1 ] received at least a good rating also in questions 2 , asking the experts to grade the degree to which the method in the trial accorded with their personally preferred style , or - question 3 - the country - specific acupuncture style . here too 6 of 10 trials that were rated poor or very poor received the same rating for all three questions . the results for each trial , differentiated by question , are presented in table 2 . questions : " please specify how you rate the quality of acupuncture treatment in this trial for chronic low back pain according to question 1 ... chinese acupuncture as you have studied it from textbooks on chinese acupuncture ? answers were to be given on a five point likert scale : 1 = very poor ; 2 = poor ; .3 = fair ; 4 = good ; 5 = very good . lbpas = low back pain acupuncture score , from minimum of 0 to a maximum of 8 points . lbpas rating : 1 to 4 = poor ; 5 and 6 = fair ; 7 and 8 = good and very good . we also counted how often each expert rated differently from the mode , which was defined as a false rating . , the experts made 8 false ratings in question 1 and 9 false ratings in questions 2 and 3 . the minimum number of false ratings was 4 for question 2 , and 5 for questions 1 and 3 . the maximum number of false ratings was 12 for question 2 , and 13 for questions 1 and 3 . the variability , as shown by the coefficient of variation , was low for all three questions and almost identical at nearly 24% . regarding papers 2 and 10 , the mode was 4 for each of the three questions in both papers . there was a high correlation between the rating of question 1 ( conformity with chinese textbook acupuncture ) , question 2 ( conformity with individual point selection ) and question 3 ( conformity with acupuncture as performed in the expert 's country ) . for 8 trials ( nos . 2 , 3 , 8 , 9 , 10 , 14 , 15 and 22 ) , the same rating was chosen for questions one , two and three . for all other trials the difference between the ratings was one mark only ( table 2 ) . the spearmann 's ranking correlation coefficient is 0.81 for question 1 to question 2 , and 0.80 for question 1 to question 3 . for question 2 to question 3 it is 0.72 ( two - sided p value < 0.0001 for all correlations ) . with the lbpas , the conformity of a given acupuncture method with the criteria derived from the low back pain survey published earlier is expressed as a single number from 0 ( very poor ) to 8 ( very good ) . two trials had an lbpas of 8 ( 15 , 25 ) , and two had an lbpas of 7 ( 2/10 , 11 ) . the lowest lbpas was 1 , for two trials ( 8 , 12 ) ( table 2 ) . a high correlation was found between the ies rating and the lbpas for questions 1 and 2 ( 0.85 , p < 0.0001 ) , and a slightly lower correlation was found for question 3 ( 0.66 , p < 0.0001 ) . using excerpts of 24 rcts of chronic low back pain , we could show that there is a high correlation between how experts outside of china rate the acupuncture treatment compared to chinese textbook acupuncture standards , personally preferred acupuncture style , and local acupuncture style of their respective country . only a minority of 6 out of 24 trials ( 25% ) were rated good according to chinese acupuncture standards ; 10 were rated poor or very poor and 8 were rated fair . on average furthermore , the international expert evaluation correlates highly significantly with the lbpas , based on our recently published acupuncture questionnaire for low back pain , thereby providing an independent international validation of the lbpas score . nevertheless , the greatest difference in ratings was found between question 1 ( chinese acupuncture ) and question 3 ( acupuncture as performed in the respective country of the expert ) . this suggests that local styles do indeed differ from chinese textbook acupuncture in some aspects but that the ( non - significant ) differences are smaller then expected . the expert ratings of the methods used in the various rcts differed most strongly for rcts where the treatment was not described in sufficient detail according to the stricta criteria , thus adding further evidence to the argument that a complete description of acupuncture treatment in rcts is essential . we also found that rcts that made use of electro - acupuncture were generally rated higher than those that used only manual needle stimulation . recently published a systematic review of acupuncture treatment regimens used for low back pain , including nine chinese expert opinions . for chronic unspecific low back pain , the main findings , which were in line with ours , were the frequent use of bl23 , bl25 and bl40 as common acupuncture points , use of ashi and trigger points , de qi sensation , needling of about 10 points per treatment , with a needle retention time of about 20 minutes and a treatment number of 10 . in general chinese experts tended to use fewer points ( median 5 ) , treated a little longer ( median 25.5 minutes ) and our data are also in line with other major systematic reviews and meta - analyses on acupuncture for clbp , although in these reviews the rating either concerned only methodological characteristics of the study design , omitting an evaluation of the acupuncture treatment per se ( van tulder et al . ) , or judged the acupuncture treatment by three experienced acupuncturists , being coauthors , only ( furlan et al . ) . 1999 found , that only 2 of 11 studies met the level of " high quality " and furlan et al . it must be said that a high rating of the acupuncture treatment is one but not the only prerequisite of a high quality rct . while this might be self - evident for the majority of readers , large , high - powered randomized controlled trials of high methodological quality are often based on the particular acupuncture treatment standards of the authors , which are not necessarily compatible with chinese acupuncture itself . results of these trials therefore do not apply to chinese textbook acupuncture techniques , although this is not always clarified . future investigations should determine whether ratings attributed by major meta - analyses and systematic reviews have introduced a bias in favour of meeting methodology against commonly accepted acupuncture standards in rcts about clbp . it was collected using a one - step standard delphi survey of 15 acupuncture experts in nine different countries outside of china . it might be argued that 15 experts is not a sufficient number to be representative , that the respondents were not selected randomly , and that they were not equally distributed across the countries involved . while this is true , and we concede that other experts , e.g. from china , might come up with different ratings , we note that other systematic reviews or meta - analyses of acupuncture for clpb have selected between 0 and 3 acupuncture experts only , drawn from the pool of authors or the local region . these limitations aside , we know of no other study to work with as many non - chinese experts , drawn from as wide a range of different countries . furthermore , the correlations among experts , professions and countries are so high for the ratings of most rcts that it is unlikely that any other selection of experts would produce essentially different results . our data is specific to chinese acupuncture for clbp . it was collected using a one - step standard delphi survey of 15 acupuncture experts in nine different countries outside of china . it might be argued that 15 experts is not a sufficient number to be representative , that the respondents were not selected randomly , and that they were not equally distributed across the countries involved . while this is true , and we concede that other experts , e.g. from china , might come up with different ratings , we note that other systematic reviews or meta - analyses of acupuncture for clpb have selected between 0 and 3 acupuncture experts only , drawn from the pool of authors or the local region . these limitations aside , we know of no other study to work with as many non - chinese experts , drawn from as wide a range of different countries . furthermore , the correlations among experts , professions and countries are so high for the ratings of most rcts that it is unlikely that any other selection of experts would produce essentially different results . this international expert survey ( ies ) showed that only in a minority of rcts of acupuncture for low back pain the acupuncture quality was rated as good . among international experts there is a high consistency in the rating of acupuncture in respect chinese acupuncture textbook standards ; rating differences with regard to personally preferred methods or local styles of acupuncture do exist but are not significant . the ratings showed a high correlation with the low back pain acupuncture score ( lbpas ) , constituting an independent international validation of the score . we are thankful for the contributions of weihong li , david s. white ( australia ) , eva maria wolkenstein ( austria ) , hyangsook lee ( korea ) , jean - marc stephan ( france ) , michael koch , friedrich molsberger ( germany ) , panos barlas , george lewith , adrian white ( great britain ) , morten srlie ( norway ) , caridad ortega garcia , jose manuel aranda regules ( spain ) , remy coeytaux and n.n .

there is increasing use of antipsychotic drugs in pediatric and psychiatry practice for a wide range of behavioral and affective disorders . these drugs have prominent side effects of interest to pediatric endocrinologists , including weight gain and associated metabolic risk factors and hyperprolactinemia . the drugs block dopamine action , thus disinhibiting prolactin secretion . hyperprolactinemia is especially prominent with first - generation antipsychotics such as haloperidol and the second - generation drugs , most commonly risperidone , with some patients developing gynecomastia or galactorrhea or , as a result of prolactin inhibition of gonadotropin releasing hormone from the hypothalamus , amenorrhea . with concern about the long - term effects of antipsychotics on bone mass and pituitary tumor formation , it is prudent to monitor serum prolactin levels in antipsychotic drug - treated pediatric patients and consider treatment with an agent less likely to induce hyperprolactinemia .

the practice of psychiatry and the shift to noninstitutional care of severe psychiatric disorders are the result of antipsychotic medications , beginning with chlorpromazine in the early 1950s . approximately 10 other drugs , known as first - generation or typical antipsychotics , followed over the subsequent 30 years . these drugs were effective in treating positive symptoms of psychosis such as hallucination and delusion but did not alleviate the negative symptoms of withdrawal , apathy , cognitive impairment , or loss of affect . furthermore , they were associated with frequent extrapyramidal symptoms , including acute dystonia , akinesia , akathisia , tardive dyskinesia , and parkinsonism . a series of newer drugs began emerging in 1989 , referred to as second generation or atypical antipsychotics , thought to be more effective than the older agents in alleviating the negative , cognitive , and affective symptoms , with fewer extrapyramidal adverse effects . the antipsychotic drugs differ in their side effect profiles , as they affect different neuroreceptors ( histamine , -adrenergic , muscarinic , dopamine , or serotonin ) . the principal concern for endocrinologists with the newer drugs has been the metabolic effects of weight gain , glucose intolerance , hyperlipidemia , and hypertension . this is particularly important with the increasing use of these agents in pediatrics to treat bipolar disorder , schizophrenia , autism , oppositional and other behavior disturbances , tourette disorder , and pervasive developmental disorder . in 2003 - 2004 the importance and implications of the metabolic side effects of atypical antipsychotics for pediatric patients have been recently reviewed [ 1 , 4 ] . this paper will examine the side effect of hyperprolactinemia in children and adolescents treated with antipsychotic drugs . prolactin , a 198-amino acid polypeptide , is secreted by the anterior pituitary lactotroph cells in a pulsatile manner with 13 - 14 peaks per day , the peak amplitude ~60% above nadir . there is also a marked circadian variation with maximum secretion ~4 hours from sleep onset and minimum ~6 hours after waking . thus , there can be as much as a fourfold variation in level depending on the time of day or night sampling is done ; there are also transient mild increases related to meals , stress , and sexual activity . prolactin levels are higher during menstrual midcycle and the 2nd half of the cycle . during pregnancy , levels rise 1020-fold , reaching 200 g / l at term and 300 g / l during nursing . prolactin stimulates breast enlargement during pregnancy and milk production during lactation , while reducing libido and fertility , which may have evolutionary / survival significance . secretion of prolactin is inhibited predominantly by dopamine produced in the tuberoinfundibular neurons of the hypothalamus , released from nerve endings in the median eminence and carried through the portal hypophyseal circulation to the pituitary , there binding to dopamine d2 receptors on lactotrophs , inhibiting prolactin gene transcription . estrogens , binding to specific intracellular receptors in lactotrophs , enhance prolactin gene transcription and synthesis . they also inhibit dopamine synthesis in the tuberoinfundibular neurons and reduce d2 receptor levels on lactotrophs in animal models [ 10 , 11 ] . gonadotropin releasing hormone ( gnrh ) , released in a pulsatile manner from the hypothalamus , stimulates release of luteinizing hormone ( lh ) and follicle stimulating hormone ( fsh ) from the anterior pituitary . consequently , estrogen levels in women and testosterone levels in men are suppressed , with marked individual variability in the prolactin level causing gonadal hypofunction . in children and adolescents , hyperprolactinemia resulting from prolactinomas , which are rare , can result in galactorrhea , amenorrhea , gynecomastia , and maturational delay with growth failure . of concern is the potential effect of the induced hypogonadotropism state on the critical peak bone formation of adolescence and the maintenance of bone density through adulthood . psychiatric disorders may be associated with modest elevations in serum prolactin concentrations as a stress phenomenon . further prolactin elevation can be measured within minutes to hours after the start of treatment with first - generation antipsychotic drugs , with levels up to 10-fold after several weeks at therapeutic dosages . levels typically fall to normal within 2 to 4 days of stopping the drugs but may take up to 3 weeks to return to normal . in adult psychiatric patients clozapine and quetiapine did not raise plasma prolactin levels at any dosage . olanzapine only did so at higher dosages , but risperidone and amisulpride caused marked , sustained increase in serum prolactin levels in a substantial number of patients . aripiprazole , a relatively new atypical antipsychotic , also does not appear to increase prolactin levels . a review published in 2004 of 14 reports of the effects of both first- and second - generation antipsychotic agents in children and adolescents included 276 patients of whom 49 had prolactin elevations . a report of 35 patients aged 919 years found no prolactin elevation with clozapine , but in 9 of 10 with haloperidol and 7 of 10 with olanzapine . in another study of 11 outpatients aged 417 years treated with risperidone , 9 developed hyperprolactinemia of whom one had amenorrhea , and one had gynecomastia . a further study reported that prolactin levels were increased in all 34 patients aged 514 years treated with risperidone . hyperprolactinemia was also noted in pediatric patients using ziprasidone and olanzapine . as with adults , ziprasidone- associated prolactin elevation was mild and transient , but associated with mild gynecomastia and in one case with galactorrhea that resolved after drug discontinuation . two studies of the effects of quetiapine led to slightly differing results : one showed no increase in prolactin levels in 10 1215-year - old children while the other study of 15 1317-year - olds found a slight increase of no clinical significance , from a mean of 11.3 to 14.4 ng / ml . clozapine was not associated with increased prolactin levels , and the single patient who developed galactorrhea with risperidone had resolution of the problem when switched to clozapine . a retrospective study analyzed prolactin levels and hyperprolactinemia attributable side effects from 5 clinical trials involving 592 children and adolescents of subaverage intelligence with conduct or other disruptive behavior disorders aged 5 to 15 years treated with risperidone . there was a weak effect of risperidone on prolactin concentrations during short - term treatment and lesser effect with long - term treatment , with side effects of gynecomastia , amenorrhea , or galactorrhea in only 2.2% . these relatively benign findings have been attributed to low drug dosage for behavioral rather than psychiatric disorders and decreasing compliance over time . quite different findings emerged from a small double - blind placebo - controlled study of the effect of relatively low dose risperidone on prolactinemia in 10 children and adolescents with mental retardation and pervasive developmental disorders . prolactin levels approximately tripled , and this increase was sustained for a mean 33 weeks of treatment . three adolescents were reported with risperidone - induced hyperprolactinemia resulting in gynecomastia in one boy that cleared and did not recur with olanzapine , gynecomastia with galactorrhea in another boy with comparable prolactinemia that resolved when he was switched to clozapine , and amenorrhea and galactorrhea in the third patient that resolved when she was changed to quetiapine . their prolactin levels were 2100 , 1670 , and 1990 miu / l ( 58 , 46 , and 55 g / l ) when they were hyperprolactinemic and reduced to 63 , 90 , and 191 miu / l ( 2 , 2.5 , and 5.3 g / l ) after resolution . among 10 psychotic adolescents treated with risperidone , holzer and eap had 3 males developing gynecomastia and 2 females developing galactorrhea , along with 3 others having hyperprolactinemia without symptoms . sixteen adolescents aged 1317 years with subaverage intelligence and disruptive behavior disorders treated with olanzapine for 8 weeks had significant elevations in serum prolactin levels , from baseline 9.7 6.1 to 24.8 19.8 ( sd ) g / l without any symptoms or signs of hyperprolactinemia . a study from italy compared short- and long - term effects on prolactin of risperidone and olanzapine in 42 children and adolescents treated for a year . they found that after adjusting for dose and the greater potency of risperidone , the increase in prolactin levels during risperidone treatment was 10.7 times higher than that during olanzapine treatment . only one subject had a symptom of hyperprolactinemia , transient mild galactorrhea with risperidone which resolved without a change in therapy . similarly , a randomized comparison of quetiapine and risperidone in 22 1518-year - old adolescents with new - onset psychosis found prolactin elevation in 91% of those treated with risperidone versus 9% of those treated with quetiapine . dutch investigators have recently conducted an extensive literature review of studies of antipsychotic medication effects on prolactin level and associated side effects in children and adolescents . twenty of these were concerning risperidone , 7 olanzapine , 5 quetiapine , 4 haloperidol , 3 pimozide , 2 clozapine , and 1 ziprasidone . they found that all antipsychotics with the exception of clozapine , ziprasidone , and quetiapine increased the mean prolactin level from 8 to 2528 ng / ml . the incidence of hyperprolactinemia was 90% with haloperidol , 80% with pimozide , 62% with risperidone , 31% with olanzapine , and 12% with quetiapine . risperidone , olanzapine , and pimozide were seen to induce a persistent elevation in prolactin levels . associated gynecomastia , galactorrhea , or irregular menses were reported in 4.8% of the children and adolescents . interpretation of the numerous studies is confounded by variation in study design , diagnoses , dosages , and age distribution , varying use of concomitant medication , short duration of some studies , compliance uncertainty , lack of prolactin baseline values ( one fourth of the studies analyzed by roke et al . ) , and other missing data as noted in the table . there may also be publication bias , as all data on prolactin values from manufacturers ' files have not been published . the effects of hyperprolactinemia may be underestimated because they depend on self - report , and may be mistaken for common adolescent problems of gynecomastia and irregular menstrual cycles . the degree of hyperprolactinemia induced by short - term risperidone treatment in children and youth is dose dependent [ 17 , 33 ] . this dose dependency is linked to plasma concentrations of both risperidone and its active metabolite 9-hydroxyrisperidone . cyp2d6 is primarily responsible for the conversion of risperidone to 9-hydroxyrisperidone . in an examination of the possible role of activity of this enzyme in risperidone - induced prolactin release in children , troost et al . found a positive correlation of the fourfold elevation in serum prolactin level at 8 and 24 weeks with dose per kilogram body weight ( r = 0.65 , p < .001 ) , number of functional cyp2d6 genes , serum 9-hydroxyrisperidone concentration ( r = 0.66 , p < .001 ) and negative correlation with the risperidone/9-hydroxyrisperidone ratio ( r = 0.57 , p = .004 ) but not with risperidone concentration ( r = 0.24 , p = .26 ) . thus , more rapid cyp2d6 metabolism may be a risk factor for hyperprolactinemia with risperidone . polymorphic variation in the dopamine d2 receptor may be another pharmacogenetic factor determining risk for risperidone - induced hyperprolactinemia in children and adolescents . two variants were identified that were associated with higher prolactin concentration in a study of 107 patients treated for up to 3 years . because second - generation antipsychotics are being increasingly prescribed for children and adolescents with conditions that are not psychoses and that are also treated with stimulants , the potential mitigating effect of the stimulants on the side effects of the antipsychotics has been examined . subjects were 153 419-year - olds treated with antipsychotics , 71 of whom were coprescribed stimulants . the antipsychotic drugs included risperidone ( 33% ) , aripiprazole ( 30% ) , quetiapine ( 18% ) , olanzapine ( 12% ) , and ziprasidone ( 6% ) . in addition to no effect of cotreatment with stimulants on the side effect of hyperprolactinemia , there was no effect on body composition , metabolic parameters , sedation , or overall efficacy of the antipsychotic agent . antipsychotic - induced hyperprolactinemia in adults with schizophrenia has been associated with reduced bmd and increased fracture risk [ 36 , 37 ] . the initial report of the effect of psychotropic drugs on bmd in children was a cross - sectional study that involved 83 boys aged 7 to 17 years treated with risperidone for an average of 3 years and selective serotonin reuptake inhibitors ( ssris ) . with adjustment for the stage of sexual maturation , height , and body mass index , a negative association was found between serum prolactin level and trabecular volumetric bmd at the distal radius . furthermore , treatment with ssris was associated with lower trabecular bmd at the radius and bmd z - score at the lumbar spine . lumbar spine bmd z - score did not correlate with prolactinemia . in females with prolactin secreting tumors , hyperprolactinemia effects on bmd are mediated by hypogonadism . however , in this study , serum testosterone concentrations , adjusted for maturational stage , were not affected by hyperprolactinemia , suggesting a direct effect of the hyperprolactinemia on bone turnover . prolactin receptors have been found in osteoblasts , and animal studies indicate that hyperprolactinemia activates the phosphoinositide 3-kinase pathways via the prolactin receptors to suppress alkaline phosphatase activity . the finding that ssri treatment was associated with reduced bmd could reflect an effect on prolactinemia . however , no independent effect of ssris on prolactin concentration was found in an earlier study by these authors , and the negative association between ssris and bmd was found after adjustment for numerous covariates including prolactinemia . the study of calarge et al . is limited by the dependence on a single measurement of serum prolactin which can vary by time of day and stress level , and absence of measures of bone turnover . the authors recognize that it is premature to make any definitive conclusions about the effect of psychotropic medications on bone mineralization . noting higher - than - expected postmarketing reports of pituitary tumors associated with risperidone , szarfman et al . analyzed patterns of these tumors in the united states food and drug administration adverse event ( ae ) reporting system database . they sought disproportionate reporting patterns of pituitary tumor reports for antipsychotics with different affinities for blocking d2 receptors ( aripiprazole , clozapine , olanzapine , quetiapine , risperidone , ziprasidone , and haloperidol ) . the rank order of the strength of the association between the drug and the development of pituitary tumors corresponded to the affinity of these 7 drugs for the d2 receptor . in children 718 years of age , there was 1 ae of pituitary tumor associated with olanzapine and 3 with risperidone . the authors note the importance of these findings for children because the symptoms of a pituitary expanding mass may not be as readily evaluated in those with mental illness or serious behavioral problems and that delayed detection can result in hemorrhage or optic nerve compression . second - generation antipsychotics are being increasingly prescribed for children and adolescents with a wide range of behavioral disturbances in addition to psychoses , resulting in metabolic and hormonal changes of importance to the consulting pediatric endocrinologist . in addition to weight gain and associated comorbidities of insulin resistance , hyperprolactinemia is a common side effect resulting from the inhibition of dopamine action . first - generation antipsychotics , particularly haloperidol , and the second - generation antipsychotic drugs , most prominently risperidone , appear to be associated with the greatest risk for hyperprolactinemia ; some treated individuals developing hyperprolactinemia will have galactorrhea , amenorrhea , or gynecomastia . hyperprolactinemia may have a deleterious effect on peak bone mass attainment and increase long - term osteopenia risk , even in the absence of overt symptoms or signs of hyperprolactinemia . the suggestion of a greater risk for pituitary tumors related to drug affinity for d2 receptors also requires continued study . thus , in addition to surveillance for signs and symptoms of hyperprolactinemia in children and adolescents taking antipsychotic medications , monitoring serum prolactin concentrations is warranted . in the presence of hyperprolactinemia , cessation of antipsychotic therapy or changing to a formulation less likely to raise prolactin levels the author is a consultant to a law firm pursuing litigation with manufacturers of second - generation antipsychotic drugs .

intrinsically disordered proteins ( idps ) represent a new frontier in structural biology since the primary characteristic of idps is that structures need to be characterized as diverse ensembles of conformational substates . we compare two general but very different ways of combining nmr spectroscopy with theoretical methods to derive structural ensembles for the disease idps amyloid- 140 and amyloid- 142 , which are associated with alzheimer s disease . we analyze the performance of de novo molecular dynamics and knowledge - based approaches for generating structural ensembles by assessing their ability to reproduce a range of nmr experimental observables . in addition to the comparison of computational methods , we also evaluate the relative value of different types of nmr data for refining or validating the idp structural ensembles for these important disease peptides .

experimental approaches such as x - ray and electron crystallography and microscopy have traditionally excelled at determining the structure of single folded proteins and large protein complexes . however , intrinsically disordered proteins ( idps ) are not amenable to these static structural determination methods . idps represent a new frontier in structural biology in that the idp structure must be characterized as a diverse ensemble of interconverting conformational substates , as opposed to a single dominant 3d structure . this necessitates an adjustment in the core methodology of protein structure determination for this class of protein . the experimental identification of proteins with global intrinsic disorder can be performed using various spectroscopic techniques including circular dichroism ( cd ) , nmr , infrared spectroscopy ( ir ) , uv spectroscopy , and fluorescence spectroscopy . cd and ir report on the amount of secondary structure , while lack of chemical shift dispersion in nmr spectra is a good indication of high flexibility . hydrodynamic techniques such as saxs , gel filtration , and dynamic light scattering can also aid in idp identification as they report on the radius of the protein , which is often larger for an idp or denatured protein than a folded protein of the same mass . lack of a cooperative folding transition , solubility at high temperatures , and proteolytic sensitivity are also attributes of idps that are useful in forming a complete picture of a certain protein s level of disorder . a subset of these techniques is generally employed to determine that a protein is an idp . recently increased importance has been placed on characterizing the conformational substates within idp ensembles since they each may have distinct functional roles or could lead to hypotheses about disease origin . in order to achieve both better ensemble classification and a detailed description of conformational substates , we must critically assess how we build these complex structural ensembles from experimental data and theoretical models . nmr is the experimental tool of choice for characterizing the solution structure and dynamics of biological molecules since it reports on the native distribution of conformations in an aqueous environment , and more importantly is a dynamical experiment that probes the nanosecond to millisecond time scales of conformational motion . observables from these experiments include chemical shifts , which are characteristic of functional groups and their surrounding environment , and spin spin couplings ( j - couplings ) , which independently report on backbone dihedral angles . dipole interactions give rise to the nuclear overhauser effect ( noe ) that reports on tertiary structure contacts , and more recently , residual dipolar couplings ( rdcs ) have been used to describe the relative orientation of spatially separated regions of a protein . paramagnetic relaxation enhancements ( pres ) , which can produce longer distance restraints than noes have also been used in the context of idps ; however , this measurement requires chemical modification of the protein with a nitroxide spin label or an amino - terminal copper binding motif , which sometimes requires sequence modification to attach the probe , and which may perturb the monomeric idp conformations . nanoseconds milliseconds time scale of the nmr experiment , leading to an averaging of the nmr observables across structural subpopulations . this uniform average hinders the structural characterization of all the conformational substates , and can even obscure the overall ensemble classification , as we will see for the amyloid peptides in this study . building the connection between the averaged nmr observables and the complete idp structural ensemble therefore depends critically on computational models . the goal of the computational model is to provide a properly weighted set of the diverse subpopulations of the idp most consistent with the nmr observables and perhaps other experimental measures such as circular dichrosim , small - angle x - ray scattering , or pres . thus , multiple types of nmr or other experimental observables are necessary for validation of the computational model . currently there are two primary but very different computational approaches to building an idp structural ensemble , which can be loosely contrasted as first principle or de novo molecular dynamics ( md ) methods versus knowledge - based approaches . the de novo approach implements md simulations based on the theoretical foundations of statistical mechanical sampling and model - derived potential energy force fields . de novo md generates a structural ensemble that is representative of given thermodynamic conditions according to the force field employed , i.e. , a boltzmann weighted ensemble of conformational subpopulations and their time scales , independent of experimental input . the md trajectories also allow calculation of the time correlation functions that underlie the nmr experiment . the complementary use of md and nmr data to determine structure and dynamics of folded and unfolded proteins has been a highly active area over the last two decades , particularly for relaxation measurements that require a dynamical interpretation of the nmr data at the picosecond and nanosecond time scales . for the de novo md method , multiple nmr or other experimental data are necessary to validate the md ensemble through direct back - calculation of observables , many of which depend on the time scales of motion , in order to directly compare to the experiment . once validated , md simulations provide a prediction of the complete idp structural ensemble , allowing overall classification as well as the study of individual conformational substates , which can be analyzed with some confidence . in contrast , we define knowledge - based approaches as those that use experimental nmr information directly to derive the structural ensemble . such methods are the foundation of nmr structure determination of folded proteins using experimentally derived conformational constraints based on chemical shifts , j - couplings , and noe data embodied in software packages such as candid , cyana , and x - plor - nih . while md is often used to generate atomistic predictions independent of nmr experimental input , as in our de novo method , a number of researchers have advanced the combination of applying knowledge from nmr to restrain the md ensemble . for example , md simulations have been combined with rdc restraint data for folded proteins that then allows for the analysis of other features of the ensemble , such as conformational fluctuations . nmr restrained md has also been applied to idps such as -synuclein , a disease protein indicted in parkinson s disease . this study incorporated distance restraints derived from pre experiments in order to guide the md so that the protein s radius of gyration distribution is in good agreement with the experimental value . other knowledge - based approaches for idps forego md simulations altogether and instead use an extensive set of statistical coil conformations ; this starting pool , which can be generated using a variety of heuristics , can be thought of as a basis set of structures . subsequently , the starting pool of structures is then culled for the subset of conformations that are in best agreement with experimental data to create the idp ensemble . in the energy - minima mapping and weighting ( emw ) method , stultz and co - workers used end - to - end distance restraints to develop a pool of conformations with varying radii of gyration ; they then selected , via monte carlo , a weighted ensemble of 15 structures to optimize the agreement with experimental c and n chemical shifts and j - couplings . blackledge and co - workers have developed the program flexible - meccano to create a pool of structures based on random coil backbone dihedral angles , on which they employ a genetic search algorithm in their asteroids software program to select structures that together best match experimental chemical shifts , pres , or rdcs . the ensemble method , developed by forman - kay and co - workers , typically defines the starting pool of idp conformational states as an ensemble of extended or random coil states generated using trades , with an option for biasing the secondary structure of the ensemble at certain places in the sequence that are known to be partially structured . structures are selected from this pool using a monte carlo selection algorithm with an energy - weighting scheme for each type of experimental input . the ensemble program includes modules for several different experimental data types including chemical shifts , rdcs , pres , j - couplings , and contact distances derived from noes , and is a user - friendly and publically available software package . although there are some specific differences , ensemble is largely representative of the knowledge - based approaches and is qualitatively equivalent to the combination of flexible - meccano and asteroids software . it is important to note that such techniques largely ignore the inherent dynamical information of certain types of the nmr data that can be important for discriminating between different idp structural ensembles . the primary objective of this work is to compare the de novo and knowledge - based approaches for deriving idp structural ensembles in context of the intrinsically disordered alzheimer s disease peptides amyloid- 140 ( a40 ) and amyloid- 142 ( a42 ) . we implement the ensemble knowledge - based method by building an ensemble from a pool of statistical coil structures , and compare this knowledge - based ensemble to md generated ensembles , which are qualitatively different in that they are comprised of mostly cooperative secondary structure and tertiary contacts . this comparison also exposes the relative utility of different types of nmr data for refining or validating the idp computational ensemble . we find that chemical shifts and j - coupling constants are not particularly useful for distinguishing between qualitatively different idp ensembles of the amyloid- peptides . finally we show that the combination of de novo md methods that provide boltzmann weighted samples with the ability to measure time correlation functions , and knowledge - based methods for conformation selection , provides the best agreement with the nmr data . in order to evaluate the alternative ensembles produced by knowledge - based and de novo approaches , we need a method of calculating the chemical shifts , j - coupling constants , rdcs , and h h noes as averages over the entire computationally generated structural ensemble for comparison with experimental values . general purpose chemical shift calculators such as shiftx and shifts describe the isotropic shielding of the applied magnetic field for the given atom , a quantity that depends sensitively on the local electronic structure environment . even for folded proteins with a dominant native conformer , each atom type can exist in many different local environments , and for disordered peptides and proteins the ensemble average reflects an even more diverse set of chemical environments . whether one uses shiftx ( used in ensemble ) or shifts to calculate chemical shifts , the results generated by the two programs are consistent when applied to amyloid- and averaged over the structural ensembles . we report results using shiftx in this work . to calculate the scalar coupling constants , jhnh , we used the karplus equation1where indicates the protein backbone dihedral angle , with coefficients a = 6.51 , b = 1.76 , and c = 1.60 corresponding to the parameter set by vuister and bax . however , sgourakis and co - workers and our own previous work on a42 found that the md results exhibited a systematic shift of calculated j - couplings with the experimental scalar couplings of wang and co - workers . we delved into this issue more deeply and derived an analytical correction , described in recent work , that must be applied to the original experimental j - coupling data from wang and co - workers . in particular , it has been corrected for a missing relaxation that makes scalar couplings determined from the hnh 3d experiment consistently lower than those from cosy splittings by a small amount ( from 15% ) . the j - coupling values are also averaged over all structures in the ensemble as in , and then the calculations can be compared to the corrected experimental jhnh values for both amyloid peptides . the standard method in the field for calculation of rdcs is the pales program , which we have used previously for the a40 and a42 md ensembles . the program computes the rdc by using steric properties of the molecule to generate a global alignment orientation . then , the angle between the backbone amide bond vectors and the external magnetic field is used to calculate the rdc for each conformation , and the rdcs are averaged over all conformations of a given ensemble . the ensemble program by contrast , evaluates rdcs using a local alignment program developed in the forman - kay lab , where 15 residue segments along the protein are aligned separately over the ensemble of structures . the local rdcs ( l - rdcs ) are also averaged over all conformations of a given ensemble . this local alignment has lower computational cost and has been shown to give similar results to pales , hence l - rdcs , rather than rdcs generated from a global alignment algorithm , are optimized in the standard implementation of the ensemble approach . similarly , the asteroids program by default employs a local alignment tensor to optimize ensemble agreement with experimental rdc data . we also note that the pales alignment and rdc calculation were developed for folded proteins , and their application to idps assumes individual idp conformations behave similarly to folded proteins during the rdc experiment , which may not be the case for idps such as amyloid-. we also evaluate the h h noesy ( or roesy ) spectra as we have described in previous studies by calculating the intensity of the noe cross - peaks2where x and are the eigenvectors and eigenvalues of the full relaxation matrix , composed of diagonal elements3aand off - diagonal elements3bthat are comprised of appropriate combinations of the spectral density functions4evaluated at the relevant larmor frequencies , , and where k is given by5h is the gyromagnetic ratio of h , 0 is the permeability of free space , and is planck s constant . reff is the distance between the hydrogen atoms raised to 6 power , averaged over all structures in the ensemble and then raised to the 1/6 power to convert back to units of distance . these calculations account for all hydrogen atoms explicitly ( including all methyl or methylene groups ) and hence reff and correlation functions for every pair of hydrogen atoms are evaluated . the spectral density function for each atom pair is calculated as the fourier transform of the correlation function for the pair vector and water proton coordinates are ignored , as is the standard assumption in the nmr experiment . n noes as we did in refs ( 14 and 48 ) by evaluating the steady state noe enhancement factor of the n spin by the h noe according to6where h and n are the gyromagnetic ratios of h and n , respectively . n cross - relaxation rate constant is given by7and the n self - relaxation by8 in this case , jhn( ) is the spectral density function for the h n covalently bonded pair . note that the homonuclear and heteronuclear noe calculations require correlation time information about the vector between each pair of atoms given by in eq 4 . this dynamic information is naturally supplied by the de novo md method , which allows direct measurement of the autocorrelation of the interatomic vector over the time of the simulation . however , dynamics are not considered in the generation of ensembles that are used in the knowledge - based approach . this is an inherent limitation of ensembles generated from a static perspective only , which we discuss further below . to determine an idp s level of disorder , we first generate several alternative ensembles , compare these ensembles to the available nmr data , and select the best validated ensemble . we consider the creation of three qualitatively different conformational ensembles that are typically used in the knowledge - based approaches for the a40 and a42 peptides . the common null hypothesis is that the disordered peptides can be well represented by a random coil ( rc ) ensemble . the second type of statistical coil ( pred - ss ) ensemble is also random , but incorporates bioinformatics - based knowledge about what secondary structure category is more likely for a given residue in the amino acid sequence . in this case , the random ensemble is biased to contain a statistical probability of predicted secondary structure on a per residue basis , but no cooperative secondary structure such as -helices , -hairpins , or -sheets are generated from the random secondary structure assignments . computational methods such as trades and flexible - meccano are used to generate these type of random or statistical coil ensembles . finally , a fully knowledge - based approach is considered , which culls the rc or , in this case , pred - ss ensembles to derive a subset of conformations that best agrees with the nmr data ( pred - ss - ens ) . the ensemble software package provides a working example of the knowledge - based approach that performs this biased selection and which has been successfully applied to a range of idps . each of the above three ensembles can then be compared against the ensembles generated by de novo md for both of the idps a40 and a42 . details of the de novo md approach applied to amyloid- can be found in other publications . we also consider an additional fifth ensemble ( md - ens ) that combines the knowledge - based and de novo md approaches , by using ensemble to select structures from the de novo md starting pool , and which is described in our recent study . table 1 shows the average radius of gyration ( rg ) values for each type of ensemble . we see that the order from most extended to most compact proceeds as pred - ss > rc > pred - ss - ens > md md - ens , and thus the alternative ensembles span a range of idp classifications by the rg measure . figure 1 provides the propensities for the pred - ss , pred - ss - ens , de novo md , and md - ens ensembles to form turns , antiparallel -strands , or helical structure by residue for a42 . we do not show the secondary structure profiles for the rc ensemble since it is similar to the pred - ss ensemble ( see the supporting information ) . percentage of a42 simulated ensemble in different types of secondary structure by residue for ( a ) the pred - ss , ( b ) pred - ss - ens , ( c ) de novo md , and ( d ) md - ens ensembles . the red line represents helix , the blue line for antiparallel sheet , and the black line for -turns . we note that the blue line represents only antiparallel sheet structure ( the most common ) and not all sheets . for the radius of gyration ( rg ) values , we report both the ensemble average and rmsd . for chemical shifts , we report that measures agreement between the computational ensembles and the experimentally measured chemical shifts : < 1 indicates no disagreement with experiment within shiftx calculator error . we also report the jhnh rmsd ( ) . some data reproduced from ref ( 14 ) . this plot emphasizes that the md - based ensembles are qualitatively different from the rc or pred - ss ensembles , in that the a40 and a42 peptides samples some type of structured conformations in 99% of the md ensemble , including complex strand formation . from this , we conclude that the radius of gyration trends stem from the much larger propensity for the md ensembles to form cooperative secondary structure and collapsed tertiary contacts , as opposed to the random or knowledge - based ensembles that do not generate contiguous blocks of secondary structure , and hence are more extended on average . although the secondary structure content of the md - ens ensemble resembles that of the md ensemble , figure 1 shows that there is some variation in the percentages with which certain residues adopt different types of secondary structure . for analysis of a40 and a42 idps considered here , we have utilized a wide range of previously published nmr data including chemical shifts from the zagorski group as well as j - coupling constants , rdcs , and heteronuclear h our group has collected h chemical shifts and noesy h h homonuclear spectra for the full length a40 and a42 peptides as reported elsewhere . the data for the longer peptides were processed as described in ref ( 48 ) in a similar approach to that used for the a2130 fragment . first we consider the chemical shift data , for which we note that the calculated chemical shifts have an uncertainty that is independent of the quality or type of structural ensemble , and results from approximations of the shiftx or shifts calculators themselves . other research groups have reported the uncertainty , ( ppm ) , for these calculators , with the value depending on the atom type and its bonding chemistry . therefore the best way validate the various idp ensembles with chemical shift data is to calculate the difference between the experimental chemical shift and the shift calculated from each of the structural ensembles , normalizing it by the calculator uncertainty , to generate values9reported uncertainties ( root mean squared difference , rmsd , from experiment ) for the shiftx calculator are = 0.23 ppm for h , = 0.49 ppm for hn , = 0.98 ppm for c , and = 1.10 ppm for c. any dominant error due to the underlying structural ensemble would then correspond to values of > 1 . table 1 displays the agreement between experimentally measured proton and carbon chemical shifts with those generated from each candidate ensemble for both a40 and a42 . experimental chemical shift data reported for the monomeric a40 and a42 peptides do not differ greatly from random coil values , and therefore the rc ensemble falls within uncertainty . since the pred - ss ensemble shows almost no dssp defined secondary structure ( figure 1a ) , it remains largely equivalent to the rc ensemble as deduced by chemical shifts . the de novo md structural ensemble is also in good agreement with the chemical shift data ; however , 99% of the md generated a conformations contain one or more elements of cooperative secondary structure somewhere along the peptide sequence ( figure 1b ) . the reason that the md ensemble is also in good agreement with the experimental chemical shifts is that averaging over a large ensemble of cooperatively formed secondary structure and tertiary contacts yields average chemical shifts that are consistent with random coil values . for example , averaging the chemical shifts of all folded proteins in the pdb results in averages very similar to random coil values . we have found that the ensemble optimization of the pred - ss and md starting pools improves the values , but all are within the calculator uncertainty . not surprisingly , if the knowledge - based ensemble approach were biased by chemical shift data alone , they would show little deviation from their starting soup , and the structural interpretation would be highly dependent on the starting ensemble . for this reason we conclude that nmr chemical shifts alone do not provide any qualitative discrimination between the alternative ensembles , at least not for the a40 and a42 disease idps . it may still be useful to apply chemical shift constraints in combination with other experimental observables to optimize an idp ensemble , as we have done when generating the md - ens ensemble . sanity check against ensembles that fit other observables , such as noes , but lead to unphysical chemical shift values . similarly , j - couplings alone also do not discriminate between random coil idps and those that are more structured with cooperative secondary structure and tertiary structure contacts . figure 2 illustrates this by plotting the agreement between experimentally measured j(hn , h ) , and those calculated from the rc , pred - ss , pred - ss - ens , de novo md , and md - ens ensembles for a40 and a4 . table 1 shows that all ensembles yield an rmsd across all residues of 0.601.09 hz , and also reports 10with j = 0.73 hz . j - couplings report on the backbone dihedral angle , and therefore could in principle distinguish between an unstructured peptide and a peptide with a defined secondary structure ; however , in the case of the disease idp a , the presence of diverse secondary structure in the md ensemble is not apparent from the calculated j - couplings . we believe that this stems from the fact that good agreement with scalar coupling data for idps can largely be predicted by sampling over the allowed regions of residue - specific ramachandran plots without needing to assume any structure adopted by the full length sequence . thus j - couplings also do not provide an experimental measure for discriminating among qualitatively different structural ensembles for the amyloid peptides . j - coupling constants for backbone amides for a40 and a42 . ( a ) a40 experimental j - coupling constants ( red squares ) compared to rc ( green triangles ) and de novo md ( solid blue circles ) . ( b ) a40 experimental j - coupling constants ( red squares ) compared to pred - ss - ens ( black diamonds ) and md - ens ( blue circles ) . ( c ) a42 experimental , rc , and de novo md j - coupling constants . ( d ) a42 experimental , pred - ss - ens , and md - ens j - coupling constants . has been corrected to account for t1sel relaxation and bring j - couplings determined from a hnh 3d experiment to be consistent with those from cosy splittings . table 2 provides the assessment of the five alternative ensembles for a40 and a42 using rdc values evaluated residue by residue using the pales program and l - rdcs based on local alignments . while the rc and pred - ss ensembles yield lower rmsd values , 1.31.5 hz , they are marginally better than the de novo md rmsd of 2.2 hz . this is in part due to the fact that experimental rdc uncertainties for idps are larger ( 0.9 hz for a40 and 0.5 hz for a42 ) than the uncertainty observed for folded proteins of 0.1 hz . in addition , there are large uncertainties in the accuracy of rdc calculators using programs such as pales . in fact , the reported rmsd of the pales calculator for folded proteins is 2.0 hz , on the same order as the rmsd for the de novo md ensemble . while the ensemble method does significantly lower the rmsd for l - rdcs for the pred - ss - ens and md - ens ensembles , the corresponding rmsd based on the global alignment using pales is marginally better than the pred - ss and de novo md starting pools . hence for this particular application on disordered amyloid peptides , we have found that rdcs are not a particularly good experimental metric for differentiating among the different ensembles , and substantial disagreement between rdcs based on local and global alignments are observed . we report rmsds for the rdc calculator pales and l - rdcs evaluated with ensemble using local alignments . the noes are back - calculated from the structural ensembles as described in section 4 . we evaluate the rmsd normalized by the largest noe intensity , rmsdn and ( correlation coefficient , r ) with the h2o and d2o experiments . some data reproduced from ref ( 14 ) . finally , we consider the performance of the different ensemble methods for reproducing h we have presented the noe data collection for the a42 peptide in which 700 cross - peaks are observed in the noe spectra , but only 200 can be uniquely assigned from experimental information alone . the remaining cross - peaks do not have a clear independent assignment ( and in fact require a computational model to interpret them ) . therefore we have only compared the different methods against the noe cross - peaks that can be assigned by experiment alone . we note that quantitatively reproducing noe intensities is a very high bar since peak volumes are extremely sensitive to r distance averaging , that also involve an appropriate time scale that is heterogeneous across proton pairs . geometric imperfections in the conformational ensemble where contact differences differ by a factor of 2 ( difference between 1 and 1.12 ) will double the corresponding intensity value , thereby driving up the rmsd error for all ensembles . large absolute noe intensities especially tend to dominate the rmsd error , and therefore we have mitigated this effect by normalizing the rmsd ( rmsdn ) by the experimental intensity for each noe as in ref ( 14 ) . table 2 shows that the predicted set of h h noes from de novo md is better than any other ensemble , with rmsdns that are lower than the rc and pred - ss ensembles values by 23 fold and with much higher correlation coefficients . the pred - ss - ens ensemble performs better for a40 and a42 than the randomly generated ensembles because the noe restraints are used in the knowledge - based ensemble selection . however , the pred - ss - ens ensemble still does not reproduce the data as well as the de novo md ensemble . the noe validation clearly indicates that the de novo md ensemble with its cooperative secondary structure is a better representation of a40 and a42 than are the rc , pred - ss , or pred - ss - ens ensembles , which have no cooperative secondary structure . since time information is not available for the static ensembles , we can only evaluate the noes for the statistical ensembles under the assumption of one uniform correlation time applied to all pairs of protons , for which we use a 1 ns correlation time , which is on the same order as those observed in the md simulations . of course the de novo md method can account for the time scales explicitly and more importantly for the fact that different pairs of hydrogen atoms do decay on different time scales . thus the statistically generated and knowledge - based ensembles agree relatively poorly with the noe observables since the heterogeneity in correlation times are unknown , and hence even the md - ens ensemble is in somewhat worse agreement with the experimental noes than the de novo md ensemble ( table 2 ) . the noe validation emphasizes that an idp s diverse set of conformations gives rise to a heterogeneous set of correlation times that must be described in order to validate against experimental noes . we further emphasize that the calculation of heteronuclear noes , being a purely dynamical measurement , is only possible with the de novo md method . n noe intensities , measured by yan and wang , and those derived from our md simulation for a42 and a40 , showing overall excellent agreement . unlike the h h noes , these assignments are unambiguous from experiment . we find that , as in the experiment , there is an increase in h n noe intensities calculated from simulation for residues 3540 for a42 compared to a40 , indicating that the longer peptide experiences slower dynamics at the c - terminus . n noes for a42 and a40 has previously been interpreted as evidence that a42 has greater structural rigidity in the c - terminus compared to a40 and we provide more analysis on this point in recently published work . agreement with experiment of simulated ( a ) a40 and ( b ) a42 h we have shown that the md and md - ens structural ensembles for the idps a40 and a42 previously characterized yield substantially better agreement with a range of nmr data than the random coil or statistical ensembles that are typically used with knowledge based approaches . the md ensembles are qualitatively different from random coil or statistical ensembles in that the subpopulations are richly structured , contain a diverse set of secondary structures including -helix , -turns , and -strands , and span the full range of compact to fully extended conformations . furthermore , while md generated ensembles are boltzmann weighted , the knowledge - based approaches give equal statistical weight to all conformations and thus are likely inconsistent with statistical mechanical weightings that are inherent to the nmr experiment . we have also shown that some types of nmr data may not be helpful for discriminating among qualitatively different structural ensemble of idps . in particular , averages over a diverse set of cooperative secondary structure conformations yield experimental values of chemical shifts that are superficially consistent with values expected from a random coil ensemble . furthermore , if the chemical shifts are not highly dispersed along the sequence of a particular idp , such as is found for the amyloid- peptides , then the chemical shifts have limited value as experimental refinement input or as a validation measure . j - couplings also do not provide discrimination between randomly generated conformations and a diverse population of cooperative secondary structure . in fact , we found that scalar couplings calculated as averages over the allowed regions of each residue - specific ramachandran plot gave as good agreement with the experimental j - couplings for a40 and a42 as did averages over the structural populations . unlike others who have used rdc data to help interpret idp or unfolded protein structural ensembles this may be due to limitations of rdc calculators such as pales , which were originally developed and successfully applied to folded proteins , but which are reported to have large uncertainties in their predicted rdc values . furthermore , calculated rdcs based on global alignment algorithms such as pales diverge significantly from rdcs evaluated from localized alignments for a40 and a42 , indicating that in cases like this the ensemble package should be employed using the pales calculator to fit rdcs , which is possible though not standard . more research may be necessary to apply programs like pales to disordered proteins , which likely do not align in an anisotropic medium in the same way as folded proteins , in part due to the time scale of interconversion of the conformational substates . for example , conventional methods for calculating rdcs can not be applied to the motion of multidomain biomolecules , and the local conformational sampling and long - range structure need to be simultaneously accounted for because they both affect the experimental rdc data . however , progress is being made in using rdcs to provide meaningful structural information for other idps . we speculate that success is greatest when all subpopulations of the idp ensemble are homogeneously classifiable ( as extended disordered conformations for example ) , so that the idp global alignment properties are uniform and resulting averages provide meaningful and consistent structural information . h noe intensities and heteronuclear n h noes are by themselves discriminating with regard to the tertiary contacts and backbone dynamics , respectively , that define the important validation of the md - based ensembles over the statistical coil ensembles . furthermore , a correct picture of the idp ensemble based on the experimental noe data would not be possible without a computational model providing both details of individual structures and the time scales for their interconversion . in turn , although the homonuclear noes are averaged over all subpopulations , they are still vital for deducing whether a given ensemble contains subpopulations of structure with the right tertiary contacts to give rise to the observed cross - peaks in the spectra . because these cross - peak intensities rely directly on the decay time scales of correlated proton distances , the noes for idps are reporting on a heterogeneous population of time scales . one of the primary limitations of the statically generated ensembles is that they are not associated with any information about motional time scales that can be used to calculate noe observables . relaxation times can be used with the ensemble method , although they are incorporated as structural rather than dynamic constraints . this dynamic information is a genuine strength of the de novo md methods , especially for n h noes , which can not be calculated from the static ensembles . we believe that the primary limitation of knowledge - based methods applied to the difficult amyloid case is 3-fold . we note that while there will be quantitative differences between ensemble and other knowledge - based approaches such as asteroids , qualitatively the problems will be similar . basis set of conformations to select the final ensemble from ; i.e. , these methods can not use the nmr data effectively to select for compact structures with elements of cooperative secondary structure if the initial pool of structures is largely composed of extended random coil structures . both ensemble and asteroids have relied on statistical coil ensembles as the starting pool of structures , and while some on - the - fly addition of new structures is possible with these methods , they do not yet support formation of complicated -sheet motifs . metrics of ensemble heterogeneity , such as those developed by the onuchic and stultz research groups , will continue to be useful as we explore the range of idps that can not be easily classified based on their level of disorder . second , for certain classes of idps such as amyloid optimization of structures to reproduce chemical shifts and scalar couplings does not discriminate among qualitatively different structural ensembles . third , the optimization phase of the knowledge - based approaches relies on approximations to nmr observables , which may diverge from a global property , as for l - rdcs , or from the dynamical origins of noe intensities . at the same time , the de novo md method is not quantitatively perfect , and therefore the md ensemble provides an excellent start state for subsequent refinement by knowledge - based approaches . an unambiguous future direction for the structural biology of idps is the combined use of knowledge - based approaches and md that supplies boltzmann weighted conformational substates as well as heterogeneous time scales of motion . all together , the productive interplay between nmr experiments , de novo md simulations , and knowledge - based approaches , along with supporting models , algorithms , and computer hardware , gives us an ability to accurately identify structures present in idp ensembles and use that knowledge to gain previously inaccessible functional insights . to further improve techniques for studying disordered proteins , we as a community could establish a high throughput computational infrastructure to predict idp structural ensembles using a combination of md and nmr . this would be similar to the establishment of x - ray crystallography beamlines for the rapid solution of folded protein structures that was launched during the structural genomics era . the ultimate goal in both cases is to use structural information to drive the formation of hypotheses about protein function . based on the success of using structural information for functional characterization of folded proteins and complexes , we hope and expect that structural knowledge of idp ensembles can provide similar insight into idp function and enable development of molecular hypotheses for disease idps .

during systematic active surveillance of the causes of diarrhea in patients admitted to the infectious diseases and beliaghata general hospital in kolkata , india , we looked for 26 known gastrointestinal pathogens in fecal samples from 2,748 patients . samples from about one - third ( 29% ) of the patients contained multiple pathogens . polymicrobial infections frequently contained vibrio cholerae o1 and rotavirus . when these agents were present , some co - infecting agents were found significantly less often ( p = 105 to 1033 ) , some were detected significantly more often ( p = 105 to 1026 ) , and others were detected equally as often as when v. cholerae o1 or rotavirus was absent . when data were stratified by patient age and season , many nonrandom associations remained statistically significant . the causes and effects of these nonrandom associations remain unknown .

the protocol has been approved by the institutional review board at the national institute of cholera and enteric disease . briefly , fecal specimens were collected systematically from patients entering the hospital from november 2007 through february 2010 . of note , the previous study analyzed data through october 2009 ; however , the systematic sampling is still ongoing . the specimens were collected from every fifth patient with diarrhea on 2 randomly selected days each week . only patients with diarrhea ( defined by world health organization guidelines as passage of > 3 loose or liquid stools per day or more frequently than is normal for the person ) were eligible for inclusion in the study . each patient contributed 1 sample , and each sample was tested for all 26 common diarrheagenic pathogens . samples were collected in mccartney bottles ( using sterile catheters or rectal swabs ) containing cary - blair medium and examined ( within 2 hours of collection ) for bacterial , viral , and parasitic pathogens by a combination of conventional , immunologic , and molecular methods . the bacterial pathogens ( v. cholerae , v. parahaemolyticus , v. fluvialis , campylobacter jejuni , campylobacter coli , salmonella spp . , species and subtypes were confirmed by serotyping ( for v. parahaemolyticus , shigella spp . , and salmonella spp . ) with commercially available antiserum ( denka seiken , tokyo , japan ; biorad , marnes - la - coquette , france ) and by pcr ( for v. cholerae , v. fluvialis , enterotoxigenic e. coli [ etec , including heat - labile and heat - stable enterotoxin producers ] , enteropathogenic e. coli [ epec , typical and atypical ] , enteroaggregative e. coli [ eaec ] , enteroinvasive e. coli , and shiga toxin producing e. coli ) . rotavirus was detected by polyacrylamide gel electrophoresis and silver staining ( 8) . noroviruses ( groups i [ nvg1 ] and ii [ nvg2 ] ) , sapovirus , and astrovirus were detected by reverse transcription pcr with random primers for reverse transcription and specific primers for pcr ( 9 ) . adenoviruses were detected by the commercially available rotaadeno vikia kit ( biomrieux , marcy letoile , france ) . all samples were screened by using a highly sensitive antigen capture elisa ( techlab , inc . , blacksburg , va , usa ) of g. lamblia , cryptosporidium parvum , e. histolytica , and blastocystis hominis . to test for possible associations , we used the fisher exact test to compare pairs of pathogens ( 1 , both , or neither ) with an independent assortment based on the overall frequency with which pathogens were detected . to establish criteria for statistical significance , we calculated p values , odds ratios ( ors ) , and 95% confidence intervals ( cis ) . additional covariates were collected and examined for confounding and interaction . these included patient age , gender , residence , and religion and season of infection . seasons were defined as summer ( march june ) , monsoon ( july october ) , and winter ( november february ) . all analyses were conducted by using sas version 9.2 ( sas institute , cary , nc , usa ) . patient demographic characteristics are listed in table 1 . a large proportion ( 44% ) of patients were 1545 years of age , 13% were < 1 year of age , 80% resided in urban areas , 74% were hindu , and 25% were muslim . the following pathogens were detected in at least 1 sample : adenovirus , aeromonas spp . , astrovirus , b. hominis , c. jejuni , c. parvum , eaec , epec , etec , e. histolytica , g. lamblia , nvg1 , nvg2 , rotavirus , salmonella spp . , sapovirus , shigella spp . , v. cholerae o1 , v. cholerae o139 , v. cholerae non - o1 , v. cholerae non - o139 , v. parahaemolyticus , and v. fluvialis . no pathogens were detected in 766 ( 28% ) of the 2,748 samples ( table 1 ) , but test results were positive for the other 72% . one pathogen was found for 1,169 ( 43% ) samples and multiple pathogens for 813 ( 29% ) ( table 1 ) . the 2 most commonly detected pathogens were v. cholerae o1 and rotavirus , which were found in 24% and 22% of samples , respectively . * mean sd patient age 26 22 y. v. cholerae o1 was detected in 661 samples . v. cholerae was the sole pathogen in 379 samples ; however , it was isolated along with another diarrheagenic pathogen from 282 samples . the co - infection of v. cholerae and rotavirus was highly significant ( p = 1.12 10 ) . co - infection with v. cholerae and rotavirus was 5-fold less likely ( or 0.18 , 95% ci 0.130.25 ; figure , panel a ) to occur among those with than among those without v. cholerae infection . a negative association might be expected if a case of severe diarrhea caused by any given pathogen excluded other pathogens . consistent with this expectation , the presence of c. parvum , adenovirus , shigella spp . , etec , and v. parahaemolyticus was decreased significantly ( p = 7.87 10 to 1.32 10 ) and was 12.5-fold ( with v. parahaemolyticus , or 0.1 , 95% ci 0.020.33 ) to 2.44-fold ( with c. parvum ) less likely to occur among those with than among those without v. cholerae infection . however , antithetically , the rate of g. lamblia co - infection was significantly higher among v. cholerae o1positive than among v. cholerae o1negative fecal samples ( or 1.71 , 95% ci 1.322.21 ) . a significant difference in infection rates among those with and without v. cholerae o1 infection was not found for eaec , c. jejuni , v. fluvialis , e. histolytica , astrovirus , nvgii , and epec . tests for association were not performed for salmonella spp . , nvgi , aeromonas spp . , b. hominis , c. coli , sapovirus , v. cholerae non - o1 , v. cholerae non - o139 , and v. cholerae o139 because the low number of patients infected with those pathogens resulted in insufficient power . odds ratios ( ors ) showing odds of a ) vibrio cholerae or b ) rotavirus co - occurring with various other pathogens relative to the odds of v. cholerae or rotavirus co - occurring independently with various other pathogens at the frequency with which each is present in the entire sample . this standard forest plot indicates the best estimate and the 95% confidence intervals ( cis ) for each co - occurring organism . eaec , enteroaggregative escherichia coli ; epec , enteropathogenic e. coli ; etec , enterotoxigenic e. coli . rotavirus was detected in 594 of the fecal samples and was the sole pathogen found in 253 of them . rotavirus and at least 1 other gastrointestinal pathogen were found in 341 samples ; 119 samples were co - infected with rotavirus and > 2 other pathogens . when the effect of rotavirus co - infection with other pathogens was tested ( figure , panel b ) , shigella spp . were significantly less likely to be found in samples with rotavirus than in samples without rotavirus ( or 0.30 , 95% ci 0.170.52 ) . adenovirus were significantly increased in samples with rotavirus ( p = 6.15 10 to 1.61 10 ; ors 2.145.80 . a significant effect was not observed for g. lamblia , c. jejuni , epec , etec , v. parahaemolyticus , v. fluvialis , e. haemolyticus , astrovirus , and nvgii . tests for association were not performed for salmonella spp . , nvg1 , aeromonas spp . , b. hominis , c. coli , sapovirus , v. cholerae non - o1 , v. cholerae non - o139 , and v. cholerae o139 because the low number of patients infected with those pathogens resulted in insufficient power . analysis of samples from patients infected simultaneously with g. lamblia , v. cholerae , and rotavirus ( n = 41 ) revealed that the frequency of co - infection with g. lamblia was not significantly affected by co - infection with v. cholerae o1 and rotavirus ( p = 0.08 ) . analysis of covariates indicated that gender , religion , and residence largely had no effect on the associations between pathogens ; however , in some instances , age and season were identified as confounders or effect modifiers ( table 2 ) . to examine the effect of these covariates , we stratified the data by age and season and found that many associations remained significant ( table a1 ) . associations between rotavirus and adenovirus remained significant for all age and season strata except among children < 1 year of age . after adjusting for age and season by using logistic regression models , we found that co - infection with rotavirus and cryptosporidium spp . and co - infection with rotavirus and shigella spp . remained significant ( table 2 ) . the negative association between v. cholerae and adenovirus remained significant after adjustment by logistic regression for age and season ( or 0.36 ; 95% ci 0.210.64 ) ; associations between v. cholerae and many other pathogens remained significant within specific strata of age and season . assessing the effect of covariates for this reason , we did not include stratified results for co - infection with v. cholerae and v. parahaemolyticus in the table a1 . considered statistically significant at p<0.05 . or , odds ratio ; ci , confidence interval ; etec , enterotoxigenic escherichia coli ; eaec , enteroaggregative e. coli . our analyses revealed that co - occurrence of gastrointestinal pathogens in feces of patients with polymicrobial infections and severe diarrhea necessitating hospitalization was not in proportion to the pathogens presence in all patients with diarrhea . tests for association were performed with v. cholerae o1 and rotavirus because they were the most commonly detected pathogens and , hence , had the greatest power to detect an association with the other pathogens . some combinations of pathogens occurred less frequently than expected ( e.g. , v. cholerae and rotavirus [ or 0.18 , 95% ci 0.130.25 ] ) ; some combinations appeared more frequently than expected ( e.g. , rotavirus and adenovirus [ or = 5.8 , 95% ci 4.207.99 ] ) , and some combinations occurred at the same frequency whether with or without v. cholerae or rotavirus . after adjustment for age and season , these variables often acted as confounders or effect modifiers , but in general the associations remained significant . however , many of the stratified analyses had small numbers for comparison . v. cholerae o1 exhibited a positive association with only g. lamblia , suggesting that something may be unique about the co - occurrence of those 2 gastrointestinal pathogens . in support of that idea are 1 ) a report that co - infection with g. lamblia and v. cholerae results in g. lamblia being present in trophozoite form rather than in the cyst form found in feces of control patients ( 10 ) , and 2 ) a previous finding that g. lamblia trophozoites can bind cholera toxin ( 11 ) . alternatively , each is a pathogen with substantial environmental reservoirs , and the positive association may simply represent acquisition of both pathogens from the same environmental source . relatedness alone does not explain the apparent competitive inhibition or negative association that we found between v. cholerae and other pathogens . for example , although the closely related v. cholerae o1 and v. parahaemolyticus exhibited a 10-fold negative association , v. fluvialis , which is phylogenetically only slightly farther from v. cholerae o1 than is v. parahaemolyticus , did not show any inhibition in the presence of v. cholerae . also , although 2 members of the family enterobacteriaceae ( shigella spp . and etec ) were found less frequently than expected in combination with v. cholerae o1 , 2 other members of that family ( epec and eaec ) occurred in the expected proportion in samples from patients with mixed v. cholerae o1 infections . rotavirus had multiple strong positive associations ( ors > 2 ) with the other gastrointestinal pathogens detected in the fecal samples , even after considering age and season . the positive association of rotavirus , an rna virus that affects cells covered in microvilli , and adenovirus , a dna virus that affects cells that are dividing to generate new cells with microvilli , may represent an interaction between pathogens to cause more severe diarrhea ( in our sample all patients were hospitalized ) in patients 1545 years of age ( or 10.81 , 95% ci 5.6320.78 ) or a way to escape or circumvent immunity from previous exposures . in a previous study , koh et al . ( 12 ) found that among children in korea with virus - caused diarrhea , adenoviruses occurred preferentially in the presence of rotavirus and that rotavirus and norovirus , although most common , occurred in polymicrobial infections in proportion to their numbers in the samples . furthermore , bilenko et al . ( 13 ) observed that among bedouins , g. lamblia was frequently found in polymicrobial infections and , when present with rotavirus , produced less severe diarrhea than rotavirus alone . ( 14 ) found that among young children in so paulo , those with rotaviral and bacterial co - infections were more likely to have severe diarrhea than were children infected with either pathogen alone . however , the study had insufficient power to examine differences among bacterial pathogens , as did the rest of the studies of rotavirus infections mentioned in a recent review ( 15 ) . the presence of multiple pathogens in one third of patients with diarrhea has potential implications for treatment and raises several questions . do cases of diarrhea caused by v. cholerae or rotavirus and a second pathogen differ from those caused by v. cholerae or rotavirus alone ? do the pathogens behave synergistically to escape immunologic detection ? because the cross - sectional nature of our study did not enable us to investigate the temporal sequence of pathogen infection , future research is needed to provide more evidence concerning the causal pathway(s ) . also , the clinical significance of our findings must be more rigorously evaluated by studies that include infected patients and controls . a more substantive investigation into how age and season might affect polymicrobial infections the results of our current study indicate that associations can occur between some pathogens affecting the human gastrointestinal tract . the observation of selective positive associations among some gastrointestinal pathogens raises the question of how they interact in vivo ; e.g. , is the critical factor a modification of gastrointestinal tract microflora ? understanding the association(s ) among various co - infecting pathogens may help direct the development of treatment strategies .

digital brain atlases are useful as references , analytical tools , and as a data integration framework . as a result , they and their supporting tools are being recognized as potentially useful resources in the movement toward data sharing . several projects are connecting infrastructure to these tools which facilitate sharing , managing , and retrieving data of different types , scale , and even location . with these in place , we have the ability to combine , analyze , and interpret these data in a manner not previously possible , opening the door to examine issues in new and exciting ways , and potentially leading to speedier discovery of answers as well as new questions about the brain . here we discuss recent efforts in the use of digital mouse atlases for data sharing .

many neuroscientists have been calling for a system for the mouse brain where digital atlases serve as the framework used to traverse the brain and information linked to it ( baldock et al . , 2003 ; bjaalie , 2002 ; boline et al . , 2007 ; mackenzie - graham et al . , 2003 ; martone et al . , 2004 ; toga , 2002 ) . in contrast to data repositories , which allow simple access to data through a single interface , sophisticated digital atlases backed by the appropriate technology can act as a neuroinformatics hub facilitating access to different databases , information sources , and related documents and annotations . they may act as the scaffold in which otherwise unrelated data may be housed and correlated , providing an intuitive interface to share , visualize , analyze , and mine data of multiple modalities , scales , and dimensions . the semantic and spatial information tied to an atlas can add a dimension to data in a manner that exponentially increases its potential use and reusability . semantic linking of data to the atlas requires the data provider to register it with an ontology or controlled vocabulary , while spatial registration requires alignment of an image to the atlas . this information is then used to place data into the context of the atlas , allowing it to inherit information tied to the spatial coordinates of the atlas . spatial information in this type of slice data is key to interpreting results , yet these images lose anatomical context during the data collection process . thus atlas - based tools for organizing and analyzing this and related types of data may be used to create a system ideal for sharing data . several projects offer access to gene expression image data with differing levels of spatial mapping in the mouse nervous system . these projects have been comprehensively reviewed ( brumwell and curran , 2006 ; koester and insel , 2007 ; sunkin , 2006 ; sunkin and hohmann , 2007 ) and among others , include the allen brain atlas ( aba , www.brain-map.org ) , bgem ( www.stjudebgem.org ) , gensat ( www.gensat.org ) , genepaint ( www.genepaint.org ) , eurexpress ( www.eurexpress.org ) , mgi ( http://www.informatics.jax.org/ ) and emap / emage ( http://genex.hgu.mrc.ac.uk ) . as illustrated by the images from gensat and mgi in figure 1 , the ability to examine spatial and temporal expression patterns is crucial for developing correlations between genotype and phenotype as well as for interpreting and comparing findings across experiments . also illustrated , is that the anatomical information tends to be sparse in these data sources , as is also the case with bgem , genepaint , and eurexpress ( sunkin and hohmann , 2007 ) . aba and emap / emage differ from these sources in that in addition to linking the images to semantic information , they have also linked their images to spatial information by registering their images to a reference atlas ( baldock et al . , 2003 ; while this extra step can be both difficult and time consuming , it adds the potential for a great deal of analytical power and the ability to generate spatial queries ( carson et al . , 2005 ; christiansen et al . , 2006 ; leergaard and bjaalie , 2007 ) . examples of databases which manage gene expression image without an anatomical framework . gensat and mgi both provide a rich repository of image data for gene expression . neither uses a standard framework for data organization , rather , both describe the pattern and location of gene expression revealed by the image . gensat ( a ) describes the expression pattern for major structures in each image , while the mgi ( b ) summarizes a batch of assays ( the mgi dataset shown here were used to derive the lef1 gene examples shown in lee et al . , 2007 ) . while these annotations aid interpretation , the ability to query or analyze these data in this format is very limited . as most resources are built for sharing a specific set of data , most discussed above are not yet set up to easily link their data to that offered by other groups , adding a barrier to analyzing data across experiments . also , with the exception of emage , mechanisms are not readily available for an individual to easily put data into a semantic and spatial framework that facilitates comparison of their own data to others . for these reasons , it still requires a great deal of research and work to compare data collected in different experiments , which is one of our communities greatest desires , but also most difficult tasks . these are some of the recent drivers for the call to create interoperability across data resources and offering access to this system to any scientist , within the context of a digital atlasing framework . integrating these and other data sources via such a framework would allow a researcher to easily query across these resources . one could look for studies of different collection modalities , strains , developmental stages , or disease models , or examine the expression patterns of genes or regions of interest across multiple studies . for example , a scientist investigating a disease model of parkinson 's with the microarray technique finds that an unexpected gene in the caudate / putamen area seems to have a reverse correlation with motor deficits . wanting to know more , he uses this system to find data from other experiments that have examined the same disease model . he finds a mri dataset illustrating a change in the shape and decrease in the volume of that area in later stages of the disease , and a high - resolution confocal dataset from this same region shows abnormal cell morphology with disease progression . in addition , he finds that expression of this gene in this region in normal animals decreases with age and that it is expressed at a higher level throughout life in a different mouse strain which shows resistance to parkinson 's . compiling and analyzing data from these different experiments , many of which he did not even realize were applicable to his situation , allows him to more fully examine the potential role of this gene and to better inform his next experiments . while individual researchers often do a similar type of information gathering on their own , it can be difficult to examine other datasets , or we miss a relevant dataset because the data producers published it in relation to a very different topic . as diverse data generation continues to grow at an accelerated rate , we are in dire need of systems that make it easy for our community to contribute , organize , and find relevant data . while we are a long way from a fully implemented system that could perform the previous example , different groups have already created many of its components . for atlases to be used to query and access spatially indexed data , they must be backed by databases , spatial and semantic registration methods , and a client that allows query and access to the data . building a digital atlas mapping framework to facilitate experimentation and as a result , atlas resources besides those discussed above , developed with the purpose of placing mouse gene image data within a context of an atlas and offering it to the public . these include a joint project between the mouse brain library ( mbl , www.mbl.org/ ) and neuroterrain ( www.neuroterrain.org ) , the mouse atlas project ( www.loni.ucla.edu/map/ ) , and the smart atlas ( https://portal.nbirn.net ) . the mbl consists of high - resolution histological images and associated databases of brains from many genetically characterized strains of mice ( rosen et al . , 2003 ) . neuroterrain offers a high - resolution 3d nissl atlas in macrovoxel format that may be accessed over the network and viewed in an arbitrary plane ( gustafson et al . , 2004 ) and has much of the mbl data registered to it . the mouse atlas project developed tools to bring together brain architecture , gene expression , and 2d and 3d imaging information into a single interface ( mackenzie - graham et al . , 2003 ) and the smart atlas integrates data using both semantic and spatial geographical information systems ( gis ) methods ( martone et al . , 2008 ) . it is based on coronal and sagittal plates from the paxinos and franklin ( 2001 ) atlas and includes functionality that allows a user to register image slices to the atlas plates and allows spatial query of these data ( zaslavsky et al . , 2004 ) . to go a step farther and build a system where atlases act as a point of access to data from multiple sources of different type and scale requires more than just connection to more databases . it requires a large community effort , and it is now a focus of collaboratives including the mouse biomedical informatics research network ( birn , www.nbirn.net and http://www.loni.ucla.edu/birn/projects/mouse ; martone et al . , 2004 ) and the international neuroinformatics coordinating facility ( incf , www.incf.org ; boline et al . , 2007 ) . the birn project has been focused on building infrastructure supporting data sharing through a federated model and the focus of mouse birn has been on developing and applying this infrastructure to promote data sharing through an atlasing framework . as a result , the mouse birn atlasing toolkit ( mbat , www.nbirn.net/tools/mbat_2.0 and http://cms.loni.ucla.edu/mbat/ ) has been built to offer easy access to this infrastructure to the public . the mbat project builds on the complementary strengths and functionality of the mbl , neuroterrain , map , and smart atlas projects and is shaped by experimental mouse models of neurodegenerative disease studied in the mouse birn project . if a system such as this is to grow in a flexible and extensible manner ( figure 2 ) , it requires support by a large set of integration resources , such as standard terminologies and/or ontologies , database integration resources ( such as the birn mediator , www.nbirn.net/research/data_integration ) , standard schemas for data exchange ( such as mage , www.mged.org/workgroups/mage/mage.html , or xcede , www.xcede.org ) , database webservices , and standard application programming interfaces ( apis ) . finally , bringing together resources that are registered to different atlases requires additional spatial and semantic integration resources that can facilitate interoperability between these atlases ( such as an atlas interoperability server and api , www.loni.ucla.edu/twiki/bin/view/mousebirn/atlasstateexchange ) . these integration resources give an entryway for groups other than birn to participate in creating interoperable data and tools which can allow linkage to data they offer in other projects . ideally , multiple methods may be employed for interested collaborators to share their data through this framework , whether it is via their own database , or through upload processes . gray elements are specialized servers , databases , tools , and standards built by the scientific community that facilitate integration of data . yellow cylinders represent data sources that offer a certain data type ( i.e. , genenetwork and the birn microarray database are both microarray databases with services that allow access to their data ) . standardized application program interfaces ( apis - blue arrows ) allow the creation of tools that access services offered by a source . however , expanding even a flexible system with a new data type requires its own community - driven approach to modify these existing resources to set standards , web - services , apis , and application of their own unique combination of integration methods . for instance , the preferred approach discussed for gene expression images has been spatial registration , but so far , the main approach for integrating microarray data is primarily semantic , with the exception of voxelized data registered to an atlas ( chin et al . , 2007 ) . moreover , bridging the gap between different developmental stages ( i.e. , embryonic and adult ) as well as that between species will require at least an initial reliance on semantic mapping due to the huge variations in spatial differences between species and some developmental stages . from the user 's point of view , the framework allows a simple , but complete cycle of data sharing . a user may enter this path by registering their data within the client or via other server resources . she manages and visualizes her data in the client , where she can compare her data to an atlas and some other sets of data . however , she also has the option to share her data set by uploading it through resources that take advantage of an integration infrastructure and stores the data and its associated information in the appropriate databases . this upload process requires semantic and spatial integration methods ( gray elements ) to give the appropriate context to the data . once the data are uploaded , the integration infrastructure allows other users to query and retrieve her dataset through a client that accesses the infrastructure through apis . the current version of mbat ( 2.0 beta ) integrates data using both semantic and spatial methods . it points a user to accessible mouse birn upload interfaces and even includes tools for a user to create their own atlas . it allows spatial - based queries , but its current focus is to allow semantic queries and access to data from multiple distributed sites ( figure 2 , yellow cylinders and table 1 ) . currently mbat does not completely fulfill the complete atlas - based framework outlined in figure 2 . however , we continue to develop , and offer access to tools and resources that aid in moving toward this goal . we recently reported the use of a high - resolution atlas as an anatomical framework for localizing gene expression data for the mouse brain at postnatal day 0 ( p0 ) ( lee et al . , 2007 ) along with new tools that help register , manage , and analyze gene expression image data ( figure 3 ) . local atlas - based data management facilitates data correlation via several atlas tools developed in the birn project . based on the intersection of the polygon and the anatomical delineation provided by the atlas , the anatomical composition within the roi can be determined , thus enabling the retrieval of information associated with the atlas coordinates or anatomical concepts . once the user 's data is brought into the atlas space , the contours outlining the regions of interest can be mapped to the brain anatomy . ( 2007 ) employed a similar vector organization as the polygon method used for spatial query in mbat ( inset ) . bringing the data into a common standard space not only facilitates data visualization , but also allows one to use the atlas as an interface for reusing and correlating other data derived from the same location . this publication shows that a management document specifying the linear data - to - atlas spatial transformation is sufficient for relating local gene expression image data to a predefined atlas space . to setup the atlas - based data management and interaction with atlas - associated resources , it only requires an application that reports the atlas transformation in an offline management document . since mbat uses a coordinate system and integration resources to associate with multiple resources ( i.e. , paxinos ' canonical templates , microarray data ) , atlas - associated data relating to the region of interest ( roi , area with enriched gene expression ) may be retrieved by a spatial query . this registration also maps the roi to the brain anatomy , thus data correlation may also be examined using anatomical structure names . it is important to note that the methods discussed in this publication are not focused on pushing forward registration methods . as the field of image registration is problematic with no simple solutions for all data types , the management tools need not be dependent on a single registration method . instead , the creators of registration tools may be able to take advantage of these management methods in order to interact with this framework . groups with components of this framework already in place may wish to contribute to , and shape the direction and specifics of the infrastructure . others may wish to make available and link tools to this framework that facilitate registration , query , visualization , or analysis . ideally the system is built in a model that allows tool - builders to easily add to or create new tools for the framework . data rich resource groups may wish to develop services for the type of data they offer so their resources may also be easily accessed by this sharing infrastructure . individuals may also wish to help shape the direction and specifics of the developing infrastructure and tools . as they begin to use these resources to share their data with the rest of the scientific community , they may find gaps that can only be filled with their expertise . finally , since the complexity of such a system must be hidden from those using it , feedback from both the research and programming community is essential to creating usable interfaces and points of integration . in addition to these technical issues it is obvious that conceptual differences related to anatomies , terminologies , and ontologies will exist as long as there are neuroscientists . however , an advantage of this framework is the flexibility offered by the integrate layer . this can be shaped by the community so scientists can map their own well - defined atlases , terms , and ontologies into this system and for a user to choose any of these to define their own data . in fact , a system such as this would be an ideal platform for facilitating these types of discussions and debates . as we begin to close in on such a framework and add accessible resources , we will find that we have developed powerful mapping tools that help us find and analyze diverse information about the brain of different scale and type from different laboratories across the world . if we succeed in building such a powerful , intuitive , and flexible framework , we may help spread access to data and speed discovery as never before possible . 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 .

we report a 60-year - old man who presented with a 2-year history of painless red raised lesions involving the tongue and scrotum . histopathology was suggestive of angiokeratoma . oral angiokeratomas are most commonly found as a component of the generalized systemic disorder in fabry 's disease or fucosidosis . our patient had isolated mucosal angiokeratomas which is very rare .

oral angiokeratomas are most commonly found as a component of the generalized systemic disorder in fabry 's disease or fucosidosis , where multiple angiokeratomas can be found on the skin and the oral mucosa . we report a rare case of a patient with multiple oral and scrotal angiokeratomas in the absence of a systemic disorder . a 60-year - old man presented with a 2-year history of painless red raised lesions involving the tongue . he complained of burning sensation in the mouth in the region of the papules . clinical examination revealed multiple erythematous to slightly bluish , shiny , vascular papules ranging from 1to3 mm size , discrete to grouped on the undersurface and lateral aspects of the tongue [ figure 1 a and b ] . biopsy from a vascular papule on the tongue showed acanthosis , and papillomatosis , with large , dilated spaces lined by normal - appearing endothelium which were filled with erythrocytes and organizing thrombi [ figure 1d ] . the patient was counseled about the benign nature of the disease after which he chose not to take any treatment . ( a - b ) multiple erythematous to bluish , shiny , vascular papules on the undersurface and lateral aspects of the tongue ; ( c ) similar vascular papules on the scrotum ; ( d ) acanthosis , and papillomatosis , with large , dilated spaces lined by normal - appearing endothelium which are filled with erythrocytes and organizing thrombi ( h and e , 200 ) angiokeratoma is an asymptomatic cutaneous vascular disorder of the papillary dermis characterized by dermal vasculsar ectasia with overlying hyperkeratosis of the epidermis . it is thought to be a telangiectatic lesion arising from local injury to papillary dermal capillaries , either from trauma or venous hypertension . they are ( 1 ) the generalized systemic type : angiokeratoma corporis diffusum of fabry characterized by multiple tiny red papules in a symmetrical distribution in the bathing area ; ( 2 ) angiokeratoma of mibelli , the bilateral form occurring on the dorsal areas of the fingers and toes ; ( 3 ) angiokeratoma of fordyce , the most common angiokeratoma which occurs on the scrotum ; ( 4 ) the solitary papular angiokeratoma which has a predilection for the legs ; and ( 5 ) angiokeratoma circumscriptum , the multiple papular and plaque- like form . oral mucosal angiokeratomas have also been associated , uncommonly , with angiokeratomas of the scrotum , gastrointestinal mucosa ( jejunum ) , or both . recently ranjan & mahajan have proposed a clinical classification for oral angiokeratomas , depending on the number of lesions , associated other muco - cutaneous involvement and systemic disease . oral angiokeratomas present as single / multiple , erythematous , shiny papules , which may be studded with keratotic tops . they are firm on palpation , nontender , show telangiectatic vessels on diascopy , and bleed occasionally . the tongue , with a predilection for the dorsal aspect , appears to be the most common site for isolated oral angiokeratoma . they may be mistaken for hemangiomas , lymph - hemangiomas , focal epithelial hyperplasia , or nevi . the histologic features are similar to their cutaneous counterparts ; hyperkeratosis , acanthosis and dilated capillaries in the papillary dermis , partly or completely enclosed by the papillomatous epidermis . organized or organizing thrombi are occasionally observed within the dilated capillaries . the only difference being the presence of parakeratosis in oral angiokeratomas . however , in fabry 's disease and fucosidosis , swollen and vacuolated ( lipid containing ) endothelial cells are characteristic differentiating features .

this prospective study aimed to estimate the incidence and associated factors of severe maternal morbidity in southeast iran . during a 9-month period in 2013 , all women referring to eight hospitals for termination of pregnancy as well as women admitted during 42 days after the termination of pregnancy were enrolled into the study . maternal near miss conditions were defined based on say et al . 's recommendations . five hundred and one cases of maternal near miss and 19,908 live births occurred in the study period , yielding a maternal near miss ratio of 25.2 per 1000 live births . this rate was 7.5 and 105 per 1000 in private and tertiary care settings , respectively . the rate of maternal death in near miss cases was 0.40% with a case : fatality ratio of 250 : 1 . the most prevalent causes of near miss were severe preeclampsia ( 27.3% ) , ectopic pregnancy ( 18.4% ) , and abruptio placentae ( 16.2% ) . higher age , higher education , and being primiparous were associated with a higher risk of near miss . considering the high rate of maternal near miss in referral hospitals , maternal near miss surveillance system should be set up in these hospitals to identify cases of severe maternal morbidity as soon as possible .

there are approximately 287,000 preventable maternal deaths annually , of which 99% occur in developing countries . as a sentinel event , maternal death is also a prime indicator in evaluating the quality of a nation 's health care delivery systems [ 2 , 3 ] . mothers are pivotal to the social , economic , and cultural development of a community ; maintaining their health elevates the physical , psychological , and social well - being of their children and families and , by extension , society as a whole . for this reason , improving maternal health has been proposed by the world health organization ( who ) as one of their eight millennium development goals ( mdg ) . however , in countries with few maternal deaths , this approach fails to provide comprehensive information , leaving policy makers to react based on current rather than past statistics . to facilitate the development of precautionary measures and safer environments that minimize maternal deaths , it is essential that near miss data are recorded and analyzed . the who defines an individual having experienced severe acute maternal morbidity ( samm ) ( i.e. , near miss ) as a woman who nearly died but survived a complication that occurred during pregnancy , childbirth or within 42 days of termination of pregnancy . in fact , maternal near miss includes those cases in which a woman nearly died but survived during pregnancy or during 42 days after the delivery . using near miss data in maternal death prevention planning first , because the number of near miss cases exceeds maternal death cases , near miss is a better predicate for preventive planning . second , because the mother survives a near miss , she can provide valuable details on what she experienced . lastly , because near miss is one step removed from death , obtaining any information about the event could prove useful in preventing maternal death [ 810 ] . the prevalence of maternal near miss varies among different countries based on health care quality and availability . nevertheless , in a systematic review using disease - specific criteria , near miss rates have been reported to be between 0.6% and 14.98% . from a global perspective , iran has been notably successful in reducing maternal mortality . between 1990 and 2008 , iran has managed to decrease its maternal mortality rate by 80% , which , at present , translates to about 25 deaths in 100,000 . the availability of emergency obstetrical care , improvements in women 's education , and the expansion of family planning services have all contributed to the decrease in maternal mortality . in iran , a national maternal mortality surveillance system examines maternal death cases by reviewing files and interviewing key parties to determine the cause of death . although some studies have been carried out in iran and the middle east regarding the causes of maternal death and risk factors of pregnancy [ 1217 ] , the authors know of no published study on severe maternal morbidities and near miss occurrences from iran or other middle eastern countries . the present study aimed to establish a profile of severe maternal morbidities in iran and their relationship with other underlying factors this prospective study encompassed eight hospitals with maternity facilities located in the two large cities of kerman and jiroft in southeast iran . the study was performed in 2013 for a period of nine months . the study protocol was approved by the ethical committee of kerman university of medical sciences ( e.c./90/518 ) . after explaining the study 's nature and aims , oral consent was obtained from the participants who all were ensured that their information would remain confidential . first , a list of maternal near miss conditions was prepared based on say et al . 's recommendations for prospective surveillance of maternal near miss cases . this list included four major groups of haemorrhagic disorders , hypertensive disorders , severe management indicators , and other systemic disorders . moreover , a category including other conditions was also considered so as not to miss any other life - threatening illness . all women admitted during the nine - month study period for delivery or completion of pregnancy as well as women admitted within 42 days after the termination of pregnancy were enrolled into the study . after the participating hospitals ' maternity , labour , general icu , emergency , and admission departments were fully coordinated , our case survey was implemented . a check list was completed for cases with potentially life - threatening conditions by a team consisting of a midwife and gynaecologist . the first part included demographic and clinical data such as age , educational level , place of residency , gravidity , parity , type of delivery , and gestational age . next , a woman admitted to the delivery room at the same hospital was randomly selected from the list of patients as the control , and the first part of the check list was completed for her . in all stages , an experienced expert supervised completion of the check lists . according to the who definition maternal near miss ratio was defined as the number of maternal near - miss cases per 1000 live births . chi - square and independent t - tests were used to compare the qualitative and quantitative variables between near miss and control groups . a stepwise logistic regression model was used to determine the relationship between underlying variables and near miss ratio . the logistic regression model 's goodness of fit was evaluated by the hosmer - lemeshow test . during the study period , there were 501 cases of near miss in 19,908 live births ( a near miss ratio of 25.2 per 1000 live births ) . the highest near miss ratio ( 104.8 in 1000 ) was observed in the referral ( educational ) hospital ( table 1 ) . the mean age of near miss cases was 28.3 6.1 years versus the control group 's 26.0 5.8 years ( p < 0.001 ) . university degrees were seen more among near miss women ( table 2 ) . in the near miss group , 208 women ( 41.5% ) were primiparous , whereas , in the control group , the number was 225 ( 45.2% ; p = 0.243 ) . the frequency of abortion in the near miss and control groups was 18.6% and 1.6% , respectively ( p < 0.001 ) . the frequency of caesarean section in the near miss and control groups was 24.7% and 54.2% , respectively ( p < 0.001 ) . in our study group , there were two cases of maternal death . one was a 19-year - old woman diagnosed with intracerebral haemorrhage ( ich ) ; the other was a 28-year - old woman who had undergone curettage due to a failed abortion and died from sepsis because of perforations in the uterus and intestine . the rate of maternal death in near miss cases was 0.40% with a case : fatality ratio of 250 : 1 . as shown in table 3 , the most prevalent causes of near miss were severe preeclampsia ( 27.3% ) , ectopic pregnancy ( 18.4% ) , and abruptio placentae ( 16.2% ) . in all , 15.2% had at least one systemic disease , and 43 women in the near miss group were hospitalised in the icu ( table 3 ) . the majority of the near miss cases were observed in the haemorrhagic disorders group ( table 3 ) . logistic regression analysis showed that four variables had significant relationship with near miss occurrence ( table 4 ) . of the 377 near miss cases ( 75.2% ) , the major problem was discerned at or during the first six hours of admission . in the present study , the near miss ratio was 25.2 per 1000 live births . in the private setting , this rate was 7.5 per 1000 , and , in the tertiary care hospital , it was approximately 105 per 1000 . the main advantages of this study were its prospective nature and its use of standard criteria for determining near miss cases . because more than 97% of deliveries in iran occur in hospitals , the present report may be considered as a population - based study . because of the differences among patients and health care delivery systems , generalizing the results of this study on a countrywide basis should be done with caution . the literature shows that maternal near miss ratios vary greatly depending on the population studied , how near miss is defined , and how the study is conducted ( prospective versus retrospective ) [ 11 , 19 ] . near miss ratios have been reported as 44.3 per 1000 in brazil , 33 per 1000 in india , 3.83 per 1000 in scotland , and 34 per 1000 in a who survey . a recent systematic review using a unique definition for near miss showed samm rates in high - income countries to be significantly lower compared with those of low- and middle - income countries . in this study , like some other studies [ 19 , 20 ] , the rate of near miss was significantly higher in the tertiary care setting ( table 1 ) . the reason might be that , because of the limited facilities at private hospitals , women with complicated pregnancies are not usually referred to these centres . in the present study , we found a higher rate of near miss and consequently a lower fatality rate ( 0.4% ) . these findings may be derived from our broader definition for near miss events , which combined disease - specific criteria with management - based criteria . in netherland , with a near miss ratio of 7.1 per 1000 , the rate was 1.9% . in brazil , with a near miss ratio of 42 per 1000 , the rate was 1.6% . it should be mentioned that the maternal death rate shows a decreasing trend in iran . in our study , haemorrhagic disorders ( 46.1% ) and hypertensive disorders ( 31.9% ) these rates are similar to those reported from scotland and indonesia . in india , the two most common causes of near miss have been preeclampsia and haemorrhage . in a study on 64 cases of maternal death in kerman , iran , the same two factors were the most prevalent causes of maternal mortality . in our study , 43 cases required intensive care ; that is , for every 1000 live births , 2.2 mothers are hospitalized in an icu , which is nearly similar to the icu admission rate in netherland . according to our logistic regression model , four variables had a relationship with near miss ( table 4 ) . in older women with university degrees , in fact , most studies demonstrate a proportional relationship between higher near miss ratio and advancing age ( particularly over 35 years ) [ 22 , 23 ] . in the who 's global survey , this finding has been attributed to the tendency among women with higher educational levels to undergo caesarean section , which increases the probability of near miss events . the present study , as in previous studies , shows that being a primipara increases the probability of near miss ratio by 1.2%1.4% [ 22 , 23 ] . although pregnancy complications are to a great extent unpredictable and unpreventable , early awareness of near miss cases can prevent the progress of disease and maternal death . in this study , the feasibility of using who - recommended near miss criteria was recognized . however , because of inadequate health care services , it is necessary that the auditing of near miss cases be considered as important as the implementation of near miss surveillance systems [ 15 , 23 ] . the present study showed that using data related to near miss cases can provide more comprehensive information when reviewing maternal death cases ; therefore , establishing near miss surveillance systems in iranian hospitals is highly recommended .

objectivewhile acetylcholinesterase inhibitors , such as donepezil , galantamine , and rivastig - mine , are beneficial in treating behavioral symptoms of patients with alzheimer s disease ( ad ) , their dose - limiting effects include gastrointestinal disturbances , such as nausea , vomiting , and diarrhea . we aimed to predict the occurrence of these gastrointestinal disturbances with rivastigmine therapy for optimal drug choice and improved compliance.materials and methodsthirty patients with mild - to - moderate ad ( scores 1022 on the minimental state examination ) were administered a rivastigmine 18 mg patch with domperidone 30 mg ( rwd ) and without domperidone ( rwod ; n = 15 each ) for 20 weeks . gastrointestinal disturbances were evaluated using a frequency scale for symptoms of gastroesophageal reflux disease ( fssg ) , bristol stool form scale , laboratory data ( hemoglobin , albumin , total cholesterol ) , body weight , and amount of food intake.resultsafter 12 weeks , fssg scores were higher in the rwod group compared to baseline scores ; however , no significant differences were noted between the rwd and rwod groups . we then subdivided each group based on high and low baseline scores ; the rwod high - score ( 4 ) subgroup showed increased fssg after 12 weeks compared with the baseline score . in both rwd and rwod groups , the low - score ( 3 ) subgroups showed no changes during the dose - escalation phase.conclusionfor ad patients with higher fssg scores at baseline , domperidone was effective in preventing rivastigmine - related gastrointestinal disturbances .

patients show increasing declines in cognition , and behavioral symptoms occur in all stages of ad . while all ad patients can present with depression , agitation , and aggressive behaviors , behavioral difficulties are most pronounced in the advanced stages of the disease.13 rivastigmine , inhibiting both acetylcholinesterase and butyrylcholinesterase , is the first transdermal treatment available for ad patients , and has been widely approved for the symptomatic treatment of ad.4,5 by providing continuous delivery of medication from the skin into the bloodstream , these patches achieve sustained , high drug concentration in the plasma over 24 hours.6 however , the investigation of transdermal exelon in alzheimer s disease ( ideal ) study revealed that ad patients receiving a 10 cm patch frequently suffered from nausea ( 7.2% ) and vomiting ( 6.2%).4 further , japanese ad patients receiving an 10 cm patch also showed similar adverse events , including nausea ( 7.0% ) and vomiting ( 8.0%).5 ad patients have been reported to stop the medication because of these adverse effects ; however , no technique is currently available to predict the occurrence of these adverse effects . the antiemetic domperidone is often administered in order to administer higher , more effective dosages of cholinesterase inhibitors ( cheis ) , and such a combination with domperidone has been shown to be effective in preventing rivastigmine - related gastrointestinal disturbances when administered orally in the rivastigmine capsule form.7 in the present study , we examined the occurrence of gastrointestinal disturbances related to the use of the rivastigmine patch using the frequency scale for the symptoms of gastroesophageal reflux disease ( fssg),8 bristol stool form scale,9 laboratory data , body weight , and amount of food intake . in addition , we compared the differences in gastrointestinal disturbances when using a rivastigmine patch with domperidone ( rwd ) and without domperidone ( rwod ) in order to examine the efficacy of domperidone orally . patients were recruited from february 2012 to january 2013 at the department of neurology , toho university omori medical center , tokyo , japan . subjects included outpatients diagnosed with ad who lived with or had regular daily visits from a responsible caregiver . the diagnosis of ad was based on the criteria from the national institute of neurological disorders and stroke and the alzheimer s association.10 the criteria have been established by clinical and neuropsychological examination . cognitive impairments have to be progressive and be present in two or more areas of cognition . the onset of the deficits has been between the ages of 40 and 90 years , and finally there must be an absence of other diseases capable of producing a dementia syndrome . a total of 30 patients with mild - to - moderate ad ( scores 1022 on the mini - mental state examination [ mmse]11 ) were enrolled and randomly allocated to two groups by the envelope method , ie , rwd and rwod ( n = 15 each ) . dose titrations of rivastigmine patches were performed every 4 weeks using 4.5 mg / day increments from 4.5 mg / day to 18 mg / day . domperidone was administered orally at a dose of 30 mg / day in the rwd group . patients received rivastigmine - patch treatment for 20 weeks , and were evaluated for gastrointestinal disturbances at baseline and at weeks 4 , 8 , 12 , 16 , and 20 . the fssg is a simplified questionnaire for evaluation of the symptoms of gastroesophageal reflux disease ( gerd ) , and has been demonstrated to be a useful tool for objectively evaluating symptoms in gerd patients.8 the questionnaire consists of twelve items that are scored as 04 based on the frequency of the symptom ( 0 = never , 1 = occasionally , 2 = sometimes , 3 = often , and 4 = always ) with total scores ranging from 0 to 48 . ( 2 ) does your stomach get bloated ? ( 3 ) does your stomach ever feel heavy after meals ? ( 4 ) do you sometimes subconsciously rub your chest with your hand ? ( 5 ) do you ever feel sick after meals ? ( 6 ) do you get heartburn after meals ? ( 7 ) ( 8) do you feel full while eating meals ? ( 9 ) do some things get stuck when you swallow ? ( 10 ) do you get bitter liquid ( acid ) coming up into your throat ? ( 11 ) do you burp a lot ? the bristol stool form scale consists of seven types ( 1 , separate hard lumps , like nuts ; 2 , sausage - shaped but lumpy ; 3 , like a sausage or snake , but with cracks on its surface ; 4 , like a sausage or snake , smooth and soft ; 5 , soft blobs with clear - cut edges ; 6 , fluffy pieces with ragged edges , a mushy stool ; and 7 , watery , no solid pieces ) . the fssg and bristol stool form scale scores were obtained not only from patients but also from their caregivers to obtain accurate information . if the scores differed between the patient and his / her caregiver , we usually took the caregiver s score , because of the patient s cognitive impairment . in addition , we evaluated the changes in laboratory data ( hemoglobin [ hb ] , albumin [ alb ] , and total cholesterol [ t - chol ] ) , body weight , and amount of food intake to evaluate the nutritional status of ad patients . the amount of food intake per day was set as a score of 10 at baseline , and any improvement or loss in appetite was reflected as increased or decreased scores , respectively , based on patient and caregiver feedback . statistical analysis was performed by statmate ( graph - pad software , la jolla , ca , usa ) , with the mann whitney u test and wilcoxon signed - rank test , with p < 0.05 used as the threshold for statistical significance . all patients and legally accepted caregivers participating in the trial were given an explanation of the trial by the investigators before the start of study , and informed consent to participate in the study was obtained . consent of patients was obtained orally or in writing , and the consent of legally accepted representatives was always obtained in writing . overall , three patients from the rwd group discontinued their participation due to skin irritation , and one patient from the rwod group withdrew because of nausea . no differences were observed between the rwd and rwod groups with respect to mean age , the sex ratio , mmse score , or body weight . the fssg scores for the rwod group showed significant increases after 12 weeks compared with baseline ( versus 12 weeks , p = 0.035 ; versus 16 weeks , p = 0.034 ; versus 20 weeks , p = 0.034 ) ; however , this was not observed in the rwd group ( figure 1 ) . before initiating rivastigmine - patch therapy , 23 of 30 patients ( rwd group , eleven of 15 ; rowd group , twelve of 15 patients ) showed the presence of some gastrointestinal symptoms based on the fssg questionnaire . the median value of the fssg at baseline was 3.8 in the rwd group and 3.1 in the rwod group ; therefore , we set the high - score threshold 4 points and low - score threshold 3 points . in the rwod group , the high - score subgroup showed increased fssg scores after 12 weeks compared with baseline scores ( versus 12 weeks , p = 0.038 ; versus 16 weeks , p = 0.035 ; versus 20 weeks , p = 0.034 ) ; however , no such changes were observed in the high - score subgroup in the rwd group ( figure 2 ) . in both rwd and rwod groups , the low - score subgroups showed no changes in fssg scores during the dose - escalation phase . the data for mmse , laboratory results ( hb , alb , and t - chol ) , body weight , amount of food intake , and bristol stool form scale scores are shown in table 2 ; no differences were noted between the baseline values and those at week 20 for either group . we compared the data for the rwd and rwod groups for all the parameters examined . no significant differences were observed between the two groups for the fssg ( figure 1 ) , mmse , laboratory data , body weight , amount of food intake , or bristol stool form scale scores ( table 2 ) . overall , three patients from the rwd group discontinued their participation due to skin irritation , and one patient from the rwod group withdrew because of nausea . no differences were observed between the rwd and rwod groups with respect to mean age , the sex ratio , mmse score , or body weight . the fssg scores for the rwod group showed significant increases after 12 weeks compared with baseline ( versus 12 weeks , p = 0.035 ; versus 16 weeks , p = 0.034 ; versus 20 weeks , p = 0.034 ) ; however , this was not observed in the rwd group ( figure 1 ) . before initiating rivastigmine - patch therapy , 23 of 30 patients ( rwd group , eleven of 15 ; rowd group , twelve of 15 patients ) showed the presence of some gastrointestinal symptoms based on the fssg questionnaire . the median value of the fssg at baseline was 3.8 in the rwd group and 3.1 in the rwod group ; therefore , we set the high - score threshold 4 points and low - score threshold 3 points . in the rwod group , the high - score subgroup showed increased fssg scores after 12 weeks compared with baseline scores ( versus 12 weeks , p = 0.038 ; versus 16 weeks , p = 0.035 ; versus 20 weeks , p = 0.034 ) ; however , no such changes were observed in the high - score subgroup in the rwd group ( figure 2 ) . in both rwd and rwod groups , the low - score subgroups showed no changes in fssg scores during the dose - escalation phase . the data for mmse , laboratory results ( hb , alb , and t - chol ) , body weight , amount of food intake , and bristol stool form scale scores are shown in table 2 ; no differences were noted between the baseline values and those at week 20 for either group . we compared the data for the rwd and rwod groups for all the parameters examined . no significant differences were observed between the two groups for the fssg ( figure 1 ) , mmse , laboratory data , body weight , amount of food intake , or bristol stool form scale scores ( table 2 ) . vomiting is a highly specific physical event , defined as the rapid , forceful evacuation of gastric contents in retrograde fashion from the stomach up to and out of the mouth . in contrast , nausea is an entirely subjective experience , defined as the sensation that immediately precedes vomiting , wherein patients feel that they are about to vomit . rivastigmine patch - related gastrointestinal disturbances have been reported in less than 10% of patients receiving this therapy.4,5 in our study , only one patient from the rwod group discontinued the therapy due to nausea . however , our fssg questionnaire findings revealed that 23 of 30 ad patients ( > 75% ) suffered from at least one gastrointestinal disturbance before the initiation of the rivastigmine patch . furthermore , ad patients with higher fssg were observed to show a further increase in the fssg score during the dose - escalation phase . our results may indicate that ad patients do not report gastrointestinal disturbances because of the memory deficits involved in ad . domperidone , a dopamine antagonist that acts by blocking dopamine receptors in the chemoreceptor trigger zone , has been previously shown to prevent gastrointestinal disturbances in rivastigmine therapy administered orally.7 in the present study , domperidone was effective in reducing or preventing gastrointestinal disturbances during the dose - escalation phase of rivastigmine - patch therapy in ad patients with higher fssg scores at baseline . the fssg is a convenient tool for evaluating the symptoms of gerd ; in our experience , patients / caregivers require approximately 1 minute to complete the questionnaire . the questionnaire consists of twelve detailed items ; therefore , the slightest gastrointestinal problem can be easily detected . in order to obtain accurate information in the present study , we confirmed the symptoms described by the ad patients using fssg questionnaires completed by their caregivers . at present , there is no technique for predicting the occurrence of gastrointestinal disturbances with rivastigmine - patch therapy . it has been suggested that pretreatment with domperidone may protect against the onset of centrally induced nausea and vomiting induced by cheis.12 however , our findings indicate that ad patients with low fssg scores at baseline may not require additional antiemetic and/or antiulcer medications . on the other hand , ad patients with initial high fssg scores showed elevations in the fssg scores during the dose - escalation phase , indicating an increased frequency of gastrointestinal disturbances . therefore , clinicians should pay careful attention to ad patients with high fssg scores at baseline during the dose - escalation phase to prevent gastrointestinal disturbances and to avoid patient discontinuation of rivastigmine - patch therapy . domperidone has , on rare occasions , been associated with acute extrapyramidal syndromes;13 however , our ad patients did not show such side effects . randomized , double - blind clinical trials conducted with other cheis , such as donepezil and galantamine , have provided evidence that these are safe and effective therapies against cognitive dysfunction and behavioral symptoms.1416 however , similar to the side effects of rivastigmine - patch therapy , gastrointestinal disturbances are the primary adverse effects associated with this therapy . we speculate that the fssg may be a useful tool for predicting and preventing chei - related gastrointestinal disturbances for these drugs . this study did not include a placebo for domperidone , and was limited by the small sample size ; therefore , improvements in cognitive dysfunction with rivastigmine - patch therapy could not be demonstrated fully . it is important to optimize the drug for ad in individuals to slow the rate of cognitive dysfunction and behavioral and psychological symptoms of dementia.35,1720 of note , behavior symptoms in patients with ad increase direct costs of care . for example , a 1-point increase in the neuropsychiatric inventory score21 was calculated to be equivalent to an increased cost of $ 247$409 in total annual direct costs.22 in addition , compared with those who never used cognitive enhancers , patients who used cheis had a significant delay in nursing home admission.23 in summary , the group receiving rivastigmine - patch therapy without domperidone showed significant increases in the fssg score after 12 weeks ( dose - escalation phase ) compared with baseline scores . domperidone was effective in preventing rivastigmine patch - related gastrointestinal disturbances in ad patients with higher fssg scores at baseline . further , the fssg questionnaire may be a useful tool for the prediction of gastrointestinal symptoms associated with rivastigminepatch therapy .

the collected comments and pathologic diagnoses of several pathologists are summarized for 18 cases in which lesions were induced in the upper respiratory tract of rats and mice . specific neoplastic and nonneoplastic lesions of the nose and trachea are described and discussed , and opinions regarding pathogenesis and biologic significance of the lesions are presented . the anatomic and pathophysiologic complexities of the rodent nose in relation to lesion development following inhalation or systemic exposure to xenobiotics are important considerations in the genesis of pathologic changes in this organ.imagesplate 1.plate 2.plate 3.plate 4.plate 5.plate 6.plate 7.plate 8.plate 9.plate 10.plate 11.plate 12.plate 13.plate 14.plate 15.plate 16.plate 17plate 18plate 19plate 20.plate 21.plate 22.plate 23.plate 24.plate 25.plate 26.plate 27.plate 28.plate 29.plate 30.plate 31.plate 32.plate 33.plate 34.plate 35.plate 36.plate 37.plate 38.plate 39.plate 40.plate 41.plate 42.plate 43.plate 44.plate 45.plate 46.plate 47.plate 48.plate 49.plate 50.plate 51.plate 52.plate 53 .

low solubility causing low dissolution in gastrointestinal tract is the major problem for drugs meant for systemic action after oral administration , like cinnarizine . pharmaceutical products of cinnarizine are commercialized globally as immediate release preparations presenting low absorption with low and erratic bioavailability . approaches to enhance bioavailability are widely cited in the literature . an attempt has been made to review the bioavailability complications and clinical therapeutics of poorly water soluble drug : cinnarizine . the interest of writing this paper is to summarize the pharmacokinetic limitations of drug with special focus on strategies to improvise bioavailability along with effectiveness of novel dosage forms to circumvent the obstacle . the paper provides insight to the approaches to overcome low and erratic bioavailability of cinnarizine by cyclodextrin complexes and novel dosage forms : self - nanoemulsifying systems and buoyant microparticulates . nanoformulations need to systematically explored in future , for their new clinical role in prophylaxis of migraine attacks in children . clinical reports have affirmed the role of cinnarizine in migraine prophylaxis . research needs to be dedicated to develop dosage forms for efficacious bioavailability and drug directly to brain .

low aqueous solubility of drug has always presented major obstacle towards the development of drug delivery systems which often compromises patient compliance . oral route is thought to be common and easy for drug administration . on oral administration of drug in its dosage form , it is expected to dissolve and release the drug into the gastrointestinal fluid before the absorption . poor solubility may limit the dissolution of drug in gastrointestinal tract resulting to low bioavailability that can pharmacologically affect the therapeutic efficacy of drug . the drugs belonging to bcs class ii and iv particularly fall in this category and have been extensively researched molecular optimization and development of novel efficacious dosage forms . cinnarizine , ( e)-1-(diphenylmethyl)-4-(3-phenylprop-2-enyl ) piperazine ( figure 1 ) , molecular formula : c26h28n2 and molecular weight : 368.51 g / mol , is white or almost white powder . originally obtained from woodreed roots ( cinna ) , cinnarizine was first synthesized by janssen pharmaceutica in 1955 and marketed in 1958 under the brand name stugeron . according to a paper the solubility of cinnarizine is highly ph dependent , that is , 0.29 mg / ml at ph 2 , 0.017 mg / ml at ph 5 , and 0.002 mg / ml at high ph of 6.5 ( all the results were at 37c ) . pharmaceutically it belongs to bcs class ii , as displays preferential absorption in stomach ( table 1 ) . after oral administration , cinnarizine is rapidly absorbed ( tmax = 24 h ) from gastrointestinal tract , with an absorption window in upper gastrointestinal tract . on repetitive dosing in healthy human , accumulation of cinnarizine occurs due to its pharmacokinetic properties . the drug can cross the blood - brain barrier by simple diffusion , due to weakly basic and lipophilic . cinnarizine is oxidized by cytochrome p450 into metabolites such as 1-(diphenylmethyl)piperazine(c1 ) , 1(diphenylmethyl)-4-[3-(4-hydroxyphenyl)-2-propenyl ] piperazine ( c2 ) , and benzophenone(c3 ) 1-[(4-hydroxyphenyl)-phenylmethyl]4-(3-phenyl-2-propenyl)piperazine(c4 ) . among the various cyp enzymes , only cyp2d6 can catalyze p - hydroxylation of the cinnamyl phenyl ring of cinnarizine ( c-2 formation ) and only cyp2b6 can catalyze p - hydroxylation of the diphenylmethyl group ( c-4 formation ) of cinnarizine . on the other hand , cyp2c9 with cyp1a1 , -1a2 , and/or -2a6 have affinity for n - desalkylation at the 1- and 4-positions of the piperazine ring that results in c-1 and c-3 formation . it has high protein binding capacity of 91% . the drug is predominantly excreted in urine as metabolites and in feces mainly as unchanged drug with elimination half - life of 36 h. the pharmacokinetic aspects are also compiled in table 1 . these agents do not show action on slow calcium channels in myocardium but have selective action on arteries . the inhibitory effects of cinnarizine on the noradrenaline and ca - induced contraction of the rabbit thoracic aorta and mesenteric arteries were compared to papaverine . cinnarizine was found to block the ca - evoked contraction of the depolarized vessels but was less effective against the noradrenaline - induced contraction of the mesenteric arteries and even failed to antagonize the response of the thoracic aorta to noradrenaline . cinnarizine was effective in mesenteric arteries exposed to noradrenaline by blocking the ca0 dependent response , while papaverine was less effective towards the ca0 dependent response but efficiently inhibited the initial fast response more . cinnarizine was less effective or even failed to inhibit the response produced by thoracic aorta to nonadrenaline . these results suggested reduction of membrane permeability to extracellular calcium was the mechanism by which cinnarizine acted . the report also suggested that papaverine inhibits the contraction to less extent than that of cinnarizine . so have wide application in treatment of cerebral apoplexy , cerebral arteriosclerosis , and posttraumatic cerebral symptoms . according to a recent paper , the effect against vestibular vertigo was due to inhibiting potassium ( k ) currents , activated by heightened hydrostatic pressure on the hair cells , instead of blocking calcium channels . reports on inhibitory action on 5 h serotonin uptake by platelets are also available . its moderate antagonistic properties at dopamine d2 receptors are known to resemble the mechanism of action of most antipsychotic drugs . the active metabolite of cinnarizine , [ 1-(diphenylmethyl)-4-[3-(4-hydroxyphenyl)-2-propenyl ] piperazine , has higher affinity for d2-receptors than the parent drug that indicates significant role in inducing parkinsonism as adverse effect on chronic medication . additionally the drug was suggested to be used in cosmeceuticals as skin whitening agents for treatment of skin hyperpigmentation . clinically , cinnarizine is used for treatment of vertigo and prevention of motion sickness , due to its calcium channel blocking activity . vertigo indicates a sensation of false movement ( generally described like rotation ) and vestibular vertigo may be induced by increase in the endolymphatic pressure that activates pressure - dependent k currents ( ik , p ) in vestibular hair cells ; this modulates transmitter release . cinnarizine acts by enhancing the transmitter release in vestibular type ii hair cells by inhibition of ( ik , p ) , thus enhancing depolarization and increasing the voltage - dependent activation of ca currents . the therapeutics of cinnarizine has been proved employing clinical trials and the reports are compiled in table 2 . additionally , cinnarizine has evoked clinical interest for other disorders . consequently , recently summarized reports suggest clinical application of drug for migraine prophylaxis . on the other hand , cinnarizine is known to aggravate parkinson 's disease . when used in average dose of 150 mg daily , it can aggravate motor function in patients with parkinson 's disease ; however , the effect was reversible and disappeared upon withdrawal of drug . commercial cinnarizine is available as tablets and capsules usually administered 2 - 3 times a day . high dose should be used with caution in patients with hypotension , owing to the possibility of further decrease in blood pressure , and is contraindicated in pregnancy due to teratogenic and embryocidal effects on the foetus . the drug can only be prescribed if the potential benefit justifies the potential risk to the foetus . cinnarizine is widely marketed as combination medication with other drugs . in the indian domestic pharmaceutical market , its combination with domperidone is predominant , whereas combination with other drugs such as dimenhydrinate , dihydroergocristine , chlorcyclizine , phenylpropanolamine , and piracetam are available with overseas pharmaceutical companies . india leads in terms of commercialization of different brands of cinnarizine comprising approximately more than 50 brands including both drug in alone as well as in combination with other drugs . among all brands stugeron it has been accounted in research that bioavailability of cinnarizine is remarkably influenced by gastric acidity , displaying its ph dependent solubility and in turn dissolution . the marketed dosage forms account for low and erratic bioavailability , mainly due to the above - mentioned pharmacokinetic reasons . also , high dosing frequency is needed because of its short elimination half - life . being a weak base it is very soluble in acidic solutions and clinical absorption of cinnarizine was found to depend on the gastric acidity of patients . cinnarizine showed rapid dissolution in individuals having high gastric acid content ( ph = 1.2 ) , providing good absorption characteristics , whereas , in case of low - gastric acid content ( ph = 6 ) , reduction was observed in cmax and auc approximately 75 to 85% . literature retrieval on enhancement in solubility and bioavailability of cinnarizine highlighted various effective approaches since 1984 till date . in 1984 , tokumura et al . , in his report , claimed 30 times improved dissolution for inclusion complex of cinnarizine with -cyclodextrin than the drug alone , whereas no difference in bioavailability was observed . further working with the cinnarizine -cyclodextrin complex , tokumura with other research devised a method for improvement of bioavailability on simultaneous administration of proposed -cyclodextrin with competing agent . it was found that the penetration rate constant of cinnarizine in complex decreased which was restored on addition of competing agents . these agents compete with -cyclodextrin in complex formation , thus keeping more available free drug to penetrate the lipid barrier . in the same content , two papers were projected . one was based on the investigation of the competing ability of dl - phenylalanine to improve the bioavailability of cinnarizine . on simultaneous administration of dl - phenylalanine with cinnarizine -cyclodextrin complex , it was concluded that a minimum effective dose of 1 g of dl - phenylalanine might be required for every 50 mg of cinnarizine in -cyclodextrin complex . on the similar lines , the ability of l - leucine and l - isoleucine was also investigated for competing effect in gastrointestinal tract . as a result the cmax was increased to 2.7 and 1.9 times as compared to that of drug alone and its -cyclodextrin , respectively . conclusively , l - isoleucine showed stronger competing effect than l - leucine coupled with significantly improved bioavailability of the drug . in the next year , in 1987 , enhancement of bioavailability of cinnarizine as its oleic acid solution in hard capsule for oral administration was reported . use of lipid vehicle for oral administration of drug was ascertained to improve the bioavailability when compared to cinnarizine tablet . when investigated in beagle dogs , the cmax and auc for oleic acid solution of cinnarizine were remarkably improved by 2.9 and 4 times than that of tablet , respectively . the results were encouraging and somewhat similar to those obtained with simultaneous administration of -cyclodextrin and competing agent discussed above . in yet another report the -cyclodextrin derivatives , sbe4-beta - cd and 2-hydroxypropyl--cyclodextrin ( hp - beta - cd ) , were used to form complex with cinnarizine to enhance the oral bioavailability . the absolute bioavailability of solution or capsule containing drug alone was compared to -cyclodextrin derivatives complex of drug as a solution and in capsule . to assess the bioavailability , cinnarizine was administered to beagle dogs in the dose of ( and an amount equivalent in case of -cyclodextrin derivatives complex ) 25 mg orally and 12.5 mg / ml intravenously . the bioavailability of cinnarizine was 8 4% for solution of drug and 0.8 0.4% for capsule filled with drug alone . this was observed to be significantly increased to 5560% and 38 12% in case of -cyclodextrin derivative complex of drug formulated as suspension and that filled in capsules , respectively . the higher bioavailability achieved in case of cinnarizine -cyclodextrin derivative complex formulated as suspension than that filled in capsules was attributed to rapid dissociation of drug from its inclusion complex , thus causing improved absorption . based on this concept , a simple technique of preparing solid dispersion of drug by fusion method was explored . the purpose was served by the use of two lipid carriers separately , gelucire 44/14 ( gastric acid soluble ) and other compritol 888ato ( gastric acid insoluble ) . in case of gelucire 44/14 , the results suggested initial rapid release of drug which then sustained for over 5 h giving a release of approximately 90% , whereas compritol 888ato at a maximum ratio of 1 : 1 ( drug : compritol888ato ) more prolonged the release when compared to pure drug and commercial tablet supported the efficiency of solid dispersion to enhance the bioavailability . the drug being a weak base exhibits higher solubility in acidic environment of stomach and hence dissolution . on the other hand it displays poor dissolution in basic environment of small intestine . the inhibited drug release of drug in basic environment can be raised by incorporation of acidifiers in the formulation that create acidic environment . incorporation of acidifiers like citric acid or fumaric acid in matrix tablet to raise the solubility and hence ph independent drug release was also suggested . the drug release under the influence of acidifiers was examined by preparing three combinations , formulation containing only citric acid ( 70 mg ) or fumaric acid ( 70 mg ) separately and in combination with citric acid and fumaric acid ( 35 : 35 mg ) too . the acidifiers are able to reduce the ph of the intestinal and maintaining it for extended period of time , thus providing sustained drug release . the drug release was 57.3% and 53.5% after 8 h which increases to 75.2% and 66.2% after 12 h for citric acid and fumaric acid , respectively , whereas it was only 26.5% after 12 h. this shows better efficiency of citric acid for maintaining low ph environment . the lack point is depletion of acidifier from the tablet matrix , the rate of which is dependent on type of acidifier . hence , the result suggested that proper selection of acidifiers may give ph independent release profiles that in turn would lead to higher bioavailability . conclusively , the problematic issue of low and erratic bioavailability of poorly aqueous soluble drug , cinnarizine , was a topic of interest to researchers . the efforts were dedicated from the use of well known -cyclodextrin complex to some recent use of liquid cubic phases and acidifiers like citric acid and fumaric acid . additional use of competing agents ( such as dl - phenylalanine and l - isoleucine ) in combination with -cyclodextrin complex and lipid vehicles like oleic acid was proved to be advantageous and further increased the bioavailability of drug . on establishment of comparison between these mentioned techniques to that of cinnarizine in alone or its marketed preparations , the results were encouraging with remarkable enhancement in bioavailability of cinnarizine . various dosage forms / delivery systems have been approached to solve the solubility and bioavailability issue . these include fast dissolving tablets , nanoemulsions , microparticles , and as gastroretentive systems . to the best of our knowledge , here is the compilation of research reports , till date , on delivery related approaches to overcome the pharmacokinetic constraints of cinnarizine . critical analysis based on in vitro results revealed improved bioavailability of cinnarizine formulated as various dosage forms , when compared to marketed preparation . in contrast to in vitro results , the in vivo data for improvement in bioavailability is limited and efforts are needed to be made to assess the bioavailability and performance of cinnarizine in vivo . cinnarizine could serve as suitable candidate for fast dissolving tablet that may improve the bioavailability and patient compliance . the in vitro dissolution rate of granules was increased ( 8090% , 60 min ) on comparison with drug alone ( less than 50% , 80 min ) , because of the higher hydrophilic character imparted by hydrophilic carriers and reduction in drug crystallinity in case of granules . the report suggested the use of fluidized hot melt granulation method as an advantageous means of producing granules of cinnarizine with peg 6000 as a melt binder , which does not need solvents or water . chitosan based orodispersible tablet of cinnarizine was reported with the aim of improving absorption of drug which steps towards improved oral bioavailability . the study guided the use of chitosan based orodispersible tablet to be a valuable alternative in respect to use of expensive superdisintegrant . the prepared tablets have advantage of disintegrating within few seconds without need of water , which lead to enhanced absorption of drug . the disintegration time of optimized formulation was 11 sec giving 90% of drug release in a time period of 5 min . according to this report , the researchers assumed that if 90% of drug is released in the dissolution media in vitro , then it will exert full effect in vivo . so based on the results , the sufficient dissolution was assumed to enhance the absorption of drug that leads to improvise the bioavailability when assessed in vivo providing effective therapy . this ultimately aims towards improving patient compliance with main focus on children and elderly patients . developed fast dissolving tablet of cinnarizine using combination of superdisintegrants sodium starch glycolate ( ssg ) and croscarmellose sodium ( ccs ) and camphor as a subliming material . ssg = 12 mg , ccs = 12 mg , and camphor = 10 mg ) when compared to widely used commercial tablet ( stugeron ) was reported to be more effective with drug release of 99% in 16 min . in another report , fast dissolving tablets of cinnarizine were prepared by sublimation technique and regarded as effective alternative approach compared to use of expensive excipients . the precompressional evaluation revealed good free flowing properties within the prescribed limit . considering the pharmacopoeial test , the drug was uniformly distributed throughout the tablet with high drug content ( 101.5% ) . the subliming agents have directly proportional effect on the disintegration of tablets which further affect the drug release . the optimized formulation was reported to have t50% of 6.25 min and t90% of 19.74 min . it was stated that upon storage the disintegration of tablet was significantly decreased ( p < 0.05 ) . the report concluded the effectiveness of sublimation method as an alternative approach for fast dissolving tablet of cinnarizine to reach the optimum point in shortest time with minimum efforts . in yet another report , oral dispersible tablets were prepared by three different methods : effervescent , superdisintegrant addition , and sublimation method . the tablets showed total release of drug in a period of 6 h with total disintegration in 25.3 sec . the report concluded that the superdisintegrant addition method with l - hydroxypropyl cellulose ( 10% w / v ) was best to fulfill the aim . recently heer et al . developed fast dissolving orodispersible film and fast dissolving tablet of cinnarizine and assessed the effect of superdisintegrants . for this purpose , three different superdisintegrants , sodium starch glycolate , crospovidone , and croscarmellose sodium , were used . the tablet was prepared by direct compression method and solvent casting method was employed for preparation of orodispersible film . the two preparations were evaluated for weight variation , thickness , drug content , folding endurance , and tensile strength ( for orodispersible film ) . as a result , among three superdisintegrants , crospovidone at 4% w / w was proved to have promising superdisintegrant property required to prepare fast dissolving formulations . fast dissolving orodispersible film was concluded to be better over fast dissolving tablet in terms of faster drug release with better flexibility suggestive to patient compliant dosage form . lipid based drug delivery systems were topic of interest to scientist as novel drug delivery platform and well known to improve the oral bioavailability of poorly water soluble drug that belongs to bcs class ii and iv . the most interesting mechanism of enhanced bioavailability was regarded as improved absorption due to enhancement of gastrointestinal solubilisation . the factors that relate to bioavailability from lipid formulations include digestion of lipid , the mean emulsion droplet diameter , the lipophilicity of drugs , and types of lipids . the lipids are preferred for oral administration as they can be formulated in solution , suspension emulsions , self - emulsifying , and microemulsions . reported lipid based formulation of drug and studied the effect of lipid on drug absorption . the in vivo absorption study revealed higher bioavailability with long chain triglyceride than medium chained triglyceride , giving a value of 32.1 2.3% and 16.6 2.3% , respectively , but was independent of quantity of lipid administered . however , the report concluded that the bioavailability was observed to increase with lipid dose of 125250 mg but reached static point at 500 mg . improvement in solubility of drug in self - nanoemulsified drug delivery system was reported [ 43 , 44 ] . snedds containing combination of brij 97 ( 17.6% w / w ) and peg 400 ( 8.8% w / w ) revealed good bioavailability in vivo with an area under curve ( auc048 h ) of 773 148 ng ml h. a double peak phenomenon was observed for plasma profile . this helped in keeping cinnarizine solubilized even at lower part of gastrointestinal tract ; thus it is available for absorption for longer duration . it was observed that the biorelevant media greatly affected the type and size of droplets formed . upon dispersion in media containing low bile acid content , vesicles were formed , whereas it was micelle formation in presence of higher bile acid content . the resulted nanostructured composition influenced the solubilization capacity of composition which potentially affected the absorption of drug from resultant media . this research report on snedds suggested the approach to be suitable for better absorption of drug . again working with snedds , the researcher published another paper this time to assess the effect of increasing loading level of cinnarizine . three formulations were developed , sneddshigh , sneddsmedium , and sneddslow with 50 , 25 , and 12.5 mg of drug , respectively . the in vitro lipolysis at ph 6.5 showed that snedds with high concentration of cinnarizine leads to precipitation of drug , whereas it decreased in drug loading . there was no significant difference in auc , tmax , and cmax for the three formulations . the tmax was 1.8 0.14 h , 23 0.64 h , and 3 0.71 h after 1 g sneddshigh , 2 g sneddsmedium , and 4 g sneddslow , respectively . the auc obtained was 355 74 , 485 68 , and 392 52 ng / mlh for the three formulations in order of their increased drug loading , respectively . the report presented that , on oral administration , the drug loading level in snedds and amount of snedds vehicle did not affect the bioavailability . a lipid based liquid crystalline matrix of cinnarizine was prepared for sustained release delivery of drug which ultimately enhances the bioavailability . the lipids used were glyceryl monooleate and oleyl glycerate . in vitro lipolysis study suggested less susceptibility of oleyl glycerate to hydrolysis by pancreatic lipase than that of glyceryl monooleate . in order to investigate the improvement in bioavailability , cinnarizine was orally administered in the form of suspension or solution in glyceryl monooleate or olely glycerate . the bioavailability was more enhanced by administering cinnarizine with oleyl glycerate and appeared to be 344% ( aqueous suspension formulation assigned a reference bioavailability of 100% ) . the report concluded the efficiency of lipids that have an ability to form liquid crystalline structures in excess water , to be set forth as an application for oral sustained delivery of drug to enhance bioavailability . the sedds with 30% w / w of capmul pg-12 when evaluated in phosphate buffer , ph 7.2 , provided remarkable enhancement in drug solubility in phosphate buffer(s ) with rapid drug release . the sedds showed least solubility of drug to be 1500 m in phosphate buffer , ph 7.2 . it was concluded that the sedds prepared by combination of capmul pg-12 , tween 20 , and cremophor rh 40 can be a suitable alternative dosage form to fulfill the aim of the research that is to improve solubility and dissolution of drug . snedds of the drug was also reported with the aim of exploring the effect of type of lipid on solubility and drug release . proper balance between rapid and efficient self - emulsifying ability , higher solubility of drug , maintaining high amount of drug in solution after aqueous dispersion ( at least 85% ) , and lower droplet size upon aqueous dilution were the selection criteria for optimal formulations . the length of lipid chain significantly influenced the solubility of drug and long chain lipids showed higher solubility than medium chained . in terms of glyceride composition furthermore , the unsaturated fatty acids with long chain lipids resulted in higher solubility than the above two . snedds showed significantly better release in vitro in simulated gastric fluid ( sgf ) ( to mimic the in vivo condition more accurately ) than marketed preparation , stugeron . the snedds showed a release of 8495% after 15 min which was only 66% in case of stugeron in simulated gastric fluid . after 15 min of shifting into simulated intestinal fluid , the marketed preparation showed comparatively much higher drug precipitation ( 83% ) than the snedds which causes only 723% of drug precipitation . a paper was also reported to enhance the oral bioavailability of cinnarizine from its medium chain lipid solution formulation . lipid based formulation of cinnarizine was administered intraduodenally and perorally to rats in order to assess the contribution of dispersion and digestion in the stomach towards bioavailability . the difference in bioavailability was also observed for medium chain and long chain triacylglycerides owing to difference in the dispersion and digestion in the gastric and intestinal compartments . the difference between the bioavailability was large in case of medium chain lipid when compared to long chain lipid in case of both peroral and intraduodenal administration . the result suggested that the passage of medium chain lipid formulation through stomach led to critical gastric digestion than long chain lipid formulations . the report concluded that rapid transfer of medium chain formulation to intestinal mixed micelles possibly was achieved by efficient dispersion and partial digestion in stomach . also absorption in the upper intestine prior to drug precipitation was preferred . in yet another report the high solubility and stability of cinnarizine loaded emulsion ( cle ) had been proved . egg lecithin was used with the aim of minimizing drug degradation from both water and oil . the drug was localized in the interfacial lecithin layer of emulsion so as not to contact with oil or water medium . on further working with the cle , in next year the researchers provided reports for pharmacokinetics and tissue distribution of drug . when assessed in vivo in rats , the novel formulation ( cle ) revealed significantly higher auc and lower clearance and distribution volume . on i.v . administration of 2 mg / kg of cinnarizine loaded emulsion , the auc was observed to be 1879.556 g / lh , whereas it was 865.725 g / lh for aqueous cinnarizine solution prepared using tween 80 ( 1 g ) and 1,2 propylene glycol ( 10 g ) . the result revealed lower clearance ( 0.947 l / h / kg ) for drug loaded emulsion . consequently , circulation in blood stream achieved for longer duration resulted in better therapeutic effects . contributing to bioavailability of drug , researchers explored self - microemulsifying system ( smedds ) . smedds is known to spread readily in the gastrointestinal tract , and the agitation provided by digestive motility of stomach and the intestine results in self - emulsification . smedds containing oleic acid ( 16.66% w / w ) , tween 80 ( 55.55% w / w ) , and transcutol p ( 27.77% w / w ) improved the solubility and drug release when compared to marketed formulation of cinnarizine . in the report , the authors have explained formation of small sized particles , leading to rapid drug release of 81.96% in 5 min which slightly increases to 87.67% in 60 min . this suggested the potential use of smedds to improve oral bioavailability by overcoming the solubility issue . in addition to this , smedds of cinnarizine was prepared to characterize a better understanding of the mechanisms behind the precipitation of cinnarizine during in vitro lipolysis of a self - microemulsifying drug delivery system . in the period of in vitro lipolysis of the smedds with or without cinnarizine , dissolution was performed in biorelevant media and initially the dissolution rate of cinnarizine from pellets containing precipitated cinnarizine was 10-fold higher than dissolution from blank pellet spiked with crystalline cinnarizine , which increased to more than 50% drug dissolved in the first minute . pellets were further characterized by x - ray powder diffraction ( xrpd ) and polarized light microscopy ( plm ) . results indicated that either amorphous form or a molecular dispersion of cinnarizine resulted in precipitation during in vitro lipolysis of smedds , thus improving dissolution profile . recently snedds was also investigated to eliminate the effect of food on cinnarizine absorption . a nutritional drink , fresubin energy ( 200 ml ) , the snedds capsules or tablets in fed state were assessed for bioavailability by administering snedds equivalent to 50 mg of drug orally to male beagle dog ( 1013.5 kg ) with one week wash - out period between dosing . the dogs were fasted overnight for about 2024 h. the nutritional drink was administered 30 min prior to dosing . pentagastrin ( 6 g / kg ) was administered to dogs in order to control the gastric ph . the result also established the effect of food to extend the tmax to 2.5 times than fasted state and increased bioavailability for conventional tablets ( ffasted = 20.9 5.7 and ffed = 53.8 30.1 ) . in contrast to this the food condition did not affect the absorption of drug from snedds capsules and tablets , while tmax was observed to extend the absorption and thus enhanced the bioavailability . the fed state showed 2.5 and 3.3 times longer tmax than fasted state for snedds tablets and snedds capsules , respectively . the bioavailability of snedds capsules was higher at both fed ( 79.3 14.7 ) and fasted state ( 58.1 16.7 ) than that of snedds tablet ( ffed = 43.7 6.7 and ffasted = 32.7 11.5 ) . snedds formulation was observed to diminish the variation in absorption at fasted and fed conditions , leading to overall increase in bioavailability compared to conventional tablets . at the end , the conclusive points were drawn that food affects the absorption of bcs class ii drug causing increased bioavailability in fed state which can be significantly reduced by the use of snedds capsules and tablets . in the same context , another report was published in the same year to predict the performance of two above discussed lipid formulations ( snedds capsules and snedds tablets ) . the two formulations were compared with conventional tablets on the basis of gastrointestinal in vitro lipolysis model in both fasted ( ph = 2 ) and fed conditions ( ph = 6 ) . the in vitro lipolysis model was supposed to simulate the digestion in the stomach and intestine . to mimic the lipolysis events in fed state conditions , fat milk ( 3.5% ) was administered with 0.4% mg / ml of pepsin . at gastric step during fasted state , a high concentration of drug was found in aqueous phase , whereas it was lower for snedds tablet which can be explained on the basis of presence of alkaline excipients that lead to increase of the ph to 3.64 and cause lower solubility . the solubilization of drug remained constant for snedds formulations upon initiation of duodenal step suggested the role of lipid to enhance the solubilization of cinnarizine . the total amount of cinnarizine recovered from fasted state lipolysis was almost similar for all formulation with an average of 84 12% . on examining fed state condition , approximately 1% of cinnarizine from snedds was shown in aqueous phase in gastric step , whereas it was only 0.1% for conventional tablet . on passing into duodenal step , snedds formulations showed an aqueous cinnarizine content of 40% after 83 min and maintained it throughout the lipolysis , while the conventional tablet lost solubilization capability decreasing to 25.4 1.7% at the end . when compared to in vivo data provided by paper discussed above , these in vitro results were in accordance with in vivo . the order was similar as snedds capsules > snedds tablet > conventional tablet for performance in vitro and in vivo . more conclusively , the in vitro fasted state model was able to predict the correlation with in vivo study , whereas the fed state model can not exactly establish a correlation but was able to predict the best formulation similar to that predicted by in vivo study . in the path of lipid based nanosized delivery of cinnarizine , a report was published to stabilize the nanosuspensions by the utilization of bead layering . the nanosuspension of drug was coated on sugar beads by the use of fluidized bed pellet coater with wurster insert . these nanosuspensions showed complete dissolution in 15 min , whereas it was only 11% in 1 h for unmilled powder . the coated nanocrystals resulted in slower release of cinnarizine than that of original nanosuspensions due to formation of reagglomeration upon release from coating . the conclusive point suggested influence of drug physicochemical properties on in vitro dissolution after bead layering . utilization of lipids for pharmaceutical and biomedical applications was known to overcome the issues related to biocompatibility and biodegradability . in this respect the unique mesophasic structure of lipids such as cubic , hexagonal , and sponge phase structures provides advantage of stability and production feasibility . the cubic structure has advantage of improved stability , bioadhesivity , and biocompatibility , whereas the hexagonal arrangement exhibited hexasomes accounts for encapsulation of different drugs with high stability . the similar reports on cinnarizine are discussed further . the contribution towards improved absorption of cinnarizine in addition to this , another highlight of cubic phase is its temperature sensitivity in the range of 2037c suggesting it to be a putative material for the purpose of in situ gelation . the in vivo absorption study was performed on anaesthetized male sprague - dawley rats ( 280320 g ) . the result illustrated large difference in the rate of cinnarizine absorption from cubic phase and suspension . the absorption profile was more sustained and continued beyond 8 h after dose period for cubic phase , thus supporting its application to improve bioavailability . the cmax obtained for cubic phase was 26.2 ng / ml after 4.7 h , whereas it was 99.9 ng / ml after 2.2 h for suspension proving sustained absorption for former case . the report was the first to reveal the ability of liquid crystalline nanostructured particles ( cubosomes ) to attain sustained absorption of cinnarizine . these cubosomes were developed using phytantriol ( an alcohol ) and glyceryl monooleate ( a lipid ) . phytantriol cubosomes showed improved results with sustained absorption beyond 48 h. the oral bioavailability was improved to 21% compared to suspension ( f = 9% ) and oleic acid emulsion ( f = 12% ) . in such cases of nondigestible phytantriol cubosomes , the slow release can be facilitated by stomach that acts as a nonsink reservoir . thus , the report suggested the potential use of nondigestible liquid crystalline nanostructured particles as a novel approach for sustained oral drug delivery systems leading to improvise the bioavailability of poorly water soluble drugs . in the same year hexagonal liquid crystals ( hexosomes ) of cinnarizine were prepared with the aim of sustaining the absorption for increment in bioavailability . the pharmacokinetics was evaluated on oral administration of bolus lipid solution of drug in selachyl alcohol to rats . the results revealed that , after oral administration , sustained plasma profile with 2040 ng / ml of drug maintained over the first 24 h was observed . when compared to suspension , the tmax was longer for hexosomes ( 1 h and 23.5 5.9 h resp . ) . the absolute oral bioavailability was improved for hexasomes ( 17% ) compared to that of suspension ( 9% ) . selachyl alcohol is not susceptible to digestion which attributed to prolong the absorption as similar to nondigestible phytantriol cubic system discussed above . the report concluded the potential use of nondigestible liquid crystalline systems with additional advantages of nanostructured particles as novel system for sustained drug delivery . the research reported two preparations : one was a buoyant tablet containing powdered soyabean , drug , and sodium bicarbonate and the other was laminated floating film comprising of a drug film , an effervescing film of sodium bicarbonate , and an outer drug release regulating film . the in vitro studies assured the suitable floating ability and sustained release property of the two formulations . linear dissolution was observed after 2 h in case of laminated film not similar to tablet due to structural difference . the absorption study was done by oral administration of buoyant tablet and film in beagle dogs . as a result , cinnarizine was observed in blood even 24 h after oral administration , concluding the efficiency of the two preparations as sustained delivery systems so as to upgrade the absorption and bioavailability of cinnarizine . floating microballoons were prepared by diffusion solvent evaporation technique using 2 factorial design to improve bioavailability . was performed in ph 1.2 for 8 h and then in ph 7.4 for 2 h. microballoon released approximately whole drug during 10 h period with sufficient buoyancy . the microballoons prepared with combination of eudragit s100 and eudragit rl in a ratio of 1 : 3 proved most effective results , suggesting the efficient use of microballoons for oral sustained delivery of cinnarizine . the sustained drug release was achieved due to rapid hydration of polymer on the surface of floating tablet , thus forming a gel layer surrounding the matrix . this controls penetration of water to the center of matrix tablet and retards the release . it was reported that , with increase in viscosity of polymer , drug release rate was increased . while studying the release rate with different polymers , it was also revealed that the drug release rate depends on the surface area as well as hydration capacity of polymer . based on these results , the optimized formulation containing hpmc k100lv ( 90 mg ) was compared ( in vivo ) to oral suspension and bioavailability was studied . the absorption phase was slow and prolonged in case of matrix tablet , thus indicating prolonged plasma concentration . the overall bioavailability of floating matrix tablet was 2.85 times greater than that of oral suspension . in 2012 , floating tablet of cinnarizine in combination of domperidone maleate was developed for gastroretentive delivery . the ph independent polymer , methocel polymer of different viscosity grades , was used at concentration of 2050% w / w to prepare floating tablets . the formulation was reported to remain buoyant for 12 h with lag time of 1015 sec in 0.1 n hcl . sodium bicarbonate ( 15 mg ) and sodium alginate ( 18 mg ) were used as gas generating agent necessary to impart sufficient buoyancy . so the report suggested improved retention of tablet of combination of two antiemetic drugs at gastric ph that was supposed to have improved therapeutic effects . multiparticulate systems reported as gastroretentive floating microspheres were developed to assess the effect of formulation variables on microsphere formation . microspheres were developed using acrycoat s100 , eudragit rs100 , and ethyl cellulose as gastroretentive polymers . when assessed in vitro in 0.1 n hcl , the microspheres remained buoyant for 12 h. among these , microspheres containing acrycoat s100 ( 100 mg ) showed best results for sustained drug release ( 98.14% ) , floatability ( 72% ) in 12 h , and stability . another effort to sustain the release of cinnarizine was attained by preparation of drug loaded chitosan methylcellulose interpenetrating polymer network ( cs / mc ipn ) microspheres intended for mucoadhesive gastroretentive application . the study explores the interpenetrating network of two biopolymers , chitosan and methylcellulose , to prolong the residence of microspheres in the stomach . the microspheres were prepared using central composite design at three different levels of polymeric ratio and glutaraldehyde . the formulations were evaluated for degree of mucoadhesiveness which was correlated to amount of polymer and interpenetrating network of polymers . for optimized formulation the percent of mucoadhesion was 73.85 2.78 at 4 h with t50 of 432.21 26.15 min . the other observed results were for mean diameter ( 61.32 1.38 m ) , swelling index ( 2.38 0.06 ) , and entrapment efficiency ( 84.13 1.32% ) . the report suggested the utilization of chitosan methylcellulose interpenetrating polymer network ( cs / mc ipn ) as potential approach to impart mucoadhesive property for gastroretentive delivery of weakly basic drugs . recently in the past year , patel et al . prepared mucoadhesive microparticles by supercritical fluid method using chitosan as mucoadhesive polymer . the efficiency of this physical coating process for producing microparticles was established by x - ray powder diffractometry and differential scanning calorimetry . the prepared microparticles were proposed for better bioavailability than the suspension in test animal due to gastroretention . the comparison was done in vivo by administering both suspension and mucoadhesive microparticles to rabbit in fasted state . the mucoadhesive microparticles showed sustained drug release for more than 20 h. good mucoadhesive properties and strong adherence of microparticles to gastric mucus layer were established both in vitro and in vivo , resulting in gastroretention for an extended period of time . these formulations accounts for sustained effect of drug reducing the dosing frequencies . in the same year mucoadhesive tablets of cinnarizine were developed using eudragit rlpo as polymer with the aim of investigating the effect of iron oxide in the formulation . the mucoadhesive tablets were prepared at different concentrations of eudragit rlpo , iron oxide , and pvp k 30 and evaluated for mucoadhesive strength , t50% , t90% , and mean dissolution time effect of iron oxide was investigated . the result clarifies the synergistic effect of combination of eudragit rlpo , iron oxide , and pvp k 30 to enhance the mucoadhesive strength when compared to their individual effect . as per the results the mucoadhesive tablets containing 8.58% w / w eudragit rlpo , 6.68% w / w iron oxide , and 7.86% w / w pvp k 30 were selected to be optimized and fulfilled maximum requirements with better mean dissolution time ( 266.44 3.82 min ) , t50% ( 124.94 5.40 min ) , t90% ( 755.53 4.29 min ) , and desirability of 0.727 . the incorporation of iron oxide was found to enhance the mucoadhesive strength due to induction of ionic interaction . retardation of drug release with mean dissolution time of 229.56 3.30 min at higher concentration of iron oxide was achieved due to reduction in polymeric chain disentanglement and polymer erosion caused by the entrapment of iron oxide in the polymeric chain network . a novel approach of gastroretention with controlled release was recently negotiated by preparing an unfolding type film of cinnarizine . the polymeric film was procured by solvent casting method using ethyl cellulose and hydroxyl propyl methyl cellulose k15 and folded in hard gelatin capsule . as obtained by in vitro drug release study , the film describes fast release of drug in the first hour due to initial leaching out of drug , which was followed by slow release by diffusion through hydroxyl propyl methyl cellulose and ethyl cellulose providing more steady controlled release giving total release of 81.93% in 13 h. the result also suggested more appropriate use of unfolding film in simulated gastric fluid when compared to roll folded pattern . the research strongly indicated the gastroretentive potential of prepared dosage form which can be explored further for in vivo studies . cinnarizine is known to precipitate at high ph on gastric emptying and show slow and erratic bioavailability . microparticles prepared using ph responsive polymer eudragit l by emulsion solvent evaporation method showed improved bioavailability . the microparticles inhibited the in vitro release of drug under gastric conditions due to high threshold ph 6 . at favorable intestinal conditions , this diminishes the impact of drug precipitation and improved the absorption . in vivo comparison with drug powder suspension was done by oral dosing in rats and eudragit l ( 3 g ) loaded microparticles showed twofold increase in bioavailability , although c(max ) and t(max ) were not significantly different . despite wide therapeutic applications and worldwide commercialization of cinnarizine , the marketed formulation accounts for low and erratic bioavailability , due to gastric acidity dependent absorption . earlier complexation techniques were practiced but now recently developed dosage forms such as snedds , gastroretentive systems , are in interest . lipid based systems emerged with improved solubility and dissolution in vitro ( 8095% ) and in vivo bioavailability ( auc approximately 4 times higher than tablets ) . the purpose to circumvent the obstacles associated with drug was also achieved by the preparations aimed to longer retention at gastric environment . these provided new achievements for sustained release of cinnarizine , so as to reduce dosing frequencies and attain patient compliance . use of nanosized liquid crystalline particles ( cubosomes ) can be proved as new field of interest for sustained absorption of cinnarizine . patient compliance is the ultimate aim for any pharmaceutical dosage form . for the control of drug release or sustained benefits for patients , the microsized particles , microballoons , microspheres , and microsponges alone or in combination with other approaches like use of bioadhesive polymers or coating materials to facilitate intimate adherence to gastric mucosa and incorporation of acidifiers in order to improve ph dependent solubility of cinnarizine , might result in the availability of new product with improved potential for the therapeutics of cinnarizine . cinnarizine solubility is highly dependent on gastric acidity , so ph controlled systems can be proposed to provide constant rate release , irrespective of location of dosage form in the git . release of cinnarizine from its dosage form independent of environmental conditions can also be achieved by designing osmotically controlled gastroretentive drug delivery systems which can further be modified by coating external surface with some erodible polymer to provide alternate chances of improvement and must be assessed further . use of lipid based systems as gastroretentive systems can set forth for the new era of overcoming the pharmacokinetic constraints of cinnarizine . tablets and capsules formulated using self - emulsifying drug delivery system containing droplets size extending from few nanometers to microns can be developed , while sustaining good flowability , cohesive property , and high content uniformity . methods by which a transiently supersaturated state can be generated and maintained in situin the gi tract can be set forth for development . in this context , novel formulation approach of use of polymeric precipitation inhibitors or retarders like hydroxyl propyl methyl cellulose incorporated into self - nanoemulsifying delivery systems to produce a supersaturated drug concentration and prolong such a drug concentration for an extended period of time for an optimal absorption , thereby improving oral bioavailability , can be set forth for assessment for future development of cinnarizine . the various techniques discussed herein can be better option for companies to focus on commercializing them .

background : disease prevention is better than its cure . the role of healthcare worker s hand in the transmission and spread of an infectious disease to the patient is well acknowledged . indeed , the hands of a health care worker can easily pick potentially pathogenic bacteria and fungi from hand touch surfaces before wearing of gloves . for these microorganisms to multiply rapidly , a moist environment present underneath the gloves acts a good cultivating media . it is also reported that the multiplication rate also increases several folds with the duration of glove use.materials and methods : dentists 20 with rings and 20 without rings were considered . skin samples from the hand soon after professional hand cleaning and glove disposal were collected . the occurrence of potentially pathogenic fungi and bacteria were examined and investigated disposal were collected . the occurrence of potentially pathogenic fungi and bacteria were examined and investigated with biochemical and cultural laboratory tests.results:bacteria and fungi were significantly more frequent in dentist s hand with rings than those without rings . 63% versus 37% ( bacterial prevalence ) , among the isolated potentially pathogenic microorganisms were staphylococcus aureus , escherichia coli , and candida albicans.conclusion:in the present study potentially pathogenic microorganisms were more frequent in dentists who wore finger rings under gloves .

health - care associated infections ( hcais ) are one among the major health related hazards and account for a major global disease burden . they pose a health threat toward patients and health care workers . these have a global impact and pose a disease burden to millions of people worldwide . these promote resistance to antibiotics , complicate the delivery of patient care and also lead to an additional monetary expenditure to the and also lead to additional monetary expenditure to the patients with underlying pathology . hospital - acquired infections account for more than 1.4 million people worldwide.1 health - care delivery exposes the hospital workers toward infections such as tuberculosis , needle stick injuries , and hiv . the practice of hand hygiene ( hh ) has long been considered as a gold standard and has passed the test of time as an infection control measure successfully preventing hcais . the single most important preventive measure against hcai is to practice proper hh technique by either washing the hands with soap or with a disinfectant , but this practice is usually given minimal priority by the hospital staff.2 in general , health care workers are not carriers , but vectors . centers for disease control and prevention defines the clinical contact surfaces as the surfaces that can be contaminated directly from patient materials either by direct spray or the generated splatter during dental procedures or with contact of dental health care provider s gloved hands . examples of such surfaces include light and door handles , switches , dental x - ray equipment dental chair , pens , telephone , door knobs , etc . contamination of these surfaces is frequently reported , but the risk of infection for patients and the dental staff is not yet , determined with high accuracy . dental healthcare workers skin could transiently carry potential pathogens coming from the environment and the use of gloves does not automatically eliminate the need for hand washing , because gloves can have unapparent defects or can be torn during use , due to sharp equipment , widespread in dental healthcare . in addition , the opportunistic pathogens may multiply at a very rapid rate in a moist environment that is usually present underneath the gloves . an aspect associated with microbial contamination of gloved hands is the use of finger rings . indeed , rings can make glove donning difficult with consequent damage and loss of integrity . a large number of studies are there establishing the association of hcais with improper hh , but only a handful of studies have demonstrated that the skin underneath finger rings is more enormously colonized than other comparable areas in the finger skin . however , whether wearing of the finger rings increases the likelihood of pathogen transfer is still unknown.3 a significance level of microbial contamination on the skin under ringed finger is also least examined . hence , the present study was done to investigate the occurrence of potential pathogenic bacteria and fungi on skin of gloved hands of dental health care workers who wore finger rings with those who did not wore finger rings under gloves . the sample consisted of 40 dentists working in the department of oral medicine diagnosis and radiology , career post graduate institute of dental sciences ( cpgids ) , lucknow . this study was conducted in compliance with the protocol ; written ethical approval was taken from the head of department of oral medicine and also from the principal of cpgids , lucknow . skin samples were collected in the middle of the working day from the dominant hand soon after glove removal and professional hand cleaning . sterile swabs previously moistened with tubes containing 1 ml of sterile saline solution ( 0.9% w / v nacl ) were gently rubbed over the complete ventral surface of the hand and around the periphery of finger ring sample . swabs were immediately transferred onto the plates containing mannitol salt agar and sabouraud dextrose agar plates were incubated aerobically at 28c and 37c for 48 h ( figures 1 and 2 ) . isolates recovered from cultures were preliminarily subjected to microbiological procedures and were identified using biomerieux equipment , uk . the laboratory procedures were made by courtesy of the department of microbiology , career medical college , lucknow . prevalence of potential pathogenic microorganisms with and without rings was assessed and differences were statistically analyzed . this study was conducted in compliance with the protocol ; written ethical approval was taken from the head of department of oral medicine and also from the principal of cpgids , lucknow . skin samples were collected in the middle of the working day from the dominant hand soon after glove removal and professional hand cleaning . sterile swabs previously moistened with tubes containing 1 ml of sterile saline solution ( 0.9% w / v nacl ) were gently rubbed over the complete ventral surface of the hand and around the periphery of finger ring sample . swabs were immediately transferred onto the plates containing mannitol salt agar and sabouraud dextrose agar plates were incubated aerobically at 28c and 37c for 48 h ( figures 1 and 2 ) . isolates recovered from cultures were preliminarily subjected to microbiological procedures and were identified using biomerieux equipment , uk . the laboratory procedures were made by courtesy of the department of microbiology , career medical college , lucknow . prevalence of potential pathogenic microorganisms with and without rings was assessed and differences were statistically analyzed . a cross - sectional study was conducted to measure and identify the microbial contamination isolated from the skin under rings and on skin without rings and to compare the results among volunteered dentists of cgpids and hospital , lucknow , india . all dentists were males aged between 21 and 33 years ( mean 27 years ) , equally divided into ( n = 20 ) with finger rings ( n = 20 ) without finger rings . all the estimated values of prevalence ratio ( pr ) were quite high , ranging between lower limit of 2.03 and the upper limit of 4.74 , but were statistically not significant at 95% of level excluding the pr value for fungi ( table 1 ) . occurrence of potential pathogens in skin samples from hands with and without finger rings of dentists . the potentially pathogenic bacteria , which were isolated were staphylococcus aureus , staphylococcus epidermidis , escherichia coli , enterococcus spp . , while fungi were candida albicans , aspergillus niger , and aspergillus flavus . excluding s. aureus which was isolated only in one non - ring wearing dentist , all these microorganisms were more frequent in ring wearing dentists ( table 2 ) . list of potential pathogenic bacteria and fungi isolated from skin samples of dentists hand with and without finger rings ( prevalence values between brackets ) . the statistical analysis was done by applying systat version 10 by cranes software , bangalore , india in the study . the prevalence of potentially pathogenic microorganisms with and without finger rings was assessed and differences were statistically analyzed using 2 test with yates correction for continuity . the pr , with 95% of confidence interval and a level of significance of 95% was selected to statistically analyze the data . bacteria were non - significantly more frequent in the dentist s with ringed finger ( 63% vs. 37% ; p = 0.06 ) . while fungi were significantly more frequent in the dentist s with a ringed finger ( 79% vs. 21% ; p = 0.0002 ) ( table 1 ) the statistical analysis was done by applying systat version 10 by cranes software , bangalore , india in the study . the prevalence of potentially pathogenic microorganisms with and without finger rings was assessed and differences were statistically analyzed using 2 test with yates correction for continuity . the pr , with 95% of confidence interval and a level of significance of 95% was selected to statistically analyze the data . bacteria were non - significantly more frequent in the dentist s with ringed finger ( 63% vs. 37% ; p = 0.06 ) . while fungi were significantly more frequent in the dentist s with a ringed finger ( 79% vs. 21% ; p = 0.0002 ) ( table 1 ) potentially pathogenic bacteria , which were isolated were s. aureus , e. coli , while fungi were c. albicans . excluding s. aureus which was isolated only in one non - ring wearing dentist , all these microorganisms were more frequent in ring wearing dentists . dentists who wore rings were nearly twice more likely to harbor potential pathogenic bacteria ( pr , 2.13 - table 1 ) and almost 5 times more likely to harbor fungi ( pr , 4.74 - table 1 ) than those dentists who did not wore finger rings . in a previous study on 84 nurses working in intensive care units in a rural indian those who wore rings showed more gram - positive and gram - negative bacteria than those who did not earrings , while no difference was found between nurses wearing plane wedding rings and those wearing rings with stones.4 in a sample of 20 veterinary medical students , 10 wearing plain rings , wearing rings , no difference in viable flora was detected between ring and non - ring hands.5 in a sample of 200 healthcare workers from an intensive care unit , ring wearers showed almost double prevalence of gram - negative bacteria than non - wearers , but no difference with respect of s. aureus.6 similar results have been reported from other studies conducted by hoffman et al.,7 field et al.8 in which bacterial significantly high in ringed hands compared with control hands . in an attempt to summarize these data , we could speculate that ring wearing is associable with a higher level of bacteria and fungi , including potential pathogens . such a high contamination level could be due to the lower effectiveness of hh in ring wearers than in non - wearers . in addition , the residual and relatively high level of contamination , due to inefficient hygiene , will increase during the working day , because of bacterial and fungal multiplication on the warm and moistened environment of skin beneath gloves . every study has some or the other kind of limitations , which need to be recognized . for the present study , a small sample size and all the samples from same department so some of the reported differences between dentists who wore and did not wear rings could be due to chance , which still leaves scope for further studies . clean care : safer care is the globally accepted slogan raised as a first line of defense in the global patient safety challenge , which is a core component of who s world alliance for patient safety launched in the academic year 2004.9,10 the results of the present study establish the association of ringed finger with increased microbial contamination . hence , it shows the need that a major focus must be there toward the improvement in hh to prevent hcai worldwide . the three recommended strategies to minimize hcais are as follows : generation of awareness campaigns toward prevention of hcais like the staff involved in hospital hygiene activities must be included in education and training related to the prevention of hospital - acquired infectionleadership , commitment and transparency of high standards must be practiced like the hands must be decontaminated immediately before and after every episode of direct patient contact / care . all wrist watches and hand jewelry must be removed at the beginning of each clinical shift beforehand decontamination begins . abrasions and cuts must be completely covered with a waterproof dressing . application of an alcohol - based hand rub or washing hands with a liquid soap and water to decontaminate hands between caring for different patients or between different caring activities for the same patient must be practicedhigh - level reinforcement of the proposed strategies must be examined by frequent surprise audits by the public health specialists at local / national levels . generation of awareness campaigns toward prevention of hcais like the staff involved in hospital hygiene activities must be included in education and training related to the prevention of hospital - acquired infection leadership , commitment and transparency of high standards must be practiced like the hands must be decontaminated immediately before and after every episode of direct patient contact / care . all wrist watches and hand jewelry must be removed at the beginning of each clinical shift beforehand decontamination begins . abrasions and cuts must be completely covered with a waterproof dressing . application of an alcohol - based hand rub or washing hands with a liquid soap and water to decontaminate hands between caring for different patients or between different caring activities for the same patient must be practiced high - level reinforcement of the proposed strategies must be examined by frequent surprise audits by the public health specialists at local / national levels . clean care : safer care is the globally accepted slogan raised as a first line of defense in the global patient safety challenge , which is a core component of who s world alliance for patient safety launched in the academic year 2004.9,10 the results of the present study establish the association of ringed finger with increased microbial contamination . hence , it shows the need that a major focus must be there toward the improvement in hh to prevent hcai worldwide . the three recommended strategies to minimize hcais are as follows : generation of awareness campaigns toward prevention of hcais like the staff involved in hospital hygiene activities must be included in education and training related to the prevention of hospital - acquired infectionleadership , commitment and transparency of high standards must be practiced like the hands must be decontaminated immediately before and after every episode of direct patient contact / care . all wrist watches and hand jewelry must be removed at the beginning of each clinical shift beforehand decontamination begins . application of an alcohol - based hand rub or washing hands with a liquid soap and water to decontaminate hands between caring for different patients or between different caring activities for the same patient must be practicedhigh - level reinforcement of the proposed strategies must be examined by frequent surprise audits by the public health specialists at local / national levels . generation of awareness campaigns toward prevention of hcais like the staff involved in hospital hygiene activities must be included in education and training related to the prevention of hospital - acquired infection leadership , commitment and transparency of high standards must be practiced like the hands must be decontaminated immediately before and after every episode of direct patient contact / care . all wrist watches and hand jewelry must be removed at the beginning of each clinical shift beforehand decontamination begins . application of an alcohol - based hand rub or washing hands with a liquid soap and water to decontaminate hands between caring for different patients or between different caring activities for the same patient must be practiced high - level reinforcement of the proposed strategies must be examined by frequent surprise audits by the public health specialists at local / national levels . the challenges are enormous , but so are the reward : preventing illness , saving lives , improving patient safety , and providing an overall better quality of care to millions of patients and families . hcai are unintended , undesirable , and intolerable , but many are preventable . it is a time that hh promotion should be made a priority for public health and health care policymakers , medical and nursing schools , chief medical , and executive officers . the improvement in hh is feasible , affordable , and effective in a healthcare setting with limited resources . the who strategy represents evidence - based , ready - to - use solutions for planning and supporting hh promotion in healthcare facilities worldwide , including developing countries . however , the adoption of such a strategy on a national scale is the need of hour for patient s safety and control of hcai s . within the limitations of the present study , the general conclusion is that the potential pathogens are more likely detected in the hands of dentists who wore rings . although more such evidence is required before establishing guidelines regarding the wearing of finger rings during clinical procedures , but the results of this study adds to the evidence that wearing of finger ring can be a source of microbes and warrant more studies .

resistance to fluoroquinolones ( flq ) and second - line injectable drugs ( slid ) is steadily increasing , especially in eastern european countries , posing a serious threat to effective tuberculosis ( tb ) infection control and adequate patient management . the availability of rapid molecular tests for the detection of extensively drug - resistant tb ( xdr - tb ) is critical in areas with high rates of multidrug - resistant tb ( mdr - tb ) and xdr - tb and limited conventional drug susceptibility testing ( dst ) capacity . we conducted a multicenter study to evaluate the performance of the new version ( v2.0 ) of the genotype mtbdrsl assay compared to phenotypic dst and sequencing on a panel of 228 mycobacterium tuberculosis isolates and 231 smear - positive clinical specimens . the inclusion of probes for the detection of mutations in the eis promoter region in the mtbdrsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing ( 96% and 95.4% , respectively ) than that seen with the original version of the assay , whereas the test sensitivities for detection of flq resistance remained unchanged ( 93% and 83.6% for direct and indirect testing , respectively ) . moreover , mtbdrsl v2.0 showed better performance characteristics than v1.0 for the detection of xdr - tb , with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing , respectively . mtbdrsl v2.0 thus represents a reliable test for the rapid detection of resistance to second - line drugs and a useful screening tool to guide the initiation of appropriate mdr - tb treatment .

resistance to fluoroquinolones ( flq ) and second - line injectable drugs ( slid ) is becoming more common , especially in eastern european countries , posing a serious threat to effective tuberculosis ( tb ) infection control . according to the most recent world health organization ( who ) report , extensively drug - resistant tb ( xdr - tb ) had been reported by 100 countries by the end of 2013 . on average , an estimated 9.0% ( 95% confidence interval [ ci [ , 6.5% to 11.5% ) of people with multidrug - resistant tb ( mdr - tb ) have xdr - tb ( 1 , 2 ) . to date , only 8 countries among the 36 with a high tb and/or mdr - tb burden have established a national surveillance system for resistance to second - line drugs among patients with mdr - tb ( 1 , 2 ) . efforts should be made to ensure that all patients diagnosed with mdr - tb undergo testing of susceptibility to flq and slid in order to initiate early effective treatment and appropriate measures of infection control . conventional phenotypic methods take weeks to months to fully define the drug resistance profile of mycobacterium tuberculosis isolates due to the low growth rate of the bacterium ( 3 , 4 ) . the development and implementation of rapid molecular assays for the detection of resistance to anti - tb drugs promise more - rapid drug resistance detection , which can be critical in areas with high rates of mdr - tb and xdr - tb and settings with limited conventional dst capacity . in contrast , the choice of molecular assays for second - line drugs is far more limited . to date , genotype mtbdrsl ( hain lifescience , nehren , germany ) is one of the few commercially available molecular tests for detection of resistance to the main second - line anti - tb drugs . it is a qualitative test for the identification of mycobacterium tuberculosis complex ( mtbc ) and its resistance to flq , to aminoglycosides and cyclic peptides ( ag / cp ) , and to ethambutol ( emb ) as either clinical isolates or pulmonary smear - positive clinical specimens ( 57 ) . diagnosis of flq resistance is enabled by the detection of mutations in the quinolone resistance - determining regions ( qrdr ) of the gyra gene ( coding for the a - subunit of the dna gyrase ) . resistance to ag / cp is detected by targeting a region of the 16s rrna gene ( encoded by the rrs gene ) , whereas identification of mutations in the embb gene enable detection of emb resistance . several studies have been conducted to evaluate the test performances in different settings and have demonstrated generally good performance characteristics and high confidence for resistance results ( 2 ) . however , sensitivities varied significantly among studies depending on the genetic background of the strains and were frequently suboptimal for kanamycin ( kan ) , in particular , with eastern european strains ( 810 ) . in eastern europe and russia , where the beijing subfamily is largely diffused , assays using the rrs gene alone failed to detect a large proportion of kan - resistant isolates ( 11 ) . in line with this observation , huang et al . showed that the low sensitivity of the genotype mtbdrsl assay was due to the presence of specific mutations not detected by the test , located in the eis gene promoter region ( 12 ) . polymorphisms in the eis promoter region lead to an increased synthesis of the enzyme and the subsequent inactivation of the drug ( 13 ) . these mutations are responsible for more than 80% of low - level kan - resistant cases ( 1315 ) , making this genetic region a valuable molecular marker of kan resistance . on the basis of published evidence , the who decided in 2013 not to endorse the second - line line probe assay ( lpa ) given that , with the test 's low pooled sensitivity , negative results can not be considered to reliably rule out resistance . the who expert group thus concluded that the assay can be used only as a method to rule in diagnosis of xdr - tb but not as a replacement for conventional phenotypic dst to rule out resistance ( 16 ) . to improve the overall performance of genotype mtbdrsl , and in particular its sensitivity for kan resistance , a new version of the assay ( i.e. genotype mtbdrsl v2.0 includes two new target genetic regions : the eis promoter region 10 to 14 ( in addition to rrs ) and the gyrb quinolone resistance - determining region ( qrdr ) ( in addition to gyra ) for the detection of mutations at codons 536 to 541 . mtbdrsl v2.0 thus includes a total of 27 probes for the detection of resistance exclusively to second - line drugs . we have conducted an international study to assess the performance of the new version of the genotype mtbdrsl assay for detection of resistance to flq and slid . the performance of the test was evaluated by comparison to dst and sequencing using a panel of 228 clinical isolates and 231 clinical specimens from subjects referred to the different study sites ( three tb supranational reference laboratories [ srl ] in europe : srl milan , italy ; srl solna , sweden ; and srl borstel , germany ) from countries with a high mdr - tb prevalence . a collection of mtbc clinical isolates and specimens obtained from the national and supranational reference laboratories involved in the fp7 project pan - european network for study and clinical management of drug - resistant tuberculosis ( tb pan - net ) ( funded by a european union grant ) were considered for the evaluation of the test performances . the collection includes strains isolated in italy and sweden and clinical specimens collected in italy , germany , and moldova . all tests were performed at a tb supranational reference laboratory ( srl ) ( srl milan , milan , italy ; srl solna , solna , sweden ; or srl borstel , borstel , germany ) . the study included 228 mycobacterial strains isolated on liquid ( bbl mgit ; bd , franklin lakes , nj , usa ) or solid ( bbl lwenstein - jensen ; bd , franklin lakes , nj , usa ) media and identified according to standard biochemical test and sequencing , as previously recommended ( 3 ) . all mtbc strains were phenotypically resistant to rifampin ( rif ) and isoniazid ( inh ) , harboring different patterns of mutations in rpob and katg or inha genes . the profiles of susceptibility to second - line drugs were determined by conventional phenotypic testing . two different panels were tested and analyzed independently , one that included 128 samples at srl milan , milan , italy , and another that included 100 samples at srl solna , solna , sweden . dna was obtained from 231 clinical specimens from patients with pulmonary mdr - tb during standard clinical management who were referred to the different study sites ( srl milan , milan , italy ; srl borstel , borstel , germany ; and nrl minsk , minsk , belarus ) for retesting . all respiratory specimens were classified as acid - fast bacillus ( afb ) positive by sputum smear microscopy . samples were decontaminated according to international guidelines using an n - acetyl - cysteine naoh procedure ( 17 ) . the drug susceptibility profile of the corresponding strains was obtained using a conventional phenotypic assay according to international guidelines . the development and validation of the mtbdrsl v2.0 assay were approved by the ethical committee of the san raffaele hospital given the role of srl milan as the coordinating institution . determinations of susceptibility to flq ( ofloxacin , levofloxacin , and moxifloxacin ) and slid ( amikacin , kanamycin , and capreomycin ) were performed either on a bactec mgit 960 system ( mgit ) ( bd bioscience , erebodegem , belgium ) or on lowenstein - jensen ( lj ) media using the proportional method according to international guidelines ( 18 ) . the dst critical concentrations used for mgit are as follows : for ofloxacin , 2 g / ml ; for levofloxacin , 1.5 g / ml ; for moxifloxacin , 0.5 g / ml ; for amikacin , 1 g / ml ; for kanamycin , 2.5 g / ml ; and for capreomycin , 2.5 g / ml . the dst critical concentrations used for solid lj media are as follows : for ofloxacin , 4 g / ml ; for amikacin , 30 g / ml ; for kanamycin , 30 g / ml ; and for capreomycin , 40 g / ml . genomic dna was extracted from isolates and decontaminated specimens by thermal lysis and sonication as described elsewhere ( 19 ) . alternatively , a commercially available kit , genolyse ( hain lifescience , nehren , germany ) , was used to extract dna from clinical specimens according to the manufacturer 's instructions . amplification and hybridization of dna extracted from isolates and clinical specimens were performed according to the manufacturer 's instructions . genomic regions harboring drug resistance mutations were amplified and sequenced ( 9 ) by the sanger method using the designed internal primers for each region . results were visualized as electropherograms and analyzed by clustalw application ( bioedit software ; ibis biosciences / abbott company , carlsbad , ca ) , aligning sequences with the corresponding reference strain ( m. tuberculosis h37rv ; genbank accession no . the performance of the mtbdrsl v2.0 assay was evaluated using conventional phenotypic dst and sanger sequencing as reference standards , following the standards for reporting of diagnostic accuracy ( stard ) recommendations ( 20 ) . sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , likelihood ratio , and diagnostic accuracy values were calculated according to the wilson score . all statistical analyses were carried out using open source epidemiologic statistics for public health version 3.03 ( 21 ) . the panel included a total of 73 strains phenotypically resistant to at least one flq ( ofloxacin or moxifloxacin ) and 155 susceptible ones ( table 1 ) . the genotype mtbdrsl v2.0 assay correctly identified 61 ( 83.6% ) flq - resistant isolates , missing 12 ( 16.4% ) phenotypically resistant strains . eleven phenotypically resistant strains showed a mix of wild - type ( wt ) and mutated bands at the level of the gyra gene , suggesting a mixed infection . phenotypic drug - susceptibility testing of 228 clinical isolates and 231 specimens used in the study fqs , fluoroquinolones ; slids , second - line injectable drugs ; r , resistant ; s , susceptible . the distribution of gene mutations in the 73 flq - resistant isolates is shown in table 2 . the predominant mutations detected by mtbdrsl v2.0 as conferring flq resistance are the gyra mut1 a90v and mut3c d94 g mutations , identified in 26 ( 35.6% ) and 15 ( 20.5% ) resistant strains , respectively . nine additional strains ( 12% ) showed a mix of wild - type sequence and a90v or d94 g mutations . gyra mutations mut3a ( d94a ) and mut3b ( d94n / d94y ) were detected in 4 ( 5.5% ) and 2 ( 2.7% ) resistant strains , respectively . the gyra mutation mut2 ( s91p ) was detected in 3 ( 4.1% ) samples either alone or together with other mutations as a mix of wild - type and mutated bands . in three cases , the lack of the gyra wild - type 3 ( wt3 ) probe was used to infer a mutation at the level of codon 94 . by sequencing the gyra gene , we confirmed the presence of mutations d94y and d94a in two and one resistant strains , respectively . fluoroquinolone resistance and susceptibility results obtained by genotype mtbdrsl v2.0 assay , phenotypic dst , and sequencing on 228 clinical isolates dst , drug susceptibility testing ; wt , wild type ; r , resistant ; s , susceptible ; np , not performed . mutations in the gyrb gene were very rare and were identified in only 1 out of 73 ( 1.4% ) resistant strains . in this unique case , the mutation could be inferred by the lack of the gyrb wild - type probe on the genotype mtbdrsl v2.0 strip . dna sequencing revealed the presence of the n538 t mutation , thus confirming the mtbdrsl test result . overall , the test sensitivity for flq resistance measured against culture - based dst was 83.6% ( 95% confidence interval [ ci ] , 73.4% to 90.3% ) whereas the test specificity was 100% ( 95% ci , 97.6% to 100% ) . the concordance between phenotypic dst and the mtbdrsl v2.0 test was 94.7% ( 95% ci , 91.0% to 97.0% ) ( table 3 ) . we also calculated mtbdrsl v2.0 test performance by looking separately at each single flq tested , namely , ofloxacin or moxifloxacin . for ofloxacin , we obtained a test sensitivity and specificity of 84.7% ( 95% ci , 74.7% to 91.2% ) and 100% ( 95% ci , 97.6% to 100% ) , respectively , whereas the test sensitivity and specificity for moxifloxacin were 90.0% ( 95% ci , 69.9% to 97.2% ) and 97.4% ( 95% ci , 91.0% to 99.3% ) , respectively . performance of genotypemtbdrsl v2.0 assay compared to phenotypic dst on 228 clinical isolates flq include ofloxacin and moxifloxacin . slid ci , confidence interval ; dst , drug susceptibility testing ; ppv , positive predictive value ; npv , negative predictive value . the comparison between the mtbdrsl v2.0 test and gyra qrdr sequencing showed an overall concordance of 98.3% ( 95% ci , 95.6% to 99.3% ) ( table 4 ) . among the 11 phenotypically resistant strains showing a mix of wild - type and mutated bands at the level of the gyra gene , sequencing showed a double - pattern profile in 9 out of 11 cases ; it identified only the mutated nucleotide in 1 case , whereas it completely missed the mutation ( d94 g codon substitutions ) in another case . sequencing also missed one fully mutated sample ( i.e. , mutation at the level of codon d94 and absence of the corresponding wild - type 3 [ wt3 ] probe ) in one case . in contrast , mtbdrsl v2.0 missed a d94y codon substitution and a d89n codon substitution in the gyra gene in two distinct isolates that were instead detected as mixed populations by sequencing ( see table 2 ) . overall , the test sensitivity for flq resistance compared to gyra gene sequencing was 96.7% ( 95% ci , 88.8% to 99.1% ) and the specificity was 98.8% ( 95% ci , 95.8% to 99.7% ) ( table 4 ) . performance of genotype mtbdrsl v2.0 assay compared to sequencing on 228 clinical isolates ci , confidence interval ; ppv , positive predictive value ; npv , negative predictive value . our panel included 147 isolates resistant to at least one slid ( amikacin , kanamycin , or capreomycin ) as determined by phenotypic dst and 81 strains susceptible to all the tested injectable drugs ( table 1 ) . the genotype mtbdrsl v2.0 assay correctly identified 127 out of 147 ( 86.4% ) slid - resistant isolates and 73 out of 81 ( 90.1% ) fully susceptible ones . the distribution of gene mutations in the 127 slid - resistant isolates identified by the mtbdrsl v2.0 test is shown in table 5 . the great majority of resistant strains carried mutations only at the level of the rrs gene , namely , 98 out of 127 ( 77.2% ) , including 12 ( 9.4% ) strains with a mixed mutated and wild - type profile . among these isolates , the most frequent mutation was rrs mut1 a1401 g , which was detected in 92 out of 98 ( 93.9% ) cases ; three ( 3% ) strains carried both a1401 g and g1484 t mutations , two ( 2% ) isolates carried the c1402 t mutation , and one isolate carried the mut2 g1484 t mutation . second - line injectable drug resistance and susceptibility results obtained by genotype mtbdrsl v2.0 assay , phenotypic dst , and sequencing on 228 clinical isolates dst , drug susceptibility testing ; wt , wild type ; r , resistant ; s , susceptible . two resistant strains carried mutations in both the rrs ( mut a1401 g ) and eis promoter regions , with one sample showing both mutated and wild - type bands . a total of 27 out of 127 ( 21.3% ) slid - resistant strains carried mutations only at the level of the eis promoter region . the most frequent mutation identified was the mut1 c-14 t mutation , which was detected in 12 out of 27 ( 44.4% ) isolates , including one strain with a mixed profile . the absence of the wild - type 2 ( wt2 ) probe in the eis region correlated with the presence of mutations g-10a , g-37 t , and c-12 t in 8 , 4 , and 3 cases , respectively . all strains with a mixed mutated and wild - type profile , in either the rrs or eis promoter region , were phenotypically resistant . a total of 20 strains phenotypically resistant to slid were detected as fully wild - type by the mtbdrsl v2.0 assay : the great majority ( 13 out of 20 [ 65% ] ) were resistant to capreomycin but susceptible to both amikacin and kanamycin , 4 isolates ( 20% ) were resistant uniquely to kanamycin , 2 isolates ( 10% ) were resistant to all slid , and one isolate was resistant uniquely to amikacin . the genotype mtbdrsl v2.0 test also detected mutations in the eis promoter region of 8 phenotypically susceptible strains . among those , 6 isolates were missing the eis wt2 probe corresponding to mutations g-10a and c-12 t , as detected by gene sequencing in 5 and 1 cases , respectively , 1 sample showed the eis c-14 t mutation , and 1 sample showed the g-37 t mutation ( table 5 ) . overall , the concordance between mtbdrsl v2.0 and phenotypic dst for slid resistance was 87.7% ( 200/228 ) , whereas the test sensitivity and specificity were 86.4% ( 95% ci , 79.9% to 91.0% ) and 90.1% ( 95% ci , 81.7% to 94.9% ) , respectively . as a test for detection of kanamycin resistance measured against culture - based dst , the diagnostic accuracy of genotype mtbdrsl v2.0 was 93.8% ( 95% ci , 89.9% to 96.3% ) , whereas the sensitivity and specificity were 95.5% ( 95% ci , 90.6% to 97.9% ) and 91.4% ( 95% ci , 83.9% to 95.6% ) , respectively ( table 3 ) . the comparison between the assay and rrs sequencing showed an overall concordance of 97.8% ( 95% ci , 95.1% to 99.1% ) , with the molecular assay correctly identifying all the mutations revealed by gene sequencing ( 100% sensitivity ) ( 95% ci , 96.2% to 100% ) . our panel included 13 strains with a mixed mutated and wild - type profile either in the rrs gene or in the eis promoter region . sanger sequencing confirmed the double - pattern profile in 7 out of 13 cases , whereas it missed the mutation in 2 cases and it recognized only the mutated nucleotide in the remaining 4 cases . we excluded from the analysis one isolate showing the lack of the rrs wt1 probe since we were not able to obtain a readable sequence , which was probably due to the low quality or partial degradation of the dna . the comparison between mtbdrsl v2.0 and sequencing of the eis gene promoter region showed an overall concordance of 98.1% ( 95% ci , 94.6% to 99.4% ) . the assay missed two mutations detected by sequencing at the level of codons 10 and 37 . in both cases , the sequencing profile showed a double population with the coexistence of both wild - type and mutated nucleotides . in one case , the lack of the eis wt2 probe did not correspond to any mutation in the corresponding genetic region of the eis promoter by sequencing . overall , the test sensitivity and specificity measured against eis sequencing were 94.9% ( 95% ci , 83.1% to 98.6% ) and 99.2% ( 95% ci , 95.4% to 99.9% ) , respectively ( table 4 ) . of the 232 respiratory specimens tested with mtbdrsl v2.0 , 231 showed an interpretable result , with only one sample not providing a valid hybridization profile for any of the 4 genomic regions . gene sequencing , either direct or using the corresponding isolate , was performed uniquely on samples with a discordant genotypic / phenotypic profile . performance of genotypemtbdrsl v2.0 assay compared to phenotypic dst on 231 clinical specimens flq include ofloxacin , levofloxacin , and moxifloxacin . slid ci , confidence interval ; dst , drug susceptibility testing ; ppv , positive predictive value ; npv , negative predictive value . our panel included a total of 57 clinical specimens phenotypically resistant to at least one flq ( ofloxacin , levofloxacin , or moxifloxacin ) and 174 fully susceptible ones ( table 1 ) . the test correctly identified 53 ( 92.9% ) of the flq - resistant clinical specimens , missing four resistant cases . in contrast , 3 specimens showed a flq - resistant profile by mtbdrsl v2.0 while being susceptible by phenotypic dst . note that 2 out of 3 of these samples showed a double - pattern profile at the level of the gyra gene , suggesting a mixed infection . the most frequent mutations detected by genotype mtbdrsl among the 53 flq - resistant strains were the gyra mut3c d94 g and the mut1 a90v mutations , each identified in 13 ( 22,8% ) and 9 ( 15,8% ) cases , respectively . in addition , 10 specimens ( 17.5% ) showed a mixed profile ( i.e. , wild - type sequence and d94 g and/or a90v mutations ) . other gyra mutations , including mut2 ( s91p ) and mut3a ( d94a ) , were detected either as fully mutant or as mixed populations in 6 and 5 flq - resistant specimens , respectively . mutations at the level of the gyrb gene were very rare ( only 2 cases ) and could be inferred by the lack of the wild - type probe . dna sequencing revealed the presence of gyrb mutations n538s and t539a , thus confirming the mtbdrsl test result . however , only one of the two samples carrying the mutations in gyrb was flq resistant by phenotypic testing . fluoroquinolone resistance and susceptibility results obtained by genotype mtbdrsl v2.0 assay and phenotypic dst on 231 clinical specimens dst , drug susceptibility testing ; wt , wild type ; r , resistant ; s , susceptible . overall , the diagnostic accuracy for flq resistance detection in clinical specimens was 97.0% ( 95% ci , 93.9% to 98.5% ) , with a sensitivity and specificity of 93.0% ( 95% ci , 83.3% to 97.2% ) and 98.3% ( 95% ci , 95.1% to 99.4% ) , respectively . we also calculated mtbdrsl v2.0 test performance by looking separately at each single flq tested , namely , ofloxacin , levofloxacin , or moxifloxacin . for ofloxacin , we obtained a test sensitivity and specificity of 92.9% ( 95% ci , 83.0% to 97.2% ) and 97.7% ( 95% ci , 94.3% to 99.1% ) , respectively ; for levofloxacin , the test sensitivity and specificity were 100.0% ( 95% ci , 67.6% to 100% ) and 91.1% ( 95% ci , 79.3% to 96.5% ) , respectively , whereas the test sensitivity and specificity for moxifloxacin were 100.0% ( 95% ci , 78.5% to 100% ) and 90.9% ( 95% ci , 62.3% to 98.4% ) , respectively . our panel included a total of 63 clinical specimens phenotypically resistant to at least one slid ( kanamycin , amikacin , or capreomycin ) and 168 fully susceptible ones ( table 1 ) . the test correctly identified 56 ( 88.9% ) of the slid - resistant samples and 154 out of 168 ( 91.7% ) of the fully susceptible ones . among the 7 slid - resistant specimens missed by the assay , 5 were susceptible to kanamycin and resistant to capreomycin or amikacin or both thirty - five out of 56 ( 62.5% ) samples classified as resistant by mtbdrsl v2.0 carried the a1401 g mutation at the level of the rrs gene , with an additional 10 samples ( 17.5% ) showing a mixed wild - type and mutated ( mut a1401 g ) profile , whereas only one sample carried the rrs c1402 t substitution . mutations at the level of the eis promoter region were detected in 9 slid - resistant specimens , with an additional sample showing a double mutation at the levels of both the rrs and eis regions . mtbdrsl v2.0 test detected mutations within the eis promoter region in 11 out of 168 ( 6.5% ) slid - susceptible samples and mutations in the rrs region in 3 out of 168 ( 1.8% ) slid - susceptible specimens . the lack of an eis wt2 probe was the most prevalent mutation , detected in 8 out of 14 susceptible specimens . direct gene sequencing of these 8 samples confirmed the presence of the c-12 t and g-10a mutations in 6 and 2 cases , respectively . all three phenotypically susceptible specimens with mutations in the rrs gene showed a double ( wild - type and mutation)-pattern profile by mtbdrsl v2.0 assay , but sequencing of the corresponding isolates resulted in a wild - type profile . the full distribution of mutations detected by the genotype mtbdrsl assay is summarized in table 8 . second - line injectable drug resistance and susceptibility results obtained by genotype mtbdrsl v2.0 assay and phenotypic dst on 231 clinical specimens dst , drug susceptibility testing ; wt , wild type ; r , resistant ; s , susceptible . overall , the diagnostic accuracy of the test for slid resistance detection in clinical specimens was 90.9% ( 95% ci , 86.5% to 94.0% ) , with a sensitivity and specificity of 88.9% ( 95% ci , 78.8% to 94.5% ) and 91.7% ( 95% ci , 86.5% to 95.0% ) , respectively . as a test for detection of kanamycin resistance , the diagnostic accuracy of genotype mtbdrsl v2.0 was 93.1% ( 95% ci , 88.9% to 95.8% ) , whereas the sensitivity and specificity were 96.0% ( 95% ci , 86.5% to 98.9% ) and 92.2% ( 95% ci , 87.1% to 95.4% ) , respectively ( table 6 ) . the genotype mtbdrsl v2.0 assay correctly identified 37 out of 46 ( 80.4% ) phenotypically identified xdr - tb isolates , thus missing 9 ( 19.6% ) cases . in particular , the test detected only slid resistance , missing flq resistance , in 7 out of 9 ( 77.8% ) xdr - tb isolates and detected only flq resistance , missing slid resistance , in the remaining two cases ( 22% ) . in contrast , the test gave a positive xdr - tb result for 8 isolates resistant uniquely to flq by detecting mutations at the level of the eis promoter region . overall , the test accuracy for detection of xdr - tb was 92.5% ( 95% ci , 88.4% to 95.3% ) , whereas the test sensitivity and specificity were 80.4% ( 95% ci , 66.8% to 89.4% ) and 95.6% ( 95% ci , 91.6% to 97.8% ) , respectively ( table 9 ) . performance of genotypemtbdrsl v2.0 assay for detection of xdr - tb measured against phenotypic dst on 228 clinical isolates and 231 clinical specimens ci , confidence interval ; ppv , positive predictive value ; npv , negative predictive value . the genotype mtbdrsl v2.0 assay correctly identified 18 out of 22 ( 81.8% ) phenotypically identified xdr - tb clinical specimens , while it missed flq resistance in 2 cases , slid resistance in 1 case , and both flq resistance and slid resistance in 1 case . however , the test also gave a positive xdr - tb result for 3 specimens that were flq resistant only by detecting mutations at the level of the eis promoter region and for one sample that was slid resistant only by failing to detect the gyrb wt probe . overall , mtbdrsl v2.0 diagnostic accuracy for detection of xdr - tb was 96.5% ( 95% ci , 93.3% to 98.2% ) , with a sensitivity and specificity of 81.8% ( 95% ci , 61.5% to 92.7% ) and 98.1% ( 95% ci , 95.2% to 99.3% ) , respectively ( table 9 ) . this was the first multicenter study to assess the diagnostic accuracy of the novel version ( v2.0 ) of the genotype mtbdrsl test for detection of resistance to flq ( ofloxacin , moxifloxacin , and levofloxacin ) and slid ( amikacin , kanamycin , and capreomycin ) . similarly to genotype mtbdrsl v1.0 , the concordance between mtbdrsl v2.0 and gene sequencing was > 97% for all the genomic regions analyzed . discrepancies were mainly observed in cases of isolates showing a double - pattern profile ( 15% for gyra and 8.2% for rrs ) , which could be explained by the known lower sensitivity of sanger sequencing in detecting heteroresistance ( 22 , 23 ) . all heteroresistant strains were phenotypically resistant , thus confirming the presence of the mutated strain . the inclusion of probes for the detection of mutations in the eis promoter region increased the overall sensitivity of mtbdrsl v2.0 for the detection of slid resistance to 86.4% ( 95% ci , 79.9% to 91.0% ) and the sensitivity for the detection of kanamycin resistance to 96% for both the isolates and the clinical specimens , which is higher than the pooled sensitivity values for mtbdrsl v1.0 ( 66.9% ; 95% ci , 44.1% to 83.8 ) ( 2 ) . notably , the assay primarily missed strains that are resistant uniquely to capreomycin , namely , 13 out of 20 ( 65% ) among isolates and 4 out of 7 ( 57% ) among clinical specimens , which is expected given that the mechanisms of resistance to capreomycin partly differ from those causing resistance to aminoglycosides ( 13 ) . if , on the one side , the inclusion of the eis promoter region increased the test sensitivity , on the other , it negatively affected its specificity , which decreased from 99.5% ( 95% ci , 97.1% to 99.9% ) in mtbdrsl v1.0 ( 2 ) to 91.1% ( 95% ci , 86.43% to 94.9% ) and 90.1% ( 95% ci , 81.7% to 94.9% ) in mtbdrsl v2.0 for direct and indirect testing , respectively . as expected , the discordance between the results from the slid - resistant samples as defined by this molecular test and phenotypic dst involved primarily samples carrying mutations in the eis promoter region : 8 out of 8 among isolates and 11 out of 14 ( 78.6% ) among clinical specimens . the majority of isolates with a discordant phenotypic / genotypic profile carried the g-10a mutation ( 62.5% ) , whereas the most frequent eis mutation among the discordant clinical specimens was c-12 t ( 6 of 11 ; 54.4% ) , followed by mutations c-14 t ( 27.3% ) and g-10a ( 18.2% ) . in line with published data , the c-12 t eis mutation was more often found in sensitive strains and thus appears to be a nonspecific marker of resistance to slid ( 24 , 25 ) . in contrast , the g-10a nucleotide substitution was reported to correlate well with kanamycin resistance in several studies ( 13 , 24 , 25 ) , whereas only engstrm et al . more evidence is thus required to univocally define the role of each mutation within the eis promoter region in the development of resistance to kanamycin and of cross - resistance to the other classes of injectable drugs . this also implies the necessity to include in the assay specific probes able to discriminate between eis mutations at the level of different nucleotides , in particular , in positions 12 and 10 . in addition , since resistance to capreomycin and amikacin can not be automatically inferred on the basis of the presence of eis mutations , results from mtbdrsl v2.0 testing for slid resistance should be reported accordingly . if , on the one hand , our understanding of the role of specific mutations within the eis promoter region and slid resistance is still limited , on the other , phenotypic dst might be a suboptimal gold standard for the assessment of the test performance . data from a recent publication by the alland group strongly suggest that eis promoter mutants should be considered to have low to moderate kanamycin resistance even if resistance is not detected by lj - based or even mgit - based susceptibility tests ( 27 ) . concerning the rrs gene , in line with previous observations ( 9 , 24 , 28 ) , we found a high correlation between mutations in this genetic region and slid resistance , with 100 out of 100 mutated isolates and 47 out of 50 ( 94% ) clinical specimens being phenotypically resistant to at least one slid . notably , the three specimens giving false slid resistance results were characterized by a double - pattern profile compatible with a mixed infection , which could explain the discrepancy with the phenotypic dst result . in such cases , the presence of a predominant susceptible strain might have in fact concealed the resistance ( 29 ) . also , in line with published data ( 9 , 24 , 28 , 30 ) , we found a high correlation between rrs mutations and cross - resistance to amikacin , kanamycin , and capreomycin , with 90% of mutated strains and 98% of mutated clinical specimens being resistant to both aminoglycosides and capreomycin . notably , the two strains and one clinical specimen carrying the rrs mutation c1402 t were resistant to all injectable drugs . overall , the incidence of the rrs c1402 t and g1484 t mutations in our panel was very low , with only 3 strains out of 228 and 3 specimens out of 231 carrying uniquely the g1484 t and c1402 t substitutions . given the low prevalence of these mutations ( 24 ) , rrs codons 1484 and 1402 have a low positive predictive value of slid resistance and are thus unlikely to provide strong clinical significance . the inclusion of probes for the detection of mutations in the qrdr of the gyrb gene did not significantly improve the overall sensitivity and specificity of the mtbdrsl v2.0 test compared to mtbdrsl v1.0 . indeed , the incidence of gyrb mutations , all inferred by the lack of the gyrb wt probe , was very low , with only 1 case among 228 strains tested and 2 cases among 231 clinical specimens . mutations found in gyrb , in the absence of gyra substitutions , correlated with phenotypic flq resistance in 2 out of 3 cases . given the very low prevalence of gyrb mutations ( 3133 ) and thus their low positive predictive value for flq resistance , the inclusion of gyrb probes in the new version of the test is unlikely to be clinically relevant . to date , genotype mtbdrsl has been one of the few rapid molecular assays available on the market for the detection of resistance to second - line drugs . given its high positive predictive value ( ppv ) and specificity , a positive result for flq resistance , slid resistance , or xdr - tb can be treated with confidence ( 2 ) . however , the low sensitivity , especially for detection of kanamycin resistance and its low negative predictive value ( npv ) , makes it necessary to confirm by conventional dst all cases classified as susceptible . the inclusion of probes for the detection of mutations at the level of the eis promoter region in the new version of the assay , mtbdrsl v2.0 , leads to a significant increase in test sensitivity for the detection of kanamycin resistance compared to the original version . importantly , these mutations allow the identification of strains with low or moderate kanamycin resistance which might be erroneously classified as susceptible by conventional phenotypic testing alone . if , on the one hand , this raises the problem of dealing with a suboptimal gold standard when assessing the performance of a new diagnostic test , on the other , it highlights the necessity to understand whether this low - level resistance has a real impact on the treatment efficacy . therefore , we need to further investigate the correlation between specific mutations and the level of drug responsiveness in vivo by routinely collecting clinical data on treatment outcomes . this will be essential for improving our capacity to correctly interpret the mutations identified by genotypic testing . summarizing , the mtbdrsl v2.0 assay has better performance characteristics for the detection of xdr - tb when performed either directly or indirectly on samples , by correctly identifying more than 80% of xdr - tb cases , than version 1.0 , which misses the xdr phenotype in between one in four and one in three cases ( 2 ) . on clinical specimens , mtbdrsl v2.0 showed a ppv and a npv of 81.8% ( 95% ci , 61.5% to 92.7% ) and 98.1% ( 95% ci , 95.2 to 99.2 ) , respectively . notably , the prevalence of xdr - tb cases in our panel of clinical specimens ( i.e. , a setting with high prevalence of mdr - tb ) is around 9.5% , which corresponds to the who - estimated prevalence of xdr - tb among mdr - tb cases ( 2 ) . therefore , our sample panel provides an evaluation of the diagnostic performance of mtbdrsl v2.0 in a real - world setting . indeed , the use of this rapid assay in countries with a high rate of drug resistance , especially kanamycin resistance , will further increase the test ppv and will reduce the number of xdr - tb cases missed by the test . despite some limitations , such as the fact that we tested smear - positive clinical samples only , the fact that flq resistance was measured primarily against ofloxacin phenotypic dst , and the lack of data on the mic for isolates carrying mutations in the eis promoter region , this report provides an accurate evaluation of the diagnostic performances of the new version of the genotype mtbdrsl assay based on standardized testing of a large set of samples from different settings . we show that the mtbdrsl v2.0 test has better performance characteristics in terms of increased sensitivity for detecting slid and kanamycin resistance and better accuracy for detecting xdr - tb than the original version of the test . mtbdrsl v2.0 thus represents a better screening tool for the rapid detection of resistance to second - line drugs , and it should be recommended in countries with a high burden of mdr / xdr - tb or in settings with high reported rates of resistance to ofloxacin in new and retreatment cases . in such contexts , the test can rapidly identify patients for whom mdr treatment regimens that include earlier - generation fluoroquinolones are likely to fail or who may not be good candidates for the shorter mdr treatment currently being tested in western and central africa ( 34 ) .

the authors present a rare case of tuberculous spondylitis and a large abscess in the left psoas muscle that occurred after spinal surgery for an acute traumatic burst fracture of the l2 vertebral body . we retrospectively reviewed the patient 's first magnetic resonance imaging ( mri ) we found that some unusual findings , indicative of psoas abscess had been overlooked . as a result , diagnosis and treatment of tuberculous psoas abscess and spondylitis were considerably delayed . despite the critical condition of patients in a similar emergency , surgeons should always pay close attention to the radiological findings and clinical symptoms of the patient before considering a surgical intervention or biopsy .

psoas abscess is characterized by infrequent incidence , insidious onset , and nonspecific clinical features , which can lead to misinterpretation and delayed diagnosis ; thus , it is difficult to clinically diagnose psoas abscess1,5 ) . here , we present a rare case of tuberculous spondylitis and a large abscess in the left psoas muscle that occurred after spinal surgery for an acute burst fracture of the second lumbar ( l2 ) vertebral body caused by a traffic accident . moreover , the first radiological examination revealed some unusual findings that could have possibly indicated abscess before his spinal surgery . in may 2009 , a 44-year - old man was transferred to our hospital for lower back pain after he suffered from a blunt trauma in a pedestrian traffic accident . his medical and surgical histories were unremarkable . neurological examination and chest radiology revealed no abnormalities . laboratory evaluation revealed a erythrocyte sedimentation rate ( esr ) of 48mm / hr ( normal range , 0 - 10mm / hr ) and c - reactive protein ( crp ) of 239.52mg / l ( 0 - 5mg / l ) , and a normal wbc count of 7740/l ( 4000 - 10,000/l ) . lumbar computed tomography ( ct ) scan with bone window settings revealed a burst fracture of the l2 vertebral body with displacement of a posterior fragment into the spinal canal ( fig . lumbar magnetic resonance imaging ( mri ) revealed acute burst fracture with retropulsion of the posterior bone fragment at the l2 vertebral body and acute compression fracture of the l1 vertebral body ( fig . he underwent posterior reduction , transpedicular screw fixation , secondary anterior thoracoscopic decompression , and anterior reduction that was accomplished with a distractible cage and a modular anterior construct systemthoraco - lumbar ( macs - tl ) plate ( aesculap , tuttlingen , germany ) ( fig . the patient had no postoperative complications and was discharged on the 45 postoperative day . until july 2010 , the patient 's clinical condition was satisfactory ; thereafter , he exhibited a gradually enlarging swelling in the left flank , associated with pain extending up to the left thigh , and urinary incontinence . his esr was 120mm / h ; crp level , 59.94mg / l ; blood urea nitrogen ( bun ) level , 28.1mg / dl ; and serum creatinine level , 1.77mg / dl . abdominopelvic ct revealed the presence of a large abscess in the following sites : left psoas muscle , lower pole of the left kidney , and posterolateral abdominal wall . 2 ) . abscess drainage and irrigation and partial nephrectomy through the previous surgical route were performed under general anesthesia . the abscess and surrounding soft tissue samples yielded positive results in the acid - fast bacilli ( afb ) culture and polymerase chain reaction ( pcr ) for mycobacterium tuberculosis complex . the patient received antituberculous chemotherapy , consisting of isoniazid , rifampin , pyrazinamide , along with pyridoxine supplements , on a daily basis . in november 2010 the patient was transferred to the urological department for treatment of the left ureteral stricture . initially , the predominant pathogen was mycobacterium tuberculosis , which caused psoas abscess through either hematogenous spread or direct extension from vertebral osteomyelitis of the lumbar spine ( pott 's disease ) . with the decline of mycobacterium tuberculosis as a major pathogen of psoas abscess in developed countries , the condition was mostly detected secondary to diseases of the digestive tract , with other pathogens such as staphylococcus aureus gaining more dominance in the pathogenesis of this psoas abscess4,7 ) . however , in recent years , tuberculosis - induced psoas infections are again emerging with the recurrence of tuberculosis in industrial countries and increase in the number of human immunodeficiency virus ( hiv ) infections5 ) . mckley et al5 ) emphasized the role of the spine as the primary source of infection for secondary psoas abscess . according to the most case reports on psoas abscesses , tuberculous psoas abscess occurring secondary to direct extension from other adjacent structures or even hematogenous seeding from a distant site is less frequent8 ) . however , there have been hardly any reports on tuberculous psoas abscess presenting as a manifestation of tuberculosis , without evidence of an active infection elsewhere8 ) . even in the case of our patient , there was no evidence of a tubercle bacillus infection anywhere but except in the surgical portion . a retrospective review of the first mri of our patient revealed some unusual findings of the left psoas muscle , which had been overlooked earlier . axial mri of the l2 vertebra revealed that the left psoas muscle was irregular shaped , ill - defined , and heterogeneously enlarged compared to the right psoas muscle and both paraspinal muscles at the level of the vertebral burst fracture . in addition , the coronal t2-weighted mri revealed a thick line of high - signal intensity along the left psoas muscle ( fig . even though it is very difficult to determine whether asymmetric swelling of the left psoas muscle was due to trauma or infection , we consider that this unusual finding was a result of the infection . if it was caused by the trauma , there would have been more contusions of the paraspinal muscle than that of the psoas because the psoas is positioned anterior to the paraspinal muscle . moreover , because the abnormality was detected only in the left psoas and not in either paraspinal muscle or the right psoas , it is difficult to determine whether the condition was a result of the trauma . we also consider that the possibility of tuberculous spondylitis extending to the vertebral body before injury is low . we assume that vertebral fracture and subsequent spinal surgery can make the progress of the primary psoas abscess around surrounding tissues quicker . in the majority of earlier cases , the patients , as in our case , although fever , abdominal or back pain , and limited hip - joint mobility form the classical triad of psoas abscess , they can be inconsistently presented1 ) . retrospective examination revealed that our patient also had abdominal pain and intermittent limited hip - joint mobility , weeks before the traffic accident . therefore , suspicious clinical finding , radiological study , and microbial culture study of the pus , are crucial in the diagnosis of tuberculous psoas abscess . this report might be significant , considering the possibility of missed diagnosis of psoas abscess in patients with acute lumbar compression fracture caused by trauma , even though the unusual findings of the left psoas muscle in the first mri were not histologically examined . unfortunately , because we mainly focused on the traumatic injury , we did not conduct a medical examination through interrogation and radiology prior to surgery , and as a result , diagnosis and treatment of psoas abscess and tuberculous spondylitis were considerably delayed . therefore , despite the critical state of the patient in similar emergencies , surgeons should always pay close attention to the radiological findings and clinical symptoms of the patients , before considering a surgical intervention or biopsy .

background : the last decade has seen the emergence of the internet as the prime communication medium changing the way people live and interact . studies from various countries have reported on internet addiction and its association with mental health , but none have come from malaysia.objectives:we aimed at assessing the frequency of the use of various internet applications and exploring the association of compulsive internet use with mental health and socio - demographic factors.materials and methods : a cross - sectional online survey was carried out among participants registered for the monthly opinion poll survey of university tunku abdul rahman , malaysia . the questionnaire contained socio - demographic information , the use of various internet applications on a five - point likert scale , compulsive internet use scale ( cius ) and 12 item general health questionnaire ( ghq-12 ) . correlations and linear regression analyzes were carried out.results:of the 330 respondents , 182 were females and 148 were males . the mean age was 23.17 ( sd = 3.84 ) . mean cius score was 19.85 ( sd = 10.57 ) and mean ghq score was 15.47 ( sd = 6.29 ) . correlation coefficients of cius score with age , years of use and daily hours of internet use were 0.118 ( p = 0.03 ) , 0.014 ( p = 0.81 ) and 0.242 ( p < 0.001 ) respectively . multiple linear regression analysis showed that age ( = 0.111 , p = 0.033 ) and marital status ( = 0.124 , p = 0.018 ) were negatively associated with cius scores whereas daily hours of internet use ( = 0.269 , p = 0.001 ) and ghq score ( = 0.259 , p = 0.001 ) were positively associated with the cius score.conclusions:compulsive internet use was correlated with ghq score . more research is needed to confirm our results . psychologists may consider assessing internet addiction when evaluating young psychiatric patients .

internet user population world - wide has doubled from 1.15 billion to 2.27 billion and has grown from 418 million to 1 billion in the last 5 years in asia . rapid expansion and proliferation of the internet has provided better opportunities for communication , information and social interaction . however , the excessive undisciplined use by , some individuals has led to the emergence of the concept of internet addiction . the proposed diagnostic criteria for internet addiction are obsessive thoughts about internet and the use of the internet more than is intended or despite its negative consequences including withdrawal symptoms and tolerance . studies have shown that young internet users and males have a higher risk of addiction . this addiction has been linked to intermediate psychosocial variables such as avoidance , ( escapism ) male gender and applications such as online games . excessive use and pre - occupation about the internet has been shown to affect day - to - day life activities , time management , marriage , sexual life , productivity at work , education and academic work . studies have shown that excessive use of the internet is associated with such psychiatric conditions as anxiety , depression , social phobia , impulsiveness and substance abuse , as well as the induction of seizures , insufficient sleep and social isolation . an increasing number of young malaysians are resorting to the internet as the medium of choice for information and entertainment . a study by an international marketing firm taylor nelson sofres which observed the habits and behavior of internet users found that the largest number of social network partners belonged to malaysian internet users with the highest average number of 233 digital friends when compared with their peers from other countries . another survey by microsoft conducted among 2800 internet users in 11 countries in the asia - pacific region has shown that 71% of malaysian internet users spend more than 1 h / day on cyber social activities and that the largest number of internet friends on facebook belonged to malaysian internet users . studies from various countries have reported on internet addiction , its association with mental health , yet no reports have come from malaysia . therefore , an online survey was carried out of regular internet users to assess the frequency of the use of various internet applications and explore the association of compulsive internet use with mental health and socio - demographic factors . a cross - sectional online survey was carried out by university tunku abdul rahman ( utar ) . the utar opinion poll survey is an initiative undertaken by a committee with representatives from all faculties of the university . every month , the committee plans and executes opinion poll surveys on various topics of general interest about the youth in malaysia ( http://poll.utar.edu.my ) . the registered utar opinion poll survey participants are young adults from various parts of malaysia , most of whom are young university students with access to the internet . sampling method and sample size calculation were not used since this was an exploratory study . the survey also provided an option for participants to invite their friends to take part . the online survey questionnaire had sections on demographic information such as age , gender , marital status , monthly income , educational status , employment status , questions on availability of internet at the work place and the typical number of hours spent online per day and applications or the purpose for which the internet is used . frequency of using various internet applications such as gaming , social network , e - mails etc . , were assessed on a five - point likert scale as never , seldom , sometimes , often and always . the survey also contained a 12-item general health questionnaire ( ghq-12 ) and compulsive internet use scale ( cius ) . ghq-12 designed by goldberg , has reliability coefficients ranging from 0.78 to 0.95 in various studies with a well - established international validity . for example , the options given for each question in ghq-12 and the scores assigned ( as shown in parenthesis ) are better than usual - ( 0 ) , same as usual - ( 1 ) , less than usual - ( 2 ) and much less than usual - ( 3 ) . ghq-12 scores are calculated by summing up the scores of all 12 items giving a score range of 0 - 36 . a score > 15 suggests evidence of distress and a score > 20 suggests severe problems and psychological distress . ghq-12 and its scoring is available at http://www.workhealth.org/uci%202007/practicum%2008%20ghq%202007.pdf . cius is an easily administered , psychometrically sound , valid instrument used to assess the severity of compulsive internet use . cius originates from an analysis of criteria for dependence and obsessive - compulsive disorder as found in the dsm - iv , literature on behavioral addictions and from qualitative research on self - declared internet addicts . it has a high internal consistency and high correlations with concurrent and criterion variables demonstrating its good validity . for example , the options for each question and the scores assigned ( as shown in parenthesis ) are never - ( 0 ) , seldom - ( 1 ) , sometimes - ( 2 ) , often - ( 3 ) and always - ( 4 ) . scores are calculated by summing up the scores of all 14 items giving a score range of 0 - 36 . however , it has no cut - off point to categorize a participant as an internet addict . data extracted into microsoft excel was converted into statistical package for social sciences ( spss inc . the relationship of cius score with demographic factors and ghq was explored using bivariate statistics . correlation coefficients for ghq score and cius score with age , daily hours and years of internet use were calculated . mean ghq and cius scores were compared with categories of demographic variables and the statistical significance of the difference in mean scores was tested using independent samples t - test or anova as appropriate . the relationship of cius score with ghq , daily hours and years of internet use and socio - demographic variables was tested by linear regression analysis . a p < 0.05 was considered as significant . a cross - sectional online survey was carried out by university tunku abdul rahman ( utar ) . the utar opinion poll survey is an initiative undertaken by a committee with representatives from all faculties of the university . every month , the committee plans and executes opinion poll surveys on various topics of general interest about the youth in malaysia ( http://poll.utar.edu.my ) . the registered utar opinion poll survey participants are young adults from various parts of malaysia , most of whom are young university students with access to the internet . sampling method and sample size calculation were not used since this was an exploratory study . the survey also provided an option for participants to invite their friends to take part . the online survey questionnaire had sections on demographic information such as age , gender , marital status , monthly income , educational status , employment status , questions on availability of internet at the work place and the typical number of hours spent online per day and applications or the purpose for which the internet is used . frequency of using various internet applications such as gaming , social network , e - mails etc . , were assessed on a five - point likert scale as never , seldom , sometimes , often and always . the survey also contained a 12-item general health questionnaire ( ghq-12 ) and compulsive internet use scale ( cius ) . ghq-12 designed by goldberg , has reliability coefficients ranging from 0.78 to 0.95 in various studies with a well - established international validity . for example , the options given for each question in ghq-12 and the scores assigned ( as shown in parenthesis ) are better than usual - ( 0 ) , same as usual - ( 1 ) , less than usual - ( 2 ) and much less than usual - ( 3 ) . ghq-12 scores are calculated by summing up the scores of all 12 items giving a score range of 0 - 36 . a score > 15 suggests evidence of distress and a score > 20 suggests severe problems and psychological distress . ghq-12 and its scoring is available at http://www.workhealth.org/uci%202007/practicum%2008%20ghq%202007.pdf . cius is an easily administered , psychometrically sound , valid instrument used to assess the severity of compulsive internet use . cius originates from an analysis of criteria for dependence and obsessive - compulsive disorder as found in the dsm - iv , literature on behavioral addictions and from qualitative research on self - declared internet addicts . it has a high internal consistency and high correlations with concurrent and criterion variables demonstrating its good validity . for example , the options for each question and the scores assigned ( as shown in parenthesis ) are never - ( 0 ) , seldom - ( 1 ) , sometimes - ( 2 ) , often - ( 3 ) and always - ( 4 ) . scores are calculated by summing up the scores of all 14 items giving a score range of 0 - 36 . however , it has no cut - off point to categorize a participant as an internet addict . data extracted into microsoft excel was converted into statistical package for social sciences ( spss inc . the relationship of cius score with demographic factors and ghq was explored using bivariate statistics . correlation coefficients for ghq score and cius score with age , daily hours and years of internet use were calculated . mean ghq and cius scores were compared with categories of demographic variables and the statistical significance of the difference in mean scores was tested using independent samples t - test or anova as appropriate . the relationship of cius score with ghq , daily hours and years of internet use and socio - demographic variables was tested by linear regression analysis . a p < 0.05 was considered as significant . of the 330 respondents who completed the online survey , 185 ( 55.2% ) were females and 145 ( 44.8% ) were males with a mean age of 23.17 ( sd = 3.84 ) . two hundred and three ( 61.5% ) respondents were full - time students , 247 ( 74.8% ) were single ; 214 ( 64.8% ) had a bachelor 's degree and 204 ( 61.8% ) had monthly income of < 2000 malaysian ringgits ( 1 malaysian ringgit 0.32 us dollar ) [ table 1 ] . the average years of internet use was 8.7 ( sd = 3.85 ) and the mean hours of daily internet use was 6.56 ( sd = 3.73 ) [ table 2 ] . a majority of participants had an internet connection either at home ( 93.3% ) , or in college or at the work place ( 96.1% ) and 62.7% had an internet connection on their mobile phones ( data not shown ) . association of demographic factors with cius score and ghq score bivariate correlation coefficients of cius score and ghq scores with age , durations of internet use and ghq score the overall mean cius score was 19.85 ( sd = 10.57 ) and the mean ghq score was 15.47 ( sd = 6.29 ) . according to the recommended ghq-12 cut - off scores , 55.5% ( 183/330 ) respondents showed evidence of psychological distress and 28.2% ( 93/330 ) had severe distress . bivariate correlation between cius score and ghq score was 0.249 ( p < 0.001 ) . mean cius scores were compared with categories of socio - demographic variables of gender , education , income , marital status and job status . of these , the mean cius scores were significantly high among respondents who had a master 's or a higher degree [ table 1 ] . cius score was compared with age , years of internet use and the number of hours the internet is used daily , using bivariate correlations and the correlation coefficients were 0.118 , 0.014 and 0.242 respectively . all the correlations were weak , but statistically significant for age ( p = 0.03 ) and hours of internet use daily ( p < 0.001 ) [ table 1 ] . a comparison of the mean ghq scores among categories of gender , education , income , marital status and job status exhibited no statistically significant differences . a comparison of ghq score with age , years of internet use and daily hours of internet use by bivariate correlations gave correlation coefficients of 0.040 , 0.033 and 0.017 respectively suggesting that there was no correlation and no statistical significance ( p > 0.05 ) [ table 2 ] . a majority of the participants responded that they often / always used the internet for social networking ( 85.5% ) and personal e - mails ( 78.5% ) , followed by work - related surfing ( 67.2% ) and general information search ( 63.9% ) . the frequency of the use of the youtube and movie websites on the internet and to download ( software , movies , music etc . ) reported as often / always was 54.2% [ table 3 ] . frequency of using internet applications by multiple linear regression analysis age ( = 0.111 , p = 0.033 ) and marital status ( = 0.124 , p = 0.018 ) were negatively associated with cius score whereas daily hours of internet use ( = 0.269 , p = 0.001 ) and ghq score ( = 0.259 , p = 0.001 ) were positively associated with cius score after adjusting for the effect of socio - demographic variables [ table 4 ] . association of cius score with ghq-12 score , age , sex , marital status and hours of internet use per day social networking , personal e - mails and general information search were the most frequently used internet applications . mean cius score was not high , but its range was wide and weakly correlated with daily hours of internet use , age and marital status and ghq scores . by ghq case criterion , nearly a quarter of the respondents had severe psychological distress , but the proportion with internet addiction could not be reported since unlike the more recent internet addiction tests , cius does not have a cut - off score . however , studies using internet addiction test have reported the prevalence of internet addiction of adolescents from taiwan as 31 ( 19.8% ) , china 32 ( 6.44% ) , greece 33 ( 11.6% ) , japan 34 ( 8 - 10% ) and malaysia 35 ( 29% ) . the pattern of internet usage in our study was similar to the findings of a survey by accenture ( a multinational management consulting , technology services and outsourcing company ) which has reported that social network sites usage ( sns ) was the preferred method of communication among adolescents in malaysia , which ranks first as the most sns active country ( malaysian communications and multimedia commission ) . similar to the results of our study and those from usa and korea , the same study also showed that sns usage had a significant negative effect on psychological well - being . other studies about mental health and internet use have reported that mental health problems such as depression , low self - esteem , high level of stress , distorted view of body image , loneliness and prior addictions increased the risk of internet addiction . cius scores in the higher range are consistent with the frequency of internet addiction reported in studies from china and korea . research has suggested that the ease of access will tend to facilitate the use of internet from home , school / the work place , or mobile networks . this convenience has encouraged the use of the internet to the extent of addictive behavior . it is reasonable to argue that easy access to the internet may be another possible risk factor for addiction . the internet is an essential tool for learning in the modern education system , but a study from taiwan has reported availability of the internet as a possible risk factor for addiction among college students . nevertheless , it is not easy to determine the optimal use of internet or put restrictions on the use of various applications . in our study , the number of hours of daily internet use was associated with cius score , but years of internet use was not since most ( > 90% ) of our participants had an internet connection at home or place of work / study , increasing the likelihood of their using it for longer hours . our findings of positive correlation between cius and ghq scores are in agreement with the emerging concept of association between internet addiction and mental health . as cross - sectional design can only identify the views and reported behavior of respondents at a particular point in time . further research to observe actual behavior or a prospective study to follow - up the internet users might be helpful . despite online anonymous survey , some responses may have been biased leading to a reporting bias . the sample surveyed might have been relatively healthy , accounting for relatively low to moderate correlations found between the cius score and the ghq score . only about 30% of registered participants responded to the survey , but there was no information about those who did not complete the survey ( non - respondents ) for us to assess how non - response may have affected our results . future research directed at answering questions on the relationship between problematic or addictive use of the internet and mental health should include a more heterogeneous sample , in terms of age , occupation ( i.e. non - students ) and education . as cross - sectional design can only identify the views and reported behavior of respondents at a particular point in time . further research to observe actual behavior or a prospective study to follow - up the internet users might be helpful . despite online anonymous survey , some responses may have been biased leading to a reporting bias . the sample surveyed might have been relatively healthy , accounting for relatively low to moderate correlations found between the cius score and the ghq score . only about 30% of registered participants responded to the survey , but there was no information about those who did not complete the survey ( non - respondents ) for us to assess how non - response may have affected our results . future research directed at answering questions on the relationship between problematic or addictive use of the internet and mental health should include a more heterogeneous sample , in terms of age , occupation ( i.e. non - students ) and education . compulsive internet use was weakly correlated with ghq score and hours of internet use daily . internet users should be educated about the ill effects of over - use and ways of spending time away from internet .

backgroundchanneling bias may occur when a newly marketed drug and an established drug , despite similar indications , are prescribed to patients with different prognostic characteristics ( ie , confounding).aimto investigate channeling bias and its impact on relative effectiveness of glucagon - like peptide-1 ( glp-1 ) analogs versus basal insulin and dipeptidyl peptidase-4 inhibitors ( dpp-4i ) versus sulfonylurea.methodsin the uk clinical practice research datalink , patients with type 2 diabetes initiating treatment between 2006 and 2015 were included . analyses were stratified by years since first prescription of glp-1 and dpp-4i , respectively . the characteristics of glp-1 versus insulin and dpp-4i versus sulfonylurea initiators were compared over time . after propensity score matching , the relative effectiveness regarding 6-month changes in glycated hemoglobin ( hba1c ) and body weight was estimated.resultsin total , 8,398 glp-1 , 14,807 insulin , 24,481 dpp-4i , and 33,505 sulfonylurea initiators were identified . no major channeling was observed . considerable overlap in distributions of characteristics allowed for propensity score - matched analyses . relative effectiveness was similar across time . the overall relative effect of glp-1 versus insulin showed no difference for hba1c and relative increase in body weight ( 3.57 kg [ 95% confidence interval { ci } : 3.21 , 3.92 ] ) for insulin . the overall relative effect of dpp-4i versus sulfonylurea showed relative decrease in hba1c ( 0.34% [ 95% ci : 0.38 , 0.30 ] ) and increase in body weight ( 1.58 kg [ 95% ci : 1.38 , 1.78 ] ) for sulfonylurea.conclusionno major channeling was identified in the investigated glucose - lowering drugs . relative effectiveness could be estimated already in the first year after launch and was consistent in the years thereafter .

randomized controlled trials are primarily designed and conducted to meet the needs of regulatory bodies , so as to provide evidence on the efficacy and the safety of new drugs or other health care interventions . however , these studies are generally insufficient by themselves to meet the evidentiary needs of many health technology assessment agencies , that is , evidence on the drug s effectiveness . similarly , clinicians and payers desire evidence on comparative effectiveness of new drugs immediately after launch to take informed decisions.1 the sooner valid comparative effectiveness research results can be generated , the more useful they are to patients , clinicians , and payers . comparative effectiveness research using secondary health care data ( including electronic medical records , longitudinal claims data , and registries ) provides evidence on the benefits and risks of drugs in routine medical practice.1 however , channeling bias is a potential risk when relative effectiveness of a newly marketed drug compared to an established drug is investigated in observational data . channeling bias may occur when a newly marketed drug and an established drug , despite similar therapeutic indications , are prescribed to patients with different prognostics characteristics.2 over time , the prognostic characteristics of the patients who prescribed the two drugs may become more balanced as the newly marketed drug becomes more established . reasons for channeling bias could be a belief in extra advantages of the new drug compared to the established drug , or simply because doctors do not know how else to treat a subgroup of patients due to intolerance or low response to established drugs . it is possible that patients with a better prognostic are channeled to the newly marketed drug , but it is often theorized that a newly marketed drug is predominantly prescribed to patients with worse prognostics compared to the established drug being prescribed to patients with better prognostics.1,2 when channeling bias occurs , comparative effectiveness research becomes challenging because comparison with a drug will be confounded and knowledge of comparative effectiveness close to market entry may be biased or absent . the first glucagon - like peptide-1 analogs ( glp-1 ) , exenatide , was approved by the european medicines agency ( ema ) in november 2006 , and the first dipeptidyl peptidase-4 inhibitor ( dpp-4i ) , sitagliptin , was approved by the ema in march 2007 . a us study on prescription pattern of exenatide during the first half year after approval showed that a higher proportion of obese patients used exenatide compared to patients with other glucose - lowering drugs , indicating an awareness of weight - lowering effects of glp-1.3 three us studies46 compared the characteristics of sitagliptin users with patient receiving other oral glucose - lowering drugs ; two of the studies4,5 compared 20062007 versus 20082010 , and the third study6 investigated the first 2 years after approval of sitagliptin . the three studies46 found that dpp-4i users were older and have more comorbidities than patients receiving other oral glucose - lowering drugs , but with no change over time . these studies36 were all descriptive and did not investigate if the differences in characteristics could be taken into account in an outcome analysis . the objectives of this study were , first , to compare the characteristics of patients initiating glp-1 to those initiating basal insulin and of patients initiating dpp-4i to those initiating sulfonylurea stratified on calendar time and , second , to assess the relative effectiveness over time since the market entry of glp-1 and dpp-4i . the united kingdom clinical practice research datalink ( cprd ) was used for this study . the cprd is derived from uk primary care and holds longitudinal patient - level data such as diagnoses , mortality , laboratory results , and prescription data . it is the representative for the uk general population , and validation ensures high - quality data.710 anonymized data are available after protocol approval from the independent scientific advisory committee ( isac).11 for this study , data about patients aged 18 years with a diagnosis of type 2 diabetes and a prescription of glucose - lowering medication between january 1 , 2006 , and february 9 , 2015 , were extracted . validation studies have shown that diagnosis of type 2 diabetes alone is not always correct , so to ensure identification of patients with type 2 diabetes , patients were included if they fulfilled one of the following criteria:12 1 ) first diagnosis ( either type 1 or type 2 ) before age 35 years and no continual insulin treatment within six months of diagnosis , or 2 ) first diagnosis ( either type 1 or type 2 ) after age 35 years and not on continual insulin treatment from diagnosis . two comparison groups were used throughout the study ; initiators of glp-1 were compared with initiators of basal insulin , whereas initiators of dpp-4i were compared with initiators of sulfonylurea . the chosen comparator groups were to compare second - line ( dpp-4i and sulfonylurea ) and third - line ( glp-1 and insulin ) treatments , respectively.13 the glucose - lowering drugs were handled at the drug - class level , and combination products of metformin and dpp-4i and of rosiglitazone and sulfonylurea were considered as dpp-4i and sulfonylurea . prescriptions of drugs were used to identify episodes of treatment . for each patient , the end of a prescription was estimated from the daily dose and amount prescribed ( typically available for glp-1 , dpp-4i , and sulfonylurea ) , or as the patient s median duration of prescription of the drug ( typically the case for insulin ) . if there were < 120 days between the estimated end date of one prescription and the start date of the next prescription , the prescriptions were considered to make up a continuous episode of drug treatment . initiation was defined as patients with a prescription of one of the four glucose - lowering drugs after minimum 180 days without a prescription of the drug ( washout period ) . two cohorts were created , by analogy with the terms used in clinical trials : the intention - to - treat ( itt ) cohorts comprise subjects with a treatment duration of at least one day , whereas the per - protocol ( pp ) cohorts comprise subjects with a treatment duration of at least 180 days . if patients were identified as incident users more than one time for the same drug , only the first initiation was considered . if patients initiated glp-1 and insulin , or dpp-4i and sulfonylurea at the same time , this initiation was censored . within each of the four cohorts , time blocks of 365-day duration were defined with start date of the first initiation of glp-1 or dpp-4i , respectively . if patient initiated the comparison drugs within the same time block , only the first initiation was used and the second initiation was censored . measures of glycated hemoglobin ( hba1c ) and body weight were used to establish outcome measures . the goal was to observe change in hba1c and in body weight during a 180-day period . however , because measurements were not always available at the exact date of treatment initiation and 180 days after initiation , the following were deployed when measurements were not available : if a measurement was available within 90 days prior to initiation , this was used as the measurement at initiation , and if a measurement was available between 90 and 179 days after initiation , this was used as the measurement 180 days after initiation . if several measurements were available within these periods , the measurement closest to the initiation date and 180 days after initiation , respectively , was used . the following covariates were used : sex ; age at initiation ; weight at initiation ; body mass index ( bmi ) at initiation ; hba1c at initiation ; duration of diabetes at initiation ; number of oral glucose - lowering drugs at initiation ; any use of glucose - lowering drugs ( sulfonylurea , basal insulin , dpp-4i , metformin , and other oral glucose - lowering drugs ) during the washout period ; any use of antihypertensive , statins , and anticoagulants during the washout period ; charlson comorbidity index14 at initiation ; and any diagnosis of hypertension , end - stage renal disease , myocardial infarction , or stroke prior to initiation . the covariate number of oral glucose - lowering drugs at index was a count of prescriptions at index of any of the following glucose - lowering drugs ; acarbose , canagliflozin , canagliflozin combined with metformin , dapagliflozin , dapagliflozin combined with metformin , empagliflozin , pioglitazone , pioglitazone combined with metformin , rosiglitazone , rosiglitazone combined with metformin , nateglinide , repaglinide , troglitazone , guar gum and metformin in the comparison of dpp-4i and sulfonylurea , and in the comparison of glp-1 and basal insulin number of other oral glucose - lowering drugs at index also included dpp-4i and sulfonylurea . the covariate other oral glucose - lowering drugs during washout was a binary response if at least one of the following glucose lowering drugs were prescribed during the washout period ; acarbose , canagliflozin , canagliflozin combined with metformin , dapagliflozin , dapagliflozin combined with metformin , empagliflozin , pioglitazone , pioglitazone combined with metformin , rosiglitazone , rosiglitazone combined with metformin , nateglinide , repaglinide , troglitazone or guar gum . information on hba1c , bmi , weight , and diabetes duration was missing for some individuals . under the assumption that these missing data were missing at random , missing values were imputed . multiple imputation was applied , with 10 imputed datasets.15 in addition to imputation of missing measurements at initiation , missing measurements 180 days after initiation were also imputed.16 we used the listed covariates , treatment group and change in hba1c and weight for the imputation . the charlson comorbidity index was categorized into four groups : 01 , 2 , 3 , and more than 3 points . number of oads was categorized into four groups : 0 oads , 1 oads , 2 oads , 3 oads , and more than 3 oads . results for both the descriptive and the outcome analyses were pooled across the imputed datasets using rubin s rule.17 post hoc we noticed that a fraction of hba1c measurements was very low for glp-1 and insulin initiators , and that most of these measurements were located around the fourth year since glp-1 approval . as we suspected these measurements were incorrect , all hba1c measurements below 4% were treated as missing and were imputed as described above . propensity scores were estimated as the probability of initiating glp-1 in the comparison of glp-1 and insulin initiators and the probability of initiating dpp-4i in the comparison of dpp-4i and sulfonylurea initiators . the propensity score models included the covariates mentioned earlier , except the covariate describing use of the comparison drug during washout . standardized difference was used to assess balance on covariates and to select the appropriate propensity score model.18 the propensity score was estimated twice : in the whole cohort for the descriptive purpose of characteristics over time and in the subgroup that did not receive the comparison drug during washout to be used for matching in the outcome analyses . based on the propensity score , patients were matched in order to create treatment groups that were comparable with respect to the propensity score and thus the potential confounders . greedy matching was done in a 1:1 ratio , stratified by time block and within each imputed dataset . the greedy matching was done in up to eight iterations ; first the data were searched for matches based on eight digits of the propensity score , then seven digits , and continued to matches according to one digit ( see parson19 for details ) . to assess changes in patient characteristics over time , the covariates and propensity score were plotted stratified by time blocks and treatment groups . this shows both general differences between treatment groups and potential channeling , where characteristics change over time . the propensity score serves as a single variable to describe differences in characteristics across comparison groups . both the propensity score for all individuals and the propensity score for individuals suitable for matching ( without use of the comparison drug during washout ) were assessed . because of the many covariates available , only the propensity score over time will be shown in the result section , whereas graphs for each individual variable can be found in the supplementary material . in the subgroup of the pp cohort that did not receive the comparison drug during washout , linear regression analyses were used to estimate the relative effect regarding change in hba1c and change in body weight after 180 days of follow - up . crude analyses were performed based on all identified initiators , in addition to analyses of the propensity score - matched initiators , to take confounding into account . in the analyses of change in hba1c and body weight , baseline levels of hba1c and body weight , respectively , were included as covariates in the outcome model.20 the relative effect estimate and their 95% confidence intervals ( cis ) were plotted for graphical inspections of changes in effect estimates over time . data management was done using template programs developed by the department of epidemiology at novo nordisk for importing cprd data into sas format . access to cprd data was gained through approval of protocol by the isac ( https://www.cprd.com/isac/ ) . two comparison groups were used throughout the study ; initiators of glp-1 were compared with initiators of basal insulin , whereas initiators of dpp-4i were compared with initiators of sulfonylurea . the chosen comparator groups were to compare second - line ( dpp-4i and sulfonylurea ) and third - line ( glp-1 and insulin ) treatments , respectively.13 the glucose - lowering drugs were handled at the drug - class level , and combination products of metformin and dpp-4i and of rosiglitazone and sulfonylurea were considered as dpp-4i and sulfonylurea . the end of a prescription was estimated from the daily dose and amount prescribed ( typically available for glp-1 , dpp-4i , and sulfonylurea ) , or as the patient s median duration of prescription of the drug ( typically the case for insulin ) . if there were < 120 days between the estimated end date of one prescription and the start date of the next prescription , the prescriptions were considered to make up a continuous episode of drug treatment . initiation was defined as patients with a prescription of one of the four glucose - lowering drugs after minimum 180 days without a prescription of the drug ( washout period ) . two cohorts were created , by analogy with the terms used in clinical trials : the intention - to - treat ( itt ) cohorts comprise subjects with a treatment duration of at least one day , whereas the per - protocol ( pp ) cohorts comprise subjects with a treatment duration of at least 180 days . with the two comparisons , this makes up four cohorts in total . if patients were identified as incident users more than one time for the same drug , only the first initiation was considered . if patients initiated glp-1 and insulin , or dpp-4i and sulfonylurea at the same time , this initiation was censored . within each of the four cohorts , time blocks of 365-day duration were defined with start date of the first initiation of glp-1 or dpp-4i , respectively . if patient initiated the comparison drugs within the same time block , only the first initiation was used and the second initiation was censored . measures of glycated hemoglobin ( hba1c ) and body weight were used to establish outcome measures . the goal was to observe change in hba1c and in body weight during a 180-day period . however , because measurements were not always available at the exact date of treatment initiation and 180 days after initiation , the following were deployed when measurements were not available : if a measurement was available within 90 days prior to initiation , this was used as the measurement at initiation , and if a measurement was available between 90 and 179 days after initiation , this was used as the measurement 180 days after initiation . if several measurements were available within these periods , the measurement closest to the initiation date and 180 days after initiation , respectively , was used . the following covariates were used : sex ; age at initiation ; weight at initiation ; body mass index ( bmi ) at initiation ; hba1c at initiation ; duration of diabetes at initiation ; number of oral glucose - lowering drugs at initiation ; any use of glucose - lowering drugs ( sulfonylurea , basal insulin , dpp-4i , metformin , and other oral glucose - lowering drugs ) during the washout period ; any use of antihypertensive , statins , and anticoagulants during the washout period ; charlson comorbidity index14 at initiation ; and any diagnosis of hypertension , end - stage renal disease , myocardial infarction , or stroke prior to initiation . the covariate number of oral glucose - lowering drugs at index was a count of prescriptions at index of any of the following glucose - lowering drugs ; acarbose , canagliflozin , canagliflozin combined with metformin , dapagliflozin , dapagliflozin combined with metformin , empagliflozin , pioglitazone , pioglitazone combined with metformin , rosiglitazone , rosiglitazone combined with metformin , nateglinide , repaglinide , troglitazone , guar gum and metformin in the comparison of dpp-4i and sulfonylurea , and in the comparison of glp-1 and basal insulin number of other oral glucose - lowering drugs at index also included dpp-4i and sulfonylurea . the covariate other oral glucose - lowering drugs during washout was a binary response if at least one of the following glucose lowering drugs were prescribed during the washout period ; acarbose , canagliflozin , canagliflozin combined with metformin , dapagliflozin , dapagliflozin combined with metformin , empagliflozin , pioglitazone , pioglitazone combined with metformin , rosiglitazone , rosiglitazone combined with metformin , nateglinide , repaglinide , troglitazone or guar gum . information on hba1c , bmi , weight , and diabetes duration was missing for some individuals . under the assumption that these missing data were missing at random , missing values were imputed . multiple imputation was applied , with 10 imputed datasets.15 in addition to imputation of missing measurements at initiation , missing measurements 180 days after initiation were also imputed.16 we used the listed covariates , treatment group and change in hba1c and weight for the imputation . the charlson comorbidity index was categorized into four groups : 01 , 2 , 3 , and more than 3 points . number of oads was categorized into four groups : 0 oads , 1 oads , 2 oads , 3 oads , and more than 3 oads . results for both the descriptive and the outcome analyses were pooled across the imputed datasets using rubin s rule.17 post hoc we noticed that a fraction of hba1c measurements was very low for glp-1 and insulin initiators , and that most of these measurements were located around the fourth year since glp-1 approval . as we suspected these measurements were incorrect , all hba1c measurements below 4% were treated as missing and were imputed as described above . propensity scores were estimated as the probability of initiating glp-1 in the comparison of glp-1 and insulin initiators and the probability of initiating dpp-4i in the comparison of dpp-4i and sulfonylurea initiators . the propensity score models included the covariates mentioned earlier , except the covariate describing use of the comparison drug during washout . standardized difference was used to assess balance on covariates and to select the appropriate propensity score model.18 the propensity score was estimated twice : in the whole cohort for the descriptive purpose of characteristics over time and in the subgroup that did not receive the comparison drug during washout to be used for matching in the outcome analyses . based on the propensity score , patients were matched in order to create treatment groups that were comparable with respect to the propensity score and thus the potential confounders . greedy matching was done in a 1:1 ratio , stratified by time block and within each imputed dataset . the greedy matching was done in up to eight iterations ; first the data were searched for matches based on eight digits of the propensity score , then seven digits , and continued to matches according to one digit ( see parson19 for details ) . to assess changes in patient characteristics over time , the covariates and propensity score were plotted stratified by time blocks and treatment groups . this shows both general differences between treatment groups and potential channeling , where characteristics change over time . the propensity score serves as a single variable to describe differences in characteristics across comparison groups . both the propensity score for all individuals and the propensity score for individuals suitable for matching ( without use of the comparison drug during washout ) were assessed . because of the many covariates available , only the propensity score over time will be shown in the result section , whereas graphs for each individual variable can be found in the supplementary material . in the subgroup of the pp cohort that did not receive the comparison drug during washout , linear regression analyses were used to estimate the relative effect regarding change in hba1c and change in body weight after 180 days of follow - up . crude analyses were performed based on all identified initiators , in addition to analyses of the propensity score - matched initiators , to take confounding into account . in the analyses of change in hba1c and body weight , baseline levels of hba1c and body weight , respectively , were included as covariates in the outcome model.20 the relative effect estimate and their 95% confidence intervals ( cis ) were plotted for graphical inspections of changes in effect estimates over time . data management was done using template programs developed by the department of epidemiology at novo nordisk for importing cprd data into sas format . access to cprd data was gained through approval of protocol by the isac ( https://www.cprd.com/isac/ ) . in total , 304,953 patients with diabetes and a prescription of a glucose - lowering drug were identified . the first initiation of glp-1 was observed on june 14 , 2007 , whereas the first initiation of dpp-4i was observed on june 22 , 2007 . for the descriptive analyses of characteristics over time , 10,633 and 19,243 initiators of glp-1 and insulin , respectively , and 30,048 and 41,346 initiators of dpp-4i and sulfonylurea , respectively , were included in the itt cohorts . the numbers of individuals in the pp cohorts were a little smaller . for the outcome analyses , 8,398 and 13,286 initiators of glp-1 and insulin , respectively , and 10,847 and 30,643 initiators of dpp-4i and sulfonylurea , respectively , were identified . the exact number of propensity score - matched individuals varies within each imputed dataset , but within each time block nearly half of the initiators of glp-1 were matched with an initiator of insulin and nearly all initiators of dpp-4i were matched with an initiator of sulfonylurea . it was possible to create seven time blocks of 365 days in both the glp-1 and insulin , and the dpp-4i and sulfonylurea comparisons . the distributions of propensity score look similar for both the itt and pp cohorts of all identified initiators of glp-1 and insulin , and dpp-4i and sulfonylurea , respectively , and also for the pp cohort of initiators with no use of the comparison drug during washout , which is the subgroup used for propensity score matching . time blocks 17 are demonstrated as histograms of the propensity score of the latter in figures 1 and 2 . other propensity score plots are found in the supplementary material figure s2 . in general , the distribution of propensity score of the glp-1 and insulin initiators indicate an overlap in propensity score for all time blocks considered . the propensity score of dpp-4i and sulfonylurea initiators has an even greater overlap in all time blocks , and for both dpp-4i and sulfonylurea , the distribution moves from being centered on lower propensity scores in the early time blocks to being centered on 0.4 in later time blocks , which indicate comparable patients with a similar change in characteristics over time . figures of the individual covariates are found in figure s3s4 . in general , trends for the itt and pp cohort hence , there will be no distinction of the itt and pp cohort in the following text . for initiators of glp-1 and insulin , minor changes in hba1c at initiation both glp-1 and insulin initiators had an increase in use of dpp-4i during washout and a decrease in use of other oral glucose - lowering drugs during washout . for insulin initiators only , an increase in use of sulfonylurea , glp-1 , and metformin during washout was seen . plots of the individual covariates also show some general differences of the comparison groups across time . compared to initiators of insulin , the initiators of glp-1 tended to have higher bmi ; more number of glucose - lowering drugs at initiation ; increased use of metformin , other glucose - lowering drugs , dpp-4i , and statins during washout , increased proportion of with hypertension diagnoses ; lower age ; higher charlson comorbidity index , increased proportion of myocardial infarction and end - stage renal disease diagnoses ; and shorter duration of diabetes . for dpp-4i and sulfonylurea initiators , the plots of the individual covariates showed that initiators of both drugs had an increase in duration of diabetes and use of metformin during washout and a decrease in other oral glucose - lowering drugs during washout . for sulfonylurea initiators only , a minor increase in hba1c at initiation and an increase in use of dpp-4i and glp-1 during washout were seen . across the different time blocks , the initiators of dpp-4i , compared to initiators of sulfonylurea , tended to have higher bmi , more prevalent use of other glucose - lowering drugs and statins during washout , longer duration of diabetes , lower hba1c , and , in the late time blocks , higher charlson comorbidity index . the considerable overlap in propensity scores allowed for propensity score matching and thus propensity score - matched relative effectiveness estimation . figure 3a and 3b shows the difference in change in hba1c and body weight , respectively , for the comparison of glp-1 and insulin ; nonsignificant differences in change in hba1c , except from a peak in time block 4 , and a relative increase in body weight for the insulin initiators compared to glp-1 initiators in all time blocks were seen . across time blocks , the relative increase in body weight was 3.57 kg ( 95% ci : 3.21 , 3.92 ) for insulin initiators . similarly , figure 4a and 4b shows the difference in change in hba1c and body weight , respectively compared to dpp-4i and sulfonylurea ; a relative decrease in hba1c and a relative increase in body weight for sulfonylurea initiators compared to dpp-4i initiators in all time blocks were seen . across time blocks , the relative decrease in hba1c was 0.34% ( 95% ci : 0.38 , 0.30 ) and the relative increase in body weight was 1.58 kg ( 95% ci : 1.38 , 1.78 ) for sulfonylurea initiators . the distributions of propensity score look similar for both the itt and pp cohorts of all identified initiators of glp-1 and insulin , and dpp-4i and sulfonylurea , respectively , and also for the pp cohort of initiators with no use of the comparison drug during washout , which is the subgroup used for propensity score matching . time blocks 17 are demonstrated as histograms of the propensity score of the latter in figures 1 and 2 . other propensity score plots are found in the supplementary material figure s2 . in general , the distribution of propensity score of the glp-1 and insulin initiators indicate an overlap in propensity score for all time blocks considered . the propensity score of dpp-4i and sulfonylurea initiators has an even greater overlap in all time blocks , and for both dpp-4i and sulfonylurea , the distribution moves from being centered on lower propensity scores in the early time blocks to being centered on 0.4 in later time blocks , which indicate comparable patients with a similar change in characteristics over time . figures of the individual covariates are found in figure s3s4 . in general , trends for the itt and pp cohort were similar . hence , there will be no distinction of the itt and pp cohort in the following text . for initiators of glp-1 and insulin , minor changes in hba1c at initiation were observed ; however , all were within the range of 0.5% . both glp-1 and insulin initiators had an increase in use of dpp-4i during washout and a decrease in use of other oral glucose - lowering drugs during washout . for insulin initiators only , an increase in use of sulfonylurea , glp-1 , and metformin during washout was seen . plots of the individual covariates also show some general differences of the comparison groups across time . compared to initiators of insulin , the initiators of glp-1 tended to have higher bmi ; more number of glucose - lowering drugs at initiation ; increased use of metformin , other glucose - lowering drugs , dpp-4i , and statins during washout , increased proportion of with hypertension diagnoses ; lower age ; higher charlson comorbidity index , increased proportion of myocardial infarction and end - stage renal disease diagnoses ; and shorter duration of diabetes . for dpp-4i and sulfonylurea initiators , the plots of the individual covariates showed that initiators of both drugs had an increase in duration of diabetes and use of metformin during washout and a decrease in other oral glucose - lowering drugs during washout . for sulfonylurea initiators only , a minor increase in hba1c at initiation and an increase in use of dpp-4i and glp-1 during washout were seen . across the different time blocks , the initiators of dpp-4i , compared to initiators of sulfonylurea , tended to have higher bmi , more prevalent use of other glucose - lowering drugs and statins during washout , longer duration of diabetes , lower hba1c , and , in the late time blocks , higher charlson comorbidity index . the considerable overlap in propensity scores allowed for propensity score matching and thus propensity score - matched relative effectiveness estimation . figure 3a and 3b shows the difference in change in hba1c and body weight , respectively , for the comparison of glp-1 and insulin ; nonsignificant differences in change in hba1c , except from a peak in time block 4 , and a relative increase in body weight for the insulin initiators compared to glp-1 initiators in all time blocks were seen . across time blocks , the relative increase in body weight was 3.57 kg ( 95% ci : 3.21 , 3.92 ) for insulin initiators . similarly , figure 4a and 4b shows the difference in change in hba1c and body weight , respectively compared to dpp-4i and sulfonylurea ; a relative decrease in hba1c and a relative increase in body weight for sulfonylurea initiators compared to dpp-4i initiators in all time blocks were seen . across time blocks , the relative decrease in hba1c was 0.34% ( 95% ci : 0.38 , 0.30 ) and the relative increase in body weight was 1.58 kg ( 95% ci : 1.38 , 1.78 ) for sulfonylurea initiators . in this study , change in characteristics over time since beginning treatment with glp-1 and dpp-4i in comparison with basal insulin and sulfonylurea , respectively , was investigated with focus on potential channeling bias . propensity score matching was applied to assess the relative effect on change in hba1c and weight . channeling bias was not seen in the sense that the characteristics of glp-1 and dpp-4i initiators changed over time after market entry , whereas the characteristics of the initiators of the established drugs , insulin and sulfonylurea , were stable . distribution of propensity scores , used as an overall measure of differences in characteristics , showed that initiators of glp-1 and insulin were somewhat different , although with a substantial overlap , in all time blocks . initiators of dpp-4i and sulfonylurea were more comparable with an even greater overlap across time blocks and with a similar change in propensity scores over time . for the individual covariates , changes in characteristics were observed in both groups of glp-1 and insulin initiators , and among dpp-4i and sulfonylurea initiators , as well as among initiators of insulin or sulfonylurea only . there was sufficient overlap in distribution of characteristics so that propensity score matching was possible to conduct relative effectiveness analysis , even in the first year after launch . the outcome analyses suggest similar change in hba1c for glp-1 and insulin , except for the peak in time block 4 . overall , we consider this peak as a result of random variation given the absence of a difference in effects in the other time blocks . the outcome analyses suggest a greater reduction in hba1c and weight gain for sulfonylurea compared to dpp-4i . it is hypothesized that patients initiating a newly launched drug are those most ill and with more use of comedications , but such differences will disappear over time when the new drug becomes more established.1,2 in this study , also , it is hypothesized that patients initiating a newly launched drug have a history of suboptimal drug response or intolerance . direct information on drug response was not available in data , but hba1c level indicated how well - regulated patients with diabetes are . from the measurements of hba1c at initiation , there was not much difference over time between initiators of glp-1 and insulin , and dpp-4i initiators had lower hba1c at initiation compared to initiators of sulfonylurea . this indicates that patients initiating the newly launched drugs had similar response profiles as those who initiated the established drugs . the findings in this study are in line with us studies comparing sitagliptin ( merck & co. , inc . , whitehouse station , nj , usa ) with other oral glucose - lowering drugs where there were no changes in characteristics over time in the first years after launch.46 the use of real - world data is a necessity to investigate channeling bias , but it also brings along some general problems . the following potential limitations of the study were identified : real - world data are data of real - world patients , yet may suffer from missing information , which in this study was imputed by means of multiple imputation under the assumption that the missing data are missing at random . the quality of real - world data may be questioned as the data seldom are collected primarily for research purposes . this study concerns a period after implementation of quality outcome framework in cprd , through which the highest possible data quality is achieved . it was a concern that for the majority of patients in cprd , body weight was measured only once , and that measurement was carried forward . this was , however , not the case in the data available for this study . the risk of confounding is present in observational studies of the effects of medical treatments ; this study applied propensity score matching to take confounding into account . not all relevant potential confounding factors are captured in the cprd database ( eg , diet and ethnicity ) , but the propensity score model included a diverse set of covariates describing demographics , clinical measurements , comedication including glucose - lowering drugs , and comorbidities . immortal time bias , which is used to denote misclassification of observation time to exposure groups in survival analyses , was not an issue in this study since follow - up started at the first prescription made and was continued for the same duration for all subjects ( ie , 180 days ) . however , the risk of selection bias in the pp cohort in this study might be an issue because individuals who have survived or stayed in the dataset at least 180 days were selected . however , the analyses of a pp cohort are relevant because our outcome measure of hba1c is highly affected by exposure of the investigated drugs , which is more likely in the pp cohort . in this study , we looked at drug - class levels , and combination products of sulfonylurea and rosiglitazone , as well as products of dpp-4i and metformin , were classified as sulfonylurea and dpp-4i . change in characteristics over time may be affected by launch of specific brands and the use of monotherapy and combination products . it is possible that time blocks of shorter duration would reveal channeling in the very early period after launch , but the duration of 365 days was suitable as this will ensure some level of statistical power . studies in other medical areas have observed channeling bias in cprd data . in a study comparing new nonsteroidal anti - inflammatory drugs ( nsaids ) with old nsaids on the risk of gastrointestinal hemorrhage , baseline characteristics suggested that the new nsaid population had a higher baseline risk ( possibly explained by new nsaids having a safer profile with regard to gastrointestinal toxicity ) . outcome analyses seemed to adjust for confounding , including channeling bias , and showed a lower risk among patients treated with new nsaids.21 in the treatment of chronic obstructive pulmonary disease , corticosteroids have been compared with long - acting bronchodilators . the adjusted analyses showed higher risk of pneumonia among corticosteroid users , but it was not clear to the authors if this was related to the effect of the drug alone , or if channeling bias had occurred.22 the use of a sequential cohort design , where analyses are done at several time points as in this study , is generally proposed to take channeling bias into account.2327 despite the fact that no major channeling bias was observed in this study , some variation over time was seen . also , the characteristics of the initiators of the established drugs ( basal insulin and sulfo - nylurea ) changed over time , despite the time period observed ( 20062015 ) being more or less arbitrary for these drugs . the sequential cohort design in this study used the same propensity score model across time blocks , as standardized differences suggested that balance across treatment groups were established after matching . however , individual propensity score models may be needed within each time block in other settings . to conclude , we did not identify major channeling so that patients initiating the new glucose - lowering drugs differed importantly from patients initiating the established glucose - lowering drugs . in this study , the differences in patient characteristics and changes over time were at a magnitude where it was still possible to estimate propensity score and conduct matching to control for confounding in the outcome analyses , even in the first year after market entry . change in the characteristics of initiators of established drugs generally advocates for time - dependent analyses .

background : sleep / wake disturbances in patients with amyotrophic lateral sclerosis ( als ) are well - documented , however , no animal or mechanistic studies on these disturbances exist . orexin is a crucial neurotransmitter in promoting wakefulness in sleep / wake regulation , and may play an important role in sleep disturbances in als . in this study , we used sod1-g93a transgenic mice as an als mouse model to investigate the sleep / wake disturbances and their possible mechanisms in als.methods:electroencephalogram/electromyogram recordings were performed in sod1-g93a transgenic mice and their littermate control mice at the ages of 90 and 120 days , and the samples obtained from these groups were subjected to quantitative reverse transcriptase - polymerase chain reaction , western blotting , and enzyme - linked immunosorbent assay.results:for the first time in sod1-g93a transgenic mice , we observed significantly increased wakefulness , reduced sleep time , and up - regulated orexins ( prepro - orexin , orexin a and b ) at both 90 and 120 days . correlation analysis confirmed moderate to high correlations between sleep / wake time ( total sleep time , wakefulness time , rapid eye movement [ rem ] sleep time , non - rem sleep time , and deep sleep time ) and increase in orexins ( prepro - orexin , orexin a and b).conclusion : sleep / wake disturbances occur before disease onset in this als mouse model . increased orexins may promote wakefulness and result in these disturbances before and after disease onset , thus making them potential therapeutic targets for amelioration of sleep disturbances in als . further studies are required to elucidate the underlying mechanisms in the future .

amyotrophic lateral sclerosis ( als ) is a progressive and fatal neurodegenerative disease characterized by the loss of lower and upper motor neurons , leading to muscle atrophy , paralysis , and death . sleep disorders in patients with als are well - documented , and sleep - related complaints , such as insomnia , disturbed sleep , nightmare , and daytime sleepiness , have been frequently reported . moreover , several clinical studies on sleep disturbances in patients with als have been published , focusing on the frequency , characteristics , and severity of sleep problems . however , there are no animal or mechanistic studies on sleep disturbances in als . animal and mechanistic studies on sleep disturbances in other neurodegenerative diseases , such as alzheimer 's disease ( ad ) and parkinson disease ( pd ) , might give us insights into sleep disturbances in als . sleep problems in ad and pd involve disturbances in the neurotransmitter and hormone signaling , abnormal accumulations of neurotoxic proteins , and damage in the brain regions controlling the sleep / wake cycles , which could exist in als as well . precursor peptide prepro - orexin , which is produced in the hypothalamic neurons , matures into two peptides , orexin a and orexin b. these peptides promote wakefulness by activating wake - active neurons ( wan ) in the hypothalamus and brain stem . the actions of orexins are mediated by two receptors , orexin-1 ( ox1r ) and orexin-2 ( ox2r ) receptors . we hypothesized that there are disturbances of sleep and wakefulness in the als mouse models and that orexin is an important molecule responsible for those disturbances . in the present study with sod1-g93a transgenic mice , which are extensively used as animal model for mechanistic and therapeutic studies on als , we used sleep / wake activity recordings and molecular techniques to test our hypothesis . transgenic sod1-g93a mice used in this study were bred from male hemizygous sod1-g93 a mice ( b6sjl - tg [ sod1-g93a ] 1 gur / j ) to female b6sjl / f1 hybrids . the genotyping of sod1-g93a mice was performed by polymerase chain reaction ( pcr ) , as previously reported . male hemizygous sod1-g93a mice and female b6sjl / f1 hybrids were both purchased from the jackson laboratories ( bar harbor , me , usa ) . all mice were housed under a controlled temperature ( 22 1c ) and 12 hours : 12 hours light - dark cycle . all animal studies were approved by the institutional animal care and use committee of peking university third hospital , and conducted in accordance with the guide for the care and use of laboratory animals of peking university . to monitor disease progression , all mice were tested using the rota rod test apparatus ( ugo basile , varese , italy ) . the day on which a mouse first dropped off the rota rod within 600 seconds was designated as a day of disease onset for that mouse . using this testing criterion , we observed that all the sod1-g93a transgenic mice in this study had disease onset between 90 and 120 days of age . thus , we determined the two - time points in our study : 90-day representing the age before disease onset and 120-day representing the age after disease onset . twenty - eight mice were divided into four groups : sod1-g93a group at 90-day ( n = 8) , control group at 90-day ( n = 6 ) , sod1-g93a group at 120-day ( n = 6 ) , and control group at 120-day ( n = 8) . all mice received electroencephalogram / electromyogram ( eeg / emg ) recordings at 90 or 120 days of age . after performing the recordings , the cerebrospinal fluid ( csf ) and brain samples were collected . the hypothalamus and brain stem were isolated from the brain tissue for real - time reverse transcriptase ( rt)-pcr , western blotting , and enzyme - linked immunosorbent assay ( elisa ) . surgeries and eeg / emg recordings were performed as previously described . briefly , after anesthesia , 28 mice were implanted with eeg and emg electrodes . the eeg electrodes were placed epidurally on the cortex , and the emg electrodes were placed in the dorsal neck muscles . ten days after surgery , the electrodes were connected to recording cables attached to the mp150 system ( biopac , goleta , ca , usa ) . eeg and emg recordings were performed in this manner from the freely behaving mice for 24 hours . data were analyzed using sleep sign 2.0 software ( biopac , goleta , ca , usa ) . total rna from the hypothalamus and brainstem tissues was extracted using trizol reagent ( takara , dalian , china ) according to the manufacturer 's instructions . first - strand cdna was synthesized at 42c with fastquant rt kit ( tiangen , beijing , china ) using 1 g of total rna . the amplification was performed using a superreal premix plus kit ( tiangen ) and an abi 7500 rt - pcr system ( applied biosystems , foster city , usa ) . the cdna was amplified with an initial denaturation step ( 95c , 15 minutes ) , and then with 40 pcr cycles consisting of a denaturation step ( 95c , 10 seconds ) and an annealing / extension step ( 58c , 32 seconds ) . -actin was used as internal control to calculate the relative abundance of each mrna ( n = 46/group ) . the specific sets of primers were as follows : prepro - orexin : f : tgaactttccttctacaaaggttc , r : caacagttcgtagagacggca ; orexin1 receptor : f : cgccaaccctatcatctacaa , r : gctctgcaaggacaaggactt ; orexin2 receptor : f : gctcaccagcataagcacact , r : tatctctttgagcagacatggg ; -actin : f : cctagcaccatgaagatcaagat , r : actcatcgtactcctgcttgct . total protein was extracted from the hypothalamus samples using the total protein extraction kit ( applygen , beijing , china ) , consisting of radioimmunoprecipitation assay lysis buffer , phenylmethylsulfonyl fluoride , protease inhibitors , and phosphatase inhibitors , according to the manufacturer 's instructions . protein concentration was detected by the bicinchoninic acid protein assay using a protein assay kit ( applygen ) . samples containing equal amounts of total protein ( 20 g ) were separated by 12% sds - page and transferred to a polyvinylidene fluoride membrane ( millipore , billerica , ma , usa ) . the membrane was blocked with 3% bovine serum albumin ( bsa ) in tris buffered saline ( tbs ) containing 0.1% tween 20 ( tbs - t ) for one hour , and then incubated overnight at 4c with the primary orexin antibody ( anti - orexin - prepro , 1:500 , millipore , billerica , ma , usa ) or primary -actin antibody ( anti--actin , 1:5000 , earthox , san francisco , ca , usa ) . -actin was used as an internal loading control . the membrane was then incubated with secondary irdye 800cw goat anti - rabbit ( 1:10,000 , li - cor , lincoln , ne , usa ) or goat anti - mouse ( 1:10,000 , li - cor , lincoln , ne , usa ) antibodies at 37c for one hour . quantitation of immunoreactive bands was performed using an odyssey infrared imaging system ( li - cor , lincoln ) . levels of orexin a and orexin b in mouse csf and brain tissues were measured by orexin a / orexin b elisa kits ( bluegene biotech , shanghai , china ) . mol / l , ph 7.07.2 ) per 0.5 g tissue and centrifuged . samples ( csf or brain homogenates supernatant ) or standards were added to 96-well plates coated with anti - mouse orexin a / b antibody and elisa was performed according to manufacturer 's protocol . the independent - samples t - test and pearson correlation analyses were performed using spss 19.0 ( spss inc . , transgenic sod1-g93a mice used in this study were bred from male hemizygous sod1-g93 a mice ( b6sjl - tg [ sod1-g93a ] 1 gur / j ) to female b6sjl / f1 hybrids . the genotyping of sod1-g93a mice was performed by polymerase chain reaction ( pcr ) , as previously reported . male hemizygous sod1-g93a mice and female b6sjl / f1 hybrids were both purchased from the jackson laboratories ( bar harbor , me , usa ) . all mice were housed under a controlled temperature ( 22 1c ) and 12 hours : 12 hours light - dark cycle . all animal studies were approved by the institutional animal care and use committee of peking university third hospital , and conducted in accordance with the guide for the care and use of laboratory animals of peking university . to monitor disease progression , all mice were tested using the rota rod test apparatus ( ugo basile , varese , italy ) . the day on which a mouse first dropped off the rota rod within 600 seconds was designated as a day of disease onset for that mouse . using this testing criterion , we observed that all the sod1-g93a transgenic mice in this study had disease onset between 90 and 120 days of age . thus , we determined the two - time points in our study : 90-day representing the age before disease onset and 120-day representing the age after disease onset . twenty - eight mice were divided into four groups : sod1-g93a group at 90-day ( n = 8) , control group at 90-day ( n = 6 ) , sod1-g93a group at 120-day ( n = 6 ) , and control group at 120-day ( n = 8) . all mice received electroencephalogram / electromyogram ( eeg / emg ) recordings at 90 or 120 days of age . after performing the recordings , the cerebrospinal fluid ( csf ) and brain samples were collected . the hypothalamus and brain stem were isolated from the brain tissue for real - time reverse transcriptase ( rt)-pcr , western blotting , and enzyme - linked immunosorbent assay ( elisa ) . surgeries and eeg / emg recordings were performed as previously described . briefly , after anesthesia , 28 mice were implanted with eeg and emg electrodes . the eeg electrodes were placed epidurally on the cortex , and the emg electrodes were placed in the dorsal neck muscles . ten days after surgery , the electrodes were connected to recording cables attached to the mp150 system ( biopac , goleta , ca , usa ) . eeg and emg recordings were performed in this manner from the freely behaving mice for 24 hours . data were analyzed using sleep sign 2.0 software ( biopac , goleta , ca , usa ) . total rna from the hypothalamus and brainstem tissues was extracted using trizol reagent ( takara , dalian , china ) according to the manufacturer 's instructions . first - strand cdna was synthesized at 42c with fastquant rt kit ( tiangen , beijing , china ) using 1 g of total rna . the amplification was performed using a superreal premix plus kit ( tiangen ) and an abi 7500 rt - pcr system ( applied biosystems , foster city , usa ) . the cdna was amplified with an initial denaturation step ( 95c , 15 minutes ) , and then with 40 pcr cycles consisting of a denaturation step ( 95c , 10 seconds ) and an annealing / extension step ( 58c , 32 seconds ) . -actin was used as internal control to calculate the relative abundance of each mrna ( n = 46/group ) . the specific sets of primers were as follows : prepro - orexin : f : tgaactttccttctacaaaggttc , r : caacagttcgtagagacggca ; orexin1 receptor : f : cgccaaccctatcatctacaa , r : gctctgcaaggacaaggactt ; orexin2 receptor : f : gctcaccagcataagcacact , r : tatctctttgagcagacatggg ; -actin : f : cctagcaccatgaagatcaagat , r : actcatcgtactcctgcttgct . total protein was extracted from the hypothalamus samples using the total protein extraction kit ( applygen , beijing , china ) , consisting of radioimmunoprecipitation assay lysis buffer , phenylmethylsulfonyl fluoride , protease inhibitors , and phosphatase inhibitors , according to the manufacturer 's instructions . protein concentration was detected by the bicinchoninic acid protein assay using a protein assay kit ( applygen ) . samples containing equal amounts of total protein ( 20 g ) were separated by 12% sds - page and transferred to a polyvinylidene fluoride membrane ( millipore , billerica , ma , usa ) . the membrane was blocked with 3% bovine serum albumin ( bsa ) in tris buffered saline ( tbs ) containing 0.1% tween 20 ( tbs - t ) for one hour , and then incubated overnight at 4c with the primary orexin antibody ( anti - orexin - prepro , 1:500 , millipore , billerica , ma , usa ) or primary -actin antibody ( anti--actin , 1:5000 , earthox , san francisco , ca , usa ) . the membrane was then incubated with secondary irdye 800cw goat anti - rabbit ( 1:10,000 , li - cor , lincoln , ne , usa ) or goat anti - mouse ( 1:10,000 , li - cor , lincoln , ne , usa ) antibodies at 37c for one hour . quantitation of immunoreactive bands was performed using an odyssey infrared imaging system ( li - cor , lincoln ) . levels of orexin a and orexin b in mouse csf and brain tissues were measured by orexin a / orexin b elisa kits ( bluegene biotech , shanghai , china ) . brain tissues were homogenized in 1 ml pbs ( 0.02 mol / l , ph 7.07.2 ) per 0.5 g tissue and centrifuged . samples ( csf or brain homogenates supernatant ) or standards were added to 96-well plates coated with anti - mouse orexin a / b antibody and elisa was performed according to manufacturer 's protocol . the independent - samples t - test and pearson correlation analyses were performed using spss 19.0 ( spss inc . , sleep / wake recordings were performed in both sod1-g93a transgenic mice and their control groups . in the 90-day sod1-g93a transgenic mice , across a 24-hour recording period , the total sleep time ( tst ) was significantly decreased ( [ 443.23 40.42 minutes ] vs. [ 569.97 39.04 minutes ] , p < 0.05 ) and wakefulness was increased ( [ 996.78 40.42 minutes ] vs. [ 870.03 39.04 minutes ] , p < 0.05 ) , compared to the littermate controls . non - rapid eye movement ( nrem ) sleep ( [ 411.29 35.41 minutes ] vs. [ 533.48 37.01 minutes ] , p < 0.05 ) and deep sleep ( ds ) ( [ 47.35 9.55 minutes ] vs. [ 107.35 11.36 minutes ] , p < 0.01 ) were also reduced . no significant difference was found in rem sleep and light sleep between the two groups for the 24-hour period [ figure 1a ] . eeg / emg recordings for 24 hours from the sod1-g93a transgenic mice and littermate control mice at 90 days and 120 days of age . ( a ) tst , nrem , and ds are significantly decreased in the 90-day sod1-g93a transgenic mice . ( b ) in the 120-day sod1-g93a transgenic mice , wake is also significantly enhanced . tst : total sleep time ; wake : wakefulness ; rem : rapid eye movement sleep ; nrem : non - rapid eye movement sleep ; ls : light sleep ; ds : deep sleep ; eeg / emg : electroencephalogram / electromyogram . in the 120-day sod1-g93a transgenic mice , a remarkable increase in wakefulness ( [ 1110.33 38.16 minutes ] vs. [ 804.29 74.57 minutes ] , p < 0.01 ) , and a decrease in tst ( [ 329.67 38.16 minutes ] vs. [ 635.71 74.57 minutes ] , p < 0.01 ) , nrem ( [ 306.03 36.81 minutes ] vs. [ 534.68 82.68 minutes ] , p < 0.05 ) and ds ( [ 22.88 10.27 minutes ] vs. [ 107.90 25.21 minutes ] , p < 0.05 ) were observed . however , in contrast with the 90-day sod1-g93a transgenic mice , rem sleep was decreased ( [ 23.62 9.96 minutes ] vs. [ 101.08 13.93 minutes ] , p < 0.01 ) in the 120-day sod1-g93a transgenic mice compared to the 120-day littermate controls [ figure 1b ] . next , we evaluated the changes in the orexin system in the sod1-g93a transgenic mice . first , we tested the level of mrna and protein expression of prepro - orexin . q - pcr showed increased levels of prepro - orexin mrna in the hypothalamus of the 90-day ( 1.84 0.24 vs. 1.00 0.20 , p < 0.05 ) and 120-day ( 2.06 0.25 vs. 1.00 0.14 , p < 0.01 ) sod1-g93a transgenic mice , compared to controls [ figure 2a ] . the western blotting analysis also showed increased levels of prepro - orexin protein in the hypothalamus of both 90-day ( 2.33 0.24 vs. 1.00 0.18 , p < 0.01 ) and 120-day ( 2.30 0.14 vs. 1.00 0.19 , p < 0.01 ) sod1-g93a transgenic mice as compared to littermate controls [ figure 2b ] . prepro - orexin increases in sod1-g93a transgenic mice . ( a ) q - pcr was performed in the hypothalamic tissues from the sod1-g93a transgenic mice and control groups . prepro - orexin mrna is significantly elevated in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice , compared to control . ( b ) western blotting was performed with antibody anti - orexin - prepro in the sod1-g93a transgenic mice and control groups . prepro - orexin expression is increased in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice as compared to control . data are expressed as mean standard error of mean ( n = 46/group ) . we then tested the protein levels of orexin a and b , using elisa . in the hypothalamus , the level of orexin a was significantly enhanced in the 90-day ( [ 2126.47 70.65 pg / mg ] vs. [ 1591.52 61.25 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 2166.32 115.98 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice , compared to their respective littermate control mice [ figure 3a ] . the level of orexin a was also elevated significantly in the brain stem of the 90-day ( [ 1540.93 34.87 pg / mg ] vs. [ 1170.04 47.73 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 1583.96 21.64 pg / mg ] vs. [ 1224.87 51.62 pg / mg ] protein , p there was no significant difference in orexin a levels between the sod1-g93a transgenic mice and control groups in the csf , at either time points [ figure 3c ] . similarly , orexin b levels were also significantly increased in the hypothalamus of the 90-day ( [ 3485.35 74.62 pg / mg ] vs. [ 2352.45 142.56 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 3656.34 139.17 117.70 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice [ figure 3d ] . in the brain stem , orexin b was also significantly enhanced in both transgenic groups ( 90-day : [ 2726.79 34.06 pg / mg ] vs. [ 2411.70 64.18 pg / mg ] protein , p < 0.01 and 120-day : [ 2736.45 57.32 pg / mg ] vs. [ 2369.33 83.57 pg / mg ] protein , p < 0.05 ) as compared to littermate controls [ figure 3e ] . in the csf , no significant difference was found in orexin b levels between the respective transgenic and control groups [ figure 3f ] . orexin a and b increase in sod1-g93a transgenic mice . ( a - c ) elisa reveals that orexin a levels are enhanced in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice than controls , but there is no significant difference for orexin a in the csf . ( d - f ) elisa shows increased the level of orexin b in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice compared with control , but no significant difference is seen in the csf . ht : hypothalamus ; bs : brain stem ; csf : cerebrospinal fluid ; elisa : enzyme - linked immunosorbent assay . q - pcr analysis revealed no significant difference in the mrna levels of ox1r [ figure 4a and 4b ] and ox2r [ figure 4c and 4d ] in the hypothalamus or the brain stem between the sod1-g93a transgenic mice and controls . q - pcr of orexin-1 receptor mrna show no significant difference in the hypothalamus ( a ) and brain stem ( b ) of the 90 and 120 days sod1-g93a transgenic mice than controls . no significant difference is found in orexin-2 receptor mrna in the hypothalamus ( c ) and brain stem ( d ) of the 90 and 120 days sod1-g93a transgenic mice than controls . pearson correlation analyses were performed between sleep / wake time ( tst , wake , rem , nrem , and ds ) and expression levels of orexins ( prepro - orexin , orexin a and b ) . absolute values of the correlation coefficient confirmed high correlation between the sleep / wake time ( tst , wake , and nrem ) and expression levels of orexins . meanwhile , there was a moderate correlation between rem / ds and orexins [ table 1 ] . pearson correlation coefficient of sleep / wake time and orexins tst : total sleep time ; wake : wakefulness ; rem : rapid eyes movement sleep ; nrem : nonrapid eyes movement sleep ; ds : deep sleep ; ht : hypothalamus ; bs : brain stem . sleep / wake recordings were performed in both sod1-g93a transgenic mice and their control groups . in the 90-day sod1-g93a transgenic mice , across a 24-hour recording period , the total sleep time ( tst ) was significantly decreased ( [ 443.23 40.42 minutes ] vs. [ 569.97 39.04 minutes ] , p < 0.05 ) and wakefulness was increased ( [ 996.78 40.42 minutes ] vs. [ 870.03 39.04 minutes ] , p < 0.05 ) , compared to the littermate controls . non - rapid eye movement ( nrem ) sleep ( [ 411.29 35.41 minutes ] vs. [ 533.48 37.01 minutes ] , p < 0.05 ) and deep sleep ( ds ) ( [ 47.35 9.55 minutes ] vs. [ 107.35 11.36 minutes ] , p < 0.01 ) were also reduced . no significant difference was found in rem sleep and light sleep between the two groups for the 24-hour period [ figure 1a ] . eeg / emg recordings for 24 hours from the sod1-g93a transgenic mice and littermate control mice at 90 days and 120 days of age . ( a ) tst , nrem , and ds are significantly decreased in the 90-day sod1-g93a transgenic mice . ( b ) in the 120-day sod1-g93a transgenic mice , wake is also significantly enhanced . tst : total sleep time ; wake : wakefulness ; rem : rapid eye movement sleep ; nrem : non - rapid eye movement sleep ; ls : light sleep ; ds : deep sleep ; eeg / emg : electroencephalogram / electromyogram . in the 120-day sod1-g93a transgenic mice , a remarkable increase in wakefulness ( [ 1110.33 38.16 minutes ] vs. [ 804.29 74.57 minutes ] , p < 0.01 ) , and a decrease in tst ( [ 329.67 38.16 minutes ] vs. [ 635.71 74.57 minutes ] , p < 0.01 ) , nrem ( [ 306.03 36.81 minutes ] vs. [ 534.68 82.68 minutes ] , p < 0.05 ) and ds ( [ 22.88 10.27 minutes ] vs. [ 107.90 25.21 minutes ] , p < 0.05 ) were observed . however , in contrast with the 90-day sod1-g93a transgenic mice , rem sleep was decreased ( [ 23.62 9.96 minutes ] vs. [ 101.08 13.93 minutes ] , p < 0.01 ) in the 120-day sod1-g93a transgenic mice compared to the 120-day littermate controls [ figure 1b ] . next , we evaluated the changes in the orexin system in the sod1-g93a transgenic mice . first , we tested the level of mrna and protein expression of prepro - orexin . q - pcr showed increased levels of prepro - orexin mrna in the hypothalamus of the 90-day ( 1.84 0.24 vs. 1.00 0.20 , p < 0.05 ) and 120-day ( 2.06 0.25 vs. 1.00 0.14 , p < 0.01 ) sod1-g93a transgenic mice , compared to controls [ figure 2a ] . the western blotting analysis also showed increased levels of prepro - orexin protein in the hypothalamus of both 90-day ( 2.33 0.24 vs. 1.00 0.18 , p < 0.01 ) and 120-day ( 2.30 0.14 vs. 1.00 0.19 , p < 0.01 ) sod1-g93a transgenic mice as compared to littermate controls [ figure 2b ] . ( a ) q - pcr was performed in the hypothalamic tissues from the sod1-g93a transgenic mice and control groups . prepro - orexin mrna is significantly elevated in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice , compared to control . ( b ) western blotting was performed with antibody anti - orexin - prepro in the sod1-g93a transgenic mice and control groups . prepro - orexin expression is increased in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice as compared to control . data are expressed as mean standard error of mean ( n = 46/group ) . we then tested the protein levels of orexin a and b , using elisa . in the hypothalamus , the level of orexin a was significantly enhanced in the 90-day ( [ 2126.47 70.65 pg / mg ] vs. [ 1591.52 61.25 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 2166.32 115.98 pg / mg ] vs. [ 1446.18 41.31 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice , compared to their respective littermate control mice [ figure 3a ] . the level of orexin a was also elevated significantly in the brain stem of the 90-day ( [ 1540.93 34.87 pg / mg ] vs. [ 1170.04 47.73 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 1583.96 21.64 pg / mg ] vs. [ 1224.87 51.62 pg / mg ] protein , p < 0.01 ) transgenic groups [ figure 3b ] . there was no significant difference in orexin a levels between the sod1-g93a transgenic mice and control groups in the csf , at either time points [ figure 3c ] . similarly , orexin b levels were also significantly increased in the hypothalamus of the 90-day ( [ 3485.35 74.62 pg / mg ] vs. [ 2352.45 142.56 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 3656.34 139.17 pg / mg ] vs. [ 2411.57 117.70 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice [ figure 3d ] . in the brain stem , orexin b was also significantly enhanced in both transgenic groups ( 90-day : [ 2726.79 34.06 64.18 pg / mg ] protein , p < 0.01 and 120-day : [ 2736.45 57.32 pg / mg ] vs. [ 2369.33 83.57 pg / mg ] protein , p < 0.05 ) as compared to littermate controls [ figure 3e ] . in the csf , no significant difference was found in orexin b levels between the respective transgenic and control groups [ figure 3f ] . orexin a and b increase in sod1-g93a transgenic mice . ( a - c ) elisa reveals that orexin a levels are enhanced in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice than controls , but there is no significant difference for orexin a in the csf . ( d - f ) elisa shows increased the level of orexin b in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice compared with control , but no significant difference is seen in the csf . ht : hypothalamus ; bs : brain stem ; csf : cerebrospinal fluid ; elisa : enzyme - linked immunosorbent assay . q - pcr analysis revealed no significant difference in the mrna levels of ox1r [ figure 4a and 4b ] and ox2r [ figure 4c and 4d ] in the hypothalamus or the brain stem between the sod1-g93a transgenic mice and controls . q - pcr of orexin-1 receptor mrna show no significant difference in the hypothalamus ( a ) and brain stem ( b ) of the 90 and 120 days sod1-g93a transgenic mice than controls . no significant difference is found in orexin-2 receptor mrna in the hypothalamus ( c ) and brain stem ( d ) of the 90 and 120 days sod1-g93a transgenic mice than controls . pearson correlation analyses were performed between sleep / wake time ( tst , wake , rem , nrem , and ds ) and expression levels of orexins ( prepro - orexin , orexin a and b ) . absolute values of the correlation coefficient confirmed high correlation between the sleep / wake time ( tst , wake , and nrem ) and expression levels of orexins . meanwhile , there was a moderate correlation between rem / ds and orexins [ table 1 ] . pearson correlation coefficient of sleep / wake time and orexins tst : total sleep time ; wake : wakefulness ; rem : rapid eyes movement sleep ; nrem : nonrapid eyes movement sleep ; ds : deep sleep ; ht : hypothalamus ; bs : brain stem . in summary , we observed marked sleep disturbances in the sod1-g93a mouse model of als , and demonstrated that the increase in expression of orexins correlated with these sleep disturbances in these mice . sleep / wake disturbances are often reported by patients with als , but no related animal studies have been published . the exact mechanism behind the sleep / wake disturbances in als remains to be determined . in the present study , for the first time , we observed enhanced wakefulness and reduced sleep time ( including tst , nrem , and ds ) in both 90-day and 120-day sod1-g93a transgenic mice . these results indicate that sleep / wake disturbances are early symptoms in these sod1-g93a transgenic mice , and these disturbances may be correlated with the onset of als . in addition , rem sleep time was significantly decreased in the 120-day sod1-g93a transgenic mice as compared to the 90-day sod1-g93a transgenic mice , suggesting an aggravation and the possible link of sleep disturbances with disease progression . therefore , studies on sleep disturbances in als are of significance to elucidate the etiology and pathogenesis of als . moreover , considering the early occurrence of sleep disorders in ad and pd , we speculate that the dysregulation of sleep might be common in the early stages of neurodegenerative diseases . to further investigate the mechanism of sleep / wake disturbances in als sleep is a complicated behavior regulated by wakefulness promoting systems , sleep promoting systems , and circadian rhythms . wakefulness is promoted by multiple groups of wan , which are mainly located in the brain stem and hypothalamus , including cholinergic neurons , orexinergic neurons , and dopaminergic neurons . neurotransmitters in these wan , including acetylcholine , dopamine , glutamate , histamine , norepinephrine , orexin , and serotonin , promote wakefulness . prepro - orexin , which is produced in hypothalamic neurons , matures into two neuropeptides , orexin a and orexin b. these two peptides activate monoaminergic and cholinergic neurons in the hypothalamus and brain stem to maintain a long , consolidated awake period . in our study , prepro - orexin was increased in the hypothalamus in the 90 and 120 days sod1-g93a transgenic mice . orexin a and b were significantly enhanced in the hypothalamus and brain stem of the 90 days and 120 days sod1-g93a transgenic mice compared with littermate controls . these results suggest a strong association between increased expression of orexins and the extension of wakefulness in als mice . pearson correlation analysis , which showed moderate to high correlations between sleep / wake time and expression of orexins , further confirmed this association . these results indicate that the extension of wakefulness and sleep / wake disturbances in the als mice may be caused , at least partly , by increase in expression of orexins . according to the absolute value of the correlation coefficient in table 1 , nrem had the highest correlation with expression of orexins among all stages of sleep . however , no differences were detected in the levels of orexin receptors in the hypothalamus and brain stem between the als mice and control groups , suggesting that enhanced wakefulness and other sleep disturbances in these transgenic mice were not promoted by upregulation of orexin receptors . in addition , the levels of orexin a and b were only elevated in the brain tissue other than the csf in the sod1-g93a transgenic mice . this is consistent with the clinical study by van rooij et al . , which demonstrated that csf orexin levels were normal in patients with als . sleep disturbances occur before disease onset in the als sod1-g93a transgenic mice . increased expression of orexins further experiments need to be carried out in the future to investigate the underlying mechanisms .

combined spinal - epidural anesthesia ( cse ) is considered safe in lower limb surgeries . we report a case of sudden neurological deterioration in a stable postoperative patient who was given cse for total knee replacement and low molecular weight heparin in postoperative period . on the 4th postoperative day , she developed sudden onset weakness in left lower limb along with bladder incontinence . magnetic resonance imaging spine revealed a subdural hematoma at l2-l3 level . immediate laminectomy along with cord decompression was done and patient recovered well except for a persistent foot drop on left side .

subdural spinal hematoma is an extremely rare complication of spinal and epidural anesthesia and may lead to cauda equina syndrome , bowel bladder dysfunction or even complete permanent paraplegia . a 60-year - old female patient was diagnosed to have osteoarthritis of right knee joint and was advised knee arthroplasty . enoxaparin sodium 40 mg ( lmwh ) was started 9 h after surgery and was followed by once daily dose . foot pump and compression stockings were applied for deep vein thrombosis ( dvt ) prophylaxis . on day 2 , the epidural catheter was removed 13 h after last dose , and she was discharged on 3 postoperative day in stable condition . at the time of discharge , on 4 postoperative day , she developed sudden onset pain in the lower back which was radiating to left lower limb . the pain was followed by loss of sensation and weakness in the left lower limb . it showed a large subdural hematoma , which was compressing the thecal sac at l2-l3 level . laminectomy with decompression of spinal cord and evacuation of hematoma was done on the emergent basis . postoperatively patient recovered well with complete control over bowel and bladder functions , but foot drop on the left side persisted after 6 months of follow - up . combined spinal epidural anesthesia though safe has been associated with uncommon neurologic complications like subdural hematoma . venous stasis , endothelial injury and hypercoagulable conditions contribute to the postoperative development of dvt which can be a life - threatening complication . in surgeries as total knee and hip replacement , direct injury to the large vessels of lower limb predispose patients for increased chances of dvt especially in absence of postoperative anticoagulation prophylaxis . however , prophylaxis must be balanced against the risks for postoperative bleeding and epidural hematoma . spinal and epidural anesthetic procedures in combination with anti - coagulant therapy represent the fifth most common cause of spinal hematoma . three cases developed paraplegia due to subdural hematoma which was due to underlying coagulation disorder . and horlocker and wedel highlighted the risk of spinal hematoma which gets aggravated by concomitant administration of anticoagulants and anti - platelet drugs . fryburg et al . , reported two patients who suffered from spontaneous acute spontaneous subdural hematoma while taking fondaparinux for venous thromboembolism prophylaxis . it is recommended that lmwh should be used once daily and to be started 12 - 24 h postoperatively , especially when indwelling spinal catheter are present or when concomitant antiplatelet or oral anticoagulant medication is being given . patients who develop extra or subdural hematoma usually present with acute back or radicular pain radiating to limbs , followed by progressive sensory and motor involvement in either one or both the lower limbs along with saddle anesthesia and bowel and bladder dysfunction . in most of the cases , these symptoms usually develop shortly after the procedure with mean age of onset being 30 h after operation , but in our case , the patient developed sudden onset neurological symptoms suggestive of cauda equina syndrome on 4 postoperative day . urgent mri was done to confirm the diagnosis and patient was immediately taken to the operation theatre where laminectomy with decompression of spinal canal and evacuation of hematoma was done . , neurosurgical intervention must be sought immediately , because recovery is unlikely if surgery is postponed for more than 8 h. this case highlights that even in the absence of an existing spinal pathology and carefully administrated spinal - epidural anesthesia , cse may cause dreaded neurologic complication like subdural hematoma especially in the presence of anti - coagulant therapy . hence frequent 4 hourly neurological examination is essential for early detection of hematoma , which should be continued 24 hours postoperatively following a neuraxial block or upto 24 hours following catheter removal if patient is anticoagulated . in a case of suspected cauda equina syndrome , the patient should be investigated and urgent decompression should be done on emergent basis to prevent permanent neurological sequelae .

hypophosphatasia is a congenital disease characterized by deficiency of serum and tissue non - specific alkaline phosphatase activity . the disease occurs due to mutations in the liver / bone / kidney alkaline phosphatase gene . six clinical forms of hypophosphatasia are recognized . systemic symptoms of the disease are respiratory complications , premature craniosynostosis , widespread demineralization and rachitic changes in the metaphases , stress fractures , chondrocalcinosis and osteoarthropathy . characteristic dental symptoms include premature loss of deciduous teeth , premature exfoliation of fully rooted permanent teeth , severe dental caries and alveolar bone loss . this clinical report describes the prosthetic rehabilitation of a sixteen year old female patient with hypophosphatasia with partial anodontia and no occlusion . patient was managed clinically by saving her remaining teeth , fabricating a maxillary overdenture and mandibular cast partial denture . this not only helped her functionally in biting and chewing food and achieving a good occlusion , but also restored her confidence and self image by improving the aesthetics .

hypophosphatasia is a rare enzymopathy that normally presents within the first few years of life and often has profound effects upon the periodontium . it is characterized by low or zero levels of serum and tissue non - specific alkaline phosphatase , which is necessary for normal bone mineralization . it is a heritable disorder characterised by defective mineralisation of the skeletal and dental structures of the body , and a deficiency in liver / bone / kidney isoenzyme of alkaline phosphatase ( alp ) . the symptoms are highly variable in their clinical expression , ranging from stillbirth without mineralized bone , to early loss of tooth without any bone symptoms . in 1957 , six clinical types of hypophosphatasia are currently recognized in the forms of perinatal lethal , benign prenatal , infantile , childhood , adult and odontohypophosphatasia . some infants can only live for a few days and have respiratory complications due to hypoplastic lungs and rachitic deformities of the chest . a few oral manifestations of hypophosphatasia can be given as follows : lack of cementum formationroot resorptionenamel hypoplasiadelayed appearance and premature decay of deciduous and permanent dentition and total tooth loss before 20 years of age . lack of cementum formation delayed appearance and premature decay of deciduous and permanent dentition and total tooth loss before 20 years of age . there appears to be no male / female preference , and mean age for the first tooth to be lost is 21 - 22 months . enlarged pulp spaces and roots either fail to develop fully or there is early resorption of the apices . long bones with elongated finger like processes of uncalcified tissue extending longitudinally into the poorly formed osseous tissue at the ends of long bones can be seen radiogpahically . beaten copper appearance multiple radiolucent areas on skull called gyral or convoluted markings which result due to increase in intra cranial pressure are seen . reduced enamel thickness , enlarged pulp chamber and alveolar bone atrophy are common dental findings along with generalised reduction in bone density in jaw bones . evidence of skeletal defects is found in nearly all patients and includes hypomineralization , rachitic changes , and incomplete vertebrate ossification and occasionally lateral bony spurs on the ulnae and fibulae . the most sensitive substrate marker for hypophosphatasia is an increased pyridoxal 5-phosphate ( plp ) plasma level , which often correlates with the disease severity . and , although it remains only a research technique , quantitation of urinary inorganic pyrophosphate ( ppi ) levels , which are elevated in most hypophosphatasia patients , has been reported to accurately detect carriers . in addition , increased urinary levels of phosphaoethanalamine ( pea ) are observed in most patients . differential diagnosis for this disease include osteogenesis imperfect , rickets and achondrogenesis this clinical report describes the prosthetic rehabilitation of a sixteen year old female patient with hypophosphatasia , who reported to our department with poor oral hygiene , missing teeth , unaaesthetic appearance and low self esteem . clinical examination revealed severely resorbed maxillary and mandibular alveolar ridges with partial anodontia and several root stumps . teeth present were right maxillary central incisor , maxillary right first and second molar , maxillary left first and second molar , mandibular left first and second molar , and mandibular right first and second molar . opg revealed severe alveolar bone loss with enlarged pulp chambers and stunted root growth of molars . there were horizontally impacted third molars and reduced bone density with severe bone atrophy in both maxilla and mandible . [ figure 3 ] on examining the lateral cephalogram , a prognathic relation of mandible with maxilla was observed [ figure 4 ] . pre - operative intra - oral photograph showing missing teeth , root stumps and poor oral hygiene of the patient in the case report severely atrophied alveolar ridge and partial anodontia of the patient in the case report orthopantomogram showing horizontally impacted third molars and reduced bone density with severe bone atrophy in both maxilla and mandible of the patient in the case report lateral cephalogram showing prognathic relation of the mandibular to the maxilla of the patient in the case report the patient was tested for alp levels , which were found to be 3 times lower than the normal values confirming the diagnosis of hypophosphatasia . treatment for this patient was planned keeping in mind her psychological aspect , young age and severe bone loss with partial anodontia . it was carried out step by step in various phases : rehabilitating the periodontal condition with non - surgical intervention that included complete oral prophylaxis and curettage ; extractions of the unwanted root stumps ; and , root canal treatment of 11 , 16 and 27 . patient was thus prepared for prosthetic intervention to replace missing teeth , improve the jaw relation and establish a comfortable working occlusion for the young girl . besides , her extraoral features like sunken cheeks , depressed mentolabial sulcus and decreased nasolabial angle were also kept in mind while fabricating the dentures . as the alveolar ridges were severely resorbed , conventional complete denture or partial denture fabrication implant retained prosthesis was also not an option as bone height was very less due to resportion , and the distance between the bone crest and the occlusal level was more , which would have led to an unfavourable crown : implant ratio . hence , it was decided to restore the missing teeth and occlusion with maxillary overdenture and mandibular cast partial denture , as this was the most favourable and conservative option for this young girl with severe bone loss and lost psychological confidence . after the pre - treatment procedures , the tentative aesthetic anterior try - in was performed [ figure 5 ] and improved aesthetics with adequate lip fullness and improved inter arch relationship was observed . the root canal treated teeth was carried out for the abutment teeth ( 11 , 16 and 27 ) , followed by the fabrication of post integrated metal copings [ figure 6 ] . mandibular cast partial metal framework was planned with a ring clasp placed on 37 and embrasure clasps on 46 and 47 . the regular procedures for overdenture and cast partial denture fabrication were carried out in the lab , followed by final denture insertion [ figure 8 ] . after placement of the dentures , we could appreciate the restored aesthetics , improved occlusion and an established vertical dimension for this young girl with a better functional occlusion that improved her speech , mastication and most importantly , her self confidence . the post operative frontal and lateral profile view depicted an improvement over the depressed mentolabial sulcus , and an increased and more acceptable nasolabial angle and cheek bulging , with an improved normal interjaw relationship . [ figures 9 10 ] patient was more confident with her improved aaesthetic smile and restored occlusion . tentative aesthetic anterior try - in of the patient in the case report post intergrated metal copings for the root canal treated abutment teeth ( 11 , 16 and 27 ) of the patient in the case report mesh framework of cast partial denture for the mandibular arch of the patient in the case report intra - oral post - operative view after maxillary overdenture and mandibular cast partial denture insertion in the mouth of the patient in the case report post - operative extra - oral frontal view with aesthetic smile , and adequate lip and cheek fullness in the patient in the case report post - operative extra - oral lateral profile view showing improved nasolabial angle , mentolabial sulcus and inter jaw relationship a few oral manifestations of hypophosphatasia can be given as follows : lack of cementum formationroot resorptionenamel hypoplasiadelayed appearance and premature decay of deciduous and permanent dentition and total tooth loss before 20 years of age . lack of cementum formation delayed appearance and premature decay of deciduous and permanent dentition and total tooth loss before 20 years of age . there appears to be no male / female preference , and mean age for the first tooth to be lost is 21 - 22 months . enlarged pulp spaces and reduced cortical bone thickness of the mandibular angle are one of the common characteristics . long bones with elongated finger like processes of uncalcified tissue extending longitudinally into the poorly formed osseous tissue at the ends of long bones can be seen radiogpahically . beaten copper appearance multiple radiolucent areas on skull called gyral or convoluted markings which result due to increase in intra cranial pressure are seen . reduced enamel thickness , enlarged pulp chamber and alveolar bone atrophy are common dental findings along with generalised reduction in bone density in jaw bones . evidence of skeletal defects is found in nearly all patients and includes hypomineralization , rachitic changes , and incomplete vertebrate ossification and occasionally lateral bony spurs on the ulnae and fibulae . the most sensitive substrate marker for hypophosphatasia is an increased pyridoxal 5-phosphate ( plp ) plasma level , which often correlates with the disease severity . and , although it remains only a research technique , quantitation of urinary inorganic pyrophosphate ( ppi ) levels , which are elevated in most hypophosphatasia patients , has been reported to accurately detect carriers . in addition , increased urinary levels of phosphaoethanalamine ( pea ) are observed in most patients . this clinical report describes the prosthetic rehabilitation of a sixteen year old female patient with hypophosphatasia , who reported to our department with poor oral hygiene , missing teeth , unaaesthetic appearance and low self esteem . clinical examination revealed severely resorbed maxillary and mandibular alveolar ridges with partial anodontia and several root stumps . teeth present were right maxillary central incisor , maxillary right first and second molar , maxillary left first and second molar , mandibular left first and second molar , and mandibular right first and second molar . opg revealed severe alveolar bone loss with enlarged pulp chambers and stunted root growth of molars . there were horizontally impacted third molars and reduced bone density with severe bone atrophy in both maxilla and mandible . [ figure 3 ] on examining the lateral cephalogram , a prognathic relation of mandible with maxilla was observed [ figure 4 ] . pre - operative intra - oral photograph showing missing teeth , root stumps and poor oral hygiene of the patient in the case report severely atrophied alveolar ridge and partial anodontia of the patient in the case report orthopantomogram showing horizontally impacted third molars and reduced bone density with severe bone atrophy in both maxilla and mandible of the patient in the case report lateral cephalogram showing prognathic relation of the mandibular to the maxilla of the patient in the case report the patient was tested for alp levels , which were found to be 3 times lower than the normal values confirming the diagnosis of hypophosphatasia . treatment for this patient was planned keeping in mind her psychological aspect , young age and severe bone loss with partial anodontia . it was carried out step by step in various phases : rehabilitating the periodontal condition with non - surgical intervention that included complete oral prophylaxis and curettage ; extractions of the unwanted root stumps ; and , root canal treatment of 11 , 16 and 27 . patient was thus prepared for prosthetic intervention to replace missing teeth , improve the jaw relation and establish a comfortable working occlusion for the young girl . besides , her extraoral features like sunken cheeks , depressed mentolabial sulcus and decreased nasolabial angle were also kept in mind while fabricating the dentures . as the alveolar ridges were severely resorbed , conventional complete denture or partial denture fabrication was ruled out . implant retained prosthesis was also not an option as bone height was very less due to resportion , and the distance between the bone crest and the occlusal level was more , which would have led to an unfavourable crown : implant ratio . hence , it was decided to restore the missing teeth and occlusion with maxillary overdenture and mandibular cast partial denture , as this was the most favourable and conservative option for this young girl with severe bone loss and lost psychological confidence . after the pre - treatment procedures , the tentative aesthetic anterior try - in was performed [ figure 5 ] and improved aesthetics with adequate lip fullness and improved inter arch relationship was observed . the root canal treated teeth was carried out for the abutment teeth ( 11 , 16 and 27 ) , followed by the fabrication of post integrated metal copings [ figure 6 ] . mandibular cast partial metal framework was planned with a ring clasp placed on 37 and embrasure clasps on 46 and 47 . the regular procedures for overdenture and cast partial denture fabrication were carried out in the lab , followed by final denture insertion [ figure 8 ] . after placement of the dentures , we could appreciate the restored aesthetics , improved occlusion and an established vertical dimension for this young girl with a better functional occlusion that improved her speech , mastication and most importantly , her self confidence . the post operative frontal and lateral profile view depicted an improvement over the depressed mentolabial sulcus , and an increased and more acceptable nasolabial angle and cheek bulging , with an improved normal interjaw relationship . [ figures 9 10 ] patient was more confident with her improved aaesthetic smile and restored occlusion . tentative aesthetic anterior try - in of the patient in the case report post intergrated metal copings for the root canal treated abutment teeth ( 11 , 16 and 27 ) of the patient in the case report mesh framework of cast partial denture for the mandibular arch of the patient in the case report intra - oral post - operative view after maxillary overdenture and mandibular cast partial denture insertion in the mouth of the patient in the case report post - operative extra - oral frontal view with aesthetic smile , and adequate lip and cheek fullness in the patient in the case report post - operative extra - oral lateral profile view showing improved nasolabial angle , mentolabial sulcus and inter jaw relationship initially recognized by rathbun in 1948 , hypophosphatasia is a rare inborn error of metabolism caused by low activity of the tissue - nonspecific isoenzyme of alkaline phosphatase ( tnsalp ) . at least six clinical forms of hypophosphatasia have been reported , although form assignment is often challenging . two other forms include odontohypophosphatasia ( no clinical changes in long bones are present , only biochemical and dental manifestations ) and pseudohypophosphatasia . the latter is clinically indistinguishable from infantile hypophosphatasia , because serum alp activity is normal . the form od hypophosphatasia a patient is in , should be considered before making a dental treatment planning . there are less skeletal deformities ; however , more dental symptoms at the odontohypophosphatasia form as in our case several authors have reported that dentin structure is reduced in thickness and also mineral content of dentin is low in hypophosphatasia . also in this case as the number of teeth present were very few , it was difficult to obtain support to restore function by conventional prosthesis . the patient 's anxiety was also of concern during the treatment period , as the patient was young and apprehensive about how the treatment would proceed , and about the final outcome . however , constant motivation of the patient and her family led to the successful completion of treatment . a patient with hypophosphatasia , presenting with partial anodontia and highly resorbed ridges with severely constricted maxillary arch , was treated successfully with cast partial denture in mandibular arch , and an overdenture for the maxillary arch . the occlusion was restored to a functional form , and the patient was highly satisfied with the aaesthetic outcome of the treatment . thus , to conclude , a patient with hypophosphatasia poses a challenge for the clinician , especially when the patient is of young age as such patients have high expectations ; however , a thorough diagnosis , constant motivation and critical evaluation of all treatment options and correct treatment planning and selection can lead to a successful treatment for our patients . this case presentation is different as it presents a complete intra as well as extra oral rehabilitation of a young girl with hypophosphatasia , and with severely resorbed ridges , in whom conventional treatment planning was not possible . besides , managing the aesthetics , function and occlusion along with improving the extra - oral appearance of the patient posed a great challenge .

american trypanosomiasis or chagas disease is a debilitating disease representing an important social problem that affects , approximately , 10 million people in the world . the main aggravating factor of this situation is the lack of an effective drug to treat the different stages of this disease . in this context , the search for trypanocidal substances isolated from plants , synthetic or semi synthetic molecules , is an important strategy . here , the trypanocidal potential of gallates was assayed in epimastigotes forms of t. cruzi and also , the interference of these substances on the mitochondrial membrane potential of the parasites was assessed , allowing the study of the mechanism of action of the gallates in the t. cruzi organisms . regarding the preliminary structure - activity relationships , the side chain length of gallates plays crucial role for activity . nonyl , decyl , undecyl , and dodecyl gallates showed potent antitrypanosomal effect ( ic50 from 1.46 to 2.90 m ) in contrast with benznidazole ( ic50 = 34.0 m ) . heptyl gallate showed a strong synergistic activity with benznidazole , reducing by 105-fold the ic50 of benznidazole . loss of mitochondrial membrane potential induced by these esters was revealed . tetradecyl gallate induced a loss of 53% of the mitochondrial membrane potential , at ic50 value .

chagas disease ( or american trypanosomiasis ) was recognized by world health organization as one of thirteen neglected tropical diseases in the world . it was estimated that over 10 million people are infected , and latin america was considered the area of the highest prevalence . this parasitic disease is caused by trypanosoma cruzi ( family , trypanosomatidae and order , kinetoplastida ) , a hemoflagellate protozoa , which can be found on several strains with different mechanisms of pathogenesis , immunogenicity , treatment response , and epidemiology . the development of infection includes an acute phase which lasts up to six months after infection , an indeterminate phase with no symptoms , and a chronic phase in which approximately 30% of patients show clinical evidence of heart disease or megavisceras . the current chemotherapy uses benznidazole , a nitroimidazole derivative , which is orally administered in acute phase and short - term chronic phase [ 6 , 7 ] . the usefulness of this drug is limited by its narrow therapeutic window and due to serious side effects , such as anorexia , nausea , headache , paresthesia , peripheral neuropathies , dermatitis , central nervous system ( cns ) depression , and maniac symptoms [ 7 , 8 ] . however , several parasite subpopulations with different host tissue 's tropism contribute to the low clinical efficacy of this drug . therefore , there are efforts for the discovery and design of new therapeutic compounds to treat chagas disease , due to the complexity of this disease . gallic acid ( 3,4,5-trihydroxybenzoic acid ) is a precursor of hydrolysable tannins in the biosynthesis that occurs in plants and its natural and semisynthetic derivatives have been associated with a wide variety of biological activities , such as antiproliferative [ 1113 ] , chemopreventive , antihemolytic , antioxidant , and anti - inflammatory activities . however , the main interest in gallic acid and in its derivatives has been associated with their antimicrobial properties . it has been shown that n - octyl gallate possesses fungicidal activity against saccharomyces cerevisiae and zygosaccharomyces bailii in any stage of their growth [ 18 , 19 ] . leal and coworkers reported the potent fungitoxicity of n - nonyl gallate against yeasts , dermatophytes , and hialohyphomycetes . lauryl gallate showed antibacterial activity against gram - positive bacteria , including methicillin - resistant staphylococcus aureus ( mrsa ) [ 21 , 22 ] . thus , the aim of this study was to investigate the antitrypanosomal activity of gallic acid and its esters against epimastigote forms of trypanosoma cruzi . further analysis involving interactions generated by combinations between alkyl gallates and benznidazole was also carried out . in addition , the apoptosis - inducing activity by alkyl gallates was studied through the parasite mitochondrial membrane potential assay . the compounds tested in this study were synthesized as described previously [ 14 , 15 ] . epimastigote forms of trypanosoma cruzi of y strain , described by silva and nussenzweig ( 1953 ) , which is considered a standard strain of type i , were grown in lit media ( liver infusion tryptose media ) at 28c . the cytotoxicity assay with epimastigotes t. cruzi was performed using the mtt colorimetric method as described by muelas - serrano et al . the parasites were treated with different concentrations of the substances for 72 hours and then , the parasite viability was estimated by measuring the absorbance at 595 nm . the influence of the treatments with the gallates in the mitochondrial membrane potential was assessed . the parasites were treated with the substances during 72 hours using , as standard concentration , the ic50 previously determined by the mtt assay . the percentage of parasite cells which suffered loss in the mitochondrial membrane potential due to the treatment with the tested compounds was measured by flow cytometry using jc-1 dye ( 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide , bd mitoscreen kit ) according to the manufacturer 's instruction [ 27 , 28 ] . a facscanto i cytometer was used , and the data were recorded and analyzed on the software of the equipment . the antitrypanosomal activity of a homologous series of alkyl gallates , gallic acid , and other analogues was evaluated against epimastigote forms of trypanosoma cruzi . this study was carried out in order to correlate chemical characteristics of the molecules with the observed activity , including the importance of free hydroxyl groups and side chain length . gallic acid ( 1 ) was found to be totally inactive against t. cruzi ( ic50 > 100 m ) and its derivatives up to hexyl gallate ( 27 ) exhibited similar ic50 values ( ic50 > 100 m ) . heptyl gallate ( 8 , ic50 = 37.3 0.9 ) showed weak activity which increased in the case of longer carbon chain derivatives up to undecyl gallate ( 12 , ic50 = 1.46 0.0 ) . however , the increase in the carbon chains , becoming bigger than undecyl gallate , led to lower activity , as shown for compounds 13 and 14 ( table 1 ) . such results indicate that the 3,4,5-trihydroxyphenyl moiety appeared to be necessary but not sufficient for antiprotozoal activity . on the other hand , kubo et al . ( 2001 ) verified that the length of the alkyl chain is not a major contributor but plays an important role in eliciting the activity of the gallates . among the thirteen tested gallates ( 214 ) esterified with linear alcohols from c1 to c14 , best results were observed for esters with chain lengths ranging from 9 to 12 , which present clogp values of 4.115.30 , as reported previously by rosso et al . n - undecyl gallate ( 12 ) , the most active derivative , with ic50 of 1.46 m ( clogp = 4.90 ) , was twenty - three times more potent than benznidazole , used as positive control ( ic50 = 34.0 m ) . such results indicate that esterification led to appearance of antitrypanosomal activity , suggesting that a free carboxyl group was not crucial for protozoal death . in contrast , esters with clogp values lower than 3.32 or higher than 0.92 exhibited lower potency than the positive control . altogether , there is a clear and positive correlation among ic50 values , alkyl chain length and its contribution to the lipophilicity , which are in agreement with previous studies of this homologous series [ 19 , 29 ] . furthermore , the trypanocidal potential increased with the increase in the number of carbons found on the side chain until reaching the maximum activity , in this case at n - undecyl gallate ( 12 ) , and longer carbon chains showed lower trypanocidal activity , reaching a cutoff at n - tetradecyl gallate ( 14 ) . if the homologs longer than 14 might show antitrypanosomal activity , their ic50 values will be superior at 17.6 m , representing low potency , not corroborating for further chemical or biological investments . in previous studies , the cutoff phenomenon was observed for alkanols , which was correlated with their amphipathic properties . in the case of alkyl gallates , their amphiphilicity appeared to be dependent on the presence of two features : hydrophilic phenolic hydroxyls and hydrophobic alkyl chain . fujita and kubo ( 2002 ) reported the antifungal activity of gallates , suggesting that these compounds possessed amphiphilic capacity and surfactant properties and they might act disrupting the fluidic bilayer membrane , leading to fungal death . the synergistic effects of the combination using gallic acid and its esters with benznidazole , the antichagasic drug , were also evaluated . the definitions and mathematical determination of the formula for synergism were used as described by hellmann et al . briefly , the fractional inhibitory concentration ( fic ) index was calculated by using the formula fic index = a / b + a / c . in this formula , a , b , and c were ic50 of gallate with benznidazole , ic50 of gallate alone , and ic50 of benznidazole alone , respectively . from this calculation , a fic index 0.5 was considered evidence of synergistic effect , as this value can be generated only if the concentrations of both compounds in combination do not exceed one - quarter of the ic50 of either drug tested alone . a fic index > 4.0 indicates antagonism . when tested alone , some gallates ( 27 ) have not led to a significant number of epimastigotes toxicity , even at the highest concentration tested ( 100 m ) . therefore , to calculated fic index for these compounds , the ic50 was arbitrarily defined as 50 m ( table 2 ) . the interactions between compounds 114 and benznidazole were highly variable , indicating dependence on the gallates chain length . association between gallic acid ( 1 ) and short esters ( 24 ) with benznidazole was indifferent . additive interaction was observed for inactive short esters ( 5 and 6 ) and most potent long esters , which exhibited fic index values ranging from 0.560 to 0.765 . interestingly , medium chain length gallates ( 79 ) and the long chain length ester 14 with fic index values bellow 0.257 exhibited synergism . the most significant synergistic effect was observed for heptyl gallate ( 8 , fic = 0.084 ) , which reduced by 10-fold the concentration of benznidazole necessary to inhibit cell growth in 50% ( ic50 of 37.3 0.93 m ) . synergism was observed at m level , evidencing a strong antitrypanosomal activity and the potential of medium and long chain alkyl gallates as prototypes for further studies and development of novel therapeutic agents . it is also worth mentioning that the significant results from the association of benznidazole and alkyl gallates , which enabled the use of smaller doses of benznidazole , extended dosing intervals between consecutive administrations and short - term treatment leading to fewer and less intense side effects . urbina and coauthors ( 1988 , 1995 , 2002 , 2009 ) [ 3134 ] described systematic studies involving antifungal drugs and their associations as trypanocidal combined agents for treatment of chagas disease , such as azoles ( ketoconazole and posaconazole ) with mevinolin , terbinafine , aspirin , and amiodarone . recently , the interaction between benznidazole and parthenolide was investigated against t. cruzi , which evidenced synergistic and additive activities against epimastigotes and trypomastigotes , respectively . additionally , trypanocidal gallates ( 814 ) were investigated for their influence on the mitochondrial membrane potential . considering that the loss of mitochondrial membrane potential is a typical characteristic of apoptotic cells , it was used to elucidate a possible mode of parasite death . in living cells , the membrane of healthy mitochondria is polarized and jc-1 is rapidly internalized , increasing the concentration gradient , which may lead to its aggregation . in the flow cytometer , the jc-1 monomers were detected in the fl-1 channel ( fitcs ) , while its aggregates were observed in the red channel fl-2 ( pe ) . on the other hand , injured mitochondria exhibits depolarized membrane and jc-1 remains in the cytoplasm as monomers . then , we suggest that the cells which loss their mitochondrial membrane potential , which could be apoptotic cells , lose their fluorescence and could be captured in q2 quadrant , whereas normal cells are detected in q4 quadrant . figure 1 presents the flow cytometry analysis of n - undecyl gallate ( 12 ) on t. cruzi . among the tested gallates , compounds 8 , 9 , and 14 promoted important loss in the mitochondrial membrane potential , which maybe an apoptosis - inducing activity , with percentage of cells with reduction of mitochondrial membrane potential of 46.6 , 43.6 , and 53.2 , respectively . figure 1(a ) shows that untreated epimastigotes ( control ) did not lose their mitochondrial membrane potential which can be observed due to the high fluorescence detected in fl-2 channel ( pe ) . similarly , it is observed that n - undecyl gallate ( 12 ) ( figure 1(b ) ) did not stimulate this influence in the treated cells since it did not induce a reduction of fluorescence as shown by the control , suggesting the maintenance of the membrane potential . in contrast , the cells treated with pentamidine ( figure 1(c ) ) show intense decrease in the fluorescence represented by pe , suggesting a loss of mitochondrial membrane potential which may indicate apoptosis of the parasites analyzed . the results of flow cytometry for n - undecyl gallate ( 12 ) are presented on table 3 . the results showed that the gallate esters used in this study induced loss of the mitochondrial membrane potential and maybe apoptosis in the parasites . this indicates that one of the mechanisms of cell death induced by the gallates activity could be similar to the apoptosis . this can be visualized by comparing the results with pentamidine , an apoptosis - inducing drug , which led to different percentages of mitochondrial membrane potential relatively to the control . the treatment with some gallates , such as the substances 8 , 9 , and 14 , showed a percentage of loss of mitochondrial membrane potential similar to the pentamidine treatment . pentamidine is a dicationic drug which has been used in the last 50 years for the treatment of african trypanosomiasis and antimony - resistant leishmaniasis and is known to cause changes in mitochondrial metabolism . the drug that can cause a collapse in mitochondrial membrane potential leads to an imbalance in intracellular ca . electronic microscopy transmission studies with cells treated with pentamidine showed condensation and disruption of kinetoplast dna core and collapse of mitochondrial membrane [ 39 , 40 ] . based on the results obtained by the flow cytometry and cytotoxicity analysis , it is possible to infer that other mechanisms of action , different from apoptosis , might be associated with the death of the parasites , since the substances that caused more significant dysfunction in the mitochondrial membrane potential ( 8 , 9 , and 14 ) are not the same substances that according to the cytotoxicity assay cause higher rates of parasites death ( 10 , 11 , 12 , and 13 ) . in addition , maintenance of cell viability might be associated with low affinity or steric hindrance of the molecular target due to its physical - chemical properties . another possible mechanism of action of the gallic acid esters would be similar to the results found by abe et al . these authors showed that some gallates are potent inhibitors of the enzyme squalene epoxidase , which is involved in the biosynthesis of ergosterol and , therefore , is essential for the survival of the microorganism . ergosterol is essential to the parasite because it is the most abundant sterol found in the membrane of lower eukaryotes and is therefore found in fungi and parasitic protozoa such as leishmania and trypanosoma [ 4346 ] . the inhibition of sterol biosynthesis is associated with the loss of cell membrane fluidity , which might lead to the parasite death through the same mechanism of action of commercial drugs , such as terbinafine . mitochondria are one of the first organelles to be affected after treatment of t. cruzi with ergosterol biosynthesis inhibitors , and even stronger effect may occur when an inhibitor of squalene epoxidase is used [ 3647 ] . due to the results of our experiments that demonstrated that there are changes in the mitochondrial membrane potential of the parasites when treated with gallates , we suggest that these compounds cause the parasite death by more than one pathway , which probably includes loss of mitochondrial membrane potential , correlated with the induction of apoptosis and , also , interfering in the biosynthesis of ergosterol in the parasites . the phenol moiety is often associated with cytotoxic effects due to the possible free radical or reactive oxygen species formation , which might be deleterious to the parasite metabolism . however its esterification yielding gallate esters with different side chain lengths afforded compounds with strong trypanocidal potential , which is probably also due to a change in the mitochondrial membrane potential and an interference in the biosynthesis of ergosterol in the parasites . it is important to clarify that previous studies demonstrate a correlation between epimastigote and trypomastigote forms of t. cruzi , the last one the infective form . thus , this research encourages the development of new research aiming to apply the gallates as an alternative to current therapy against chagas disease .

backward disequilibrium is observed frequently in daily clinical practice . however , there are no epidemiological data concerning this postural disorder . defined by a posterior position of the centre of mass with respect to the base of support , backward disequilibrium is abnormal postural behavior , usually characterized by a posterior trunk tilt in standing and sitting positions , which predisposes subjects to backward falls . many afflictions whether they are somatic ( degenerative , ischemic and traumatic brain lesions ) , psychosomatic ( psychomotor disadaptation syndrome , confinement to bed , nonuse situations ) or psychological ( depression ) can cause backward disequilibrium . a vicious circle of falls , and loss of autonomy can arise and this is the main consequence of backward disequilibrium . thus , in this paper , we review backward disequilibrium in elderly subjects with regard to the causes , consequences , assessment , and management .

the prevalence and the severity of posture and gait disorders increase with ageing ( salthouse and somberg 1982 ; maki et al 1994 ; mourey et al 1998 ; rankin et al 2000 ) . indeed , balance controlis a complex sensori - motor function which requires the integration of vestibular , visual , and somesthetic information into the central nervous system ( pyramidal , extrapyramidal , and cerebellar systems ) in order to maintain antigravitar postures and to produce a suitable response to any balance perturbation ( horak and mcpherson 1996 ) . in addition , the quality of muscle and joints is important in the balance function ( alencar et al 2007 ) . increased age is associated with impaired performance in vestibular , visual and somesthesic systems , a decline in the speed of information transmission and changes in the mode of information processing in the brain , allof which lead to balance and postural disorders ( brocklehurst et al 1982 ; aniansson et al 1986 ; woollacott 1993 ; choy et al 2003 ; yordanova et al 2004 ) . concerning the vestibule , it has been reported that the number of vestibular hair cells falls by 20% to 40% in healthy subjects aged 70 years and older , when compared to young subjects , and the number of vestibular nerve fibers has also been found to decrease with age ( alpini et al 2004 ) . regarding vision , decreased visual acuity , restriction of visual field , poor depth perception , losses in contrast sensitivity at the intermediate and high spatial frequencies have been found in elderly subjects ( alpini et al 2004 ) . at the somesthesic level , various alterations in the morphology and functions of the peripheral nervous system are related to ageing . elderly subjects also suffer from a loss of myelinated and unmyelinated nerve fibers associated with axonal atrophy . aging also induces a decline in nerve conduction velocity and sensory discrimination ( verdu et al 2000 ) . moreover , the age - related decline in muscle and joint quality contributes to the proprioceptive abnormalities and also explains effector disorders found in elderly subjects . indeed , with advancing age , the frequency of sarcopenia increases and the decrease in muscle mass is associated with the replacement of muscle fibers by fat and connective tissue ( lexell 1995 ; alpini et al 2004 ; crepaldi and maggi 2005 ) . moreover , there is an increase in degenerative osteoarticular lesions and osteoporosis ( crepaldi and maggi 2005 ) . independently of sensorimotor system , postural controlis also enabled by spatial references such as the subjective vertical ( massion 1992 ) . the subjective vertical ( which can be haptic , postural , or visual ) corresponds to an individual s perception of the vertical and is therefore different from the true or gravitational vertical ( manckoundia et al 2007a ) . concerning gait , physiological ageing is associated with a reduction in the length and the height of step , an increase in dual foot contact time , and a resulting reduction in gait speed ( craik et al 1992 ; alexander 1996 ) . the negative effects of certain chronic afflictions on posture and gait will compound those of physiological ageing . these chronic diseases may be neurological ( parkinson s disease or syndrome , the after - effects of stroke , neuropathy , normal pressure hydrocephalus and amyotrophic lateral sclerosis , for example ) or osteoarticular ( osteoarthritis of the lower limbs ) , muscular , visual ( cataract , age - related macular degeneration ) , vestibular and can be associated with certain geriatric syndromes such as psychomotor disadaptation syndrome ( knutsson 1972 ; fisher 1982 , pfitzenmeyer et al 1999 ; hausdorff 2000 ; lee and scudds 2003 ; corriveau et al 2004 ; manckoundia et al 2008 ) . these balance and postural disorders make elderly people vulnerable , accelerate functional decline and contribute to the loss of autonomy ( rankin et al 2000 ) . backward disequilibrium ( bd ) is a postural disorder which is characterized by a posterior position of the centre of mass with respect to the base of support in the standing and sitting position predisposing subjects to backward falls . this postural disorder is defined by a posterior body tilt ( mourey et al 2004 ) . the pathophysiological mechanisms leading to bd behavior , however , have not yet been clearly identified . nevertheless , as observed in patients with stroke , deterioration in the subjective vertical could be associated with the appearance of bd . indeed , the literature reports that the pusher behavior due to hemiplegia is associated with deterioration in the representation of the subjective verticalin the frontal plane ( lopez et al 1997 ; prennou et al 1998 ) . a similar patho - physiological hypothesis may be involved in bd . to test this hypothesis we conducted a pilot study and found a direct correlation between the subjective vertical and the bd ( manckoundia et al 2007a ) . in this pilot study , the subjective vertical was assessed by measuring the postural vertical , which corresponds to the self - perception of the body s orientation in space and in the sagittal plane ( manckoundia et al 2007a ) . moreover , bd could be related to accentuated postural tone of the extensor muscles due to a disorder in the excitability of the corresponding descending pathways ( porter and lemon 1993 ) , or to the fear of the void in front of the subject linked to the fear of falling in the context of post - fall syndrome ( murphy and isaacs 1982 ; pfitzenmeyer et al 1999 ; mourey et al 2004 ) . although the geriatrician can recognize bd , which is not uncommon in daily clinical practice , there are no tools to evaluate the severity . moreover , only a few authors have examined this postural disorder in elderly subjects ( mourey et al 2004 ) and these studies concern mainly parkinsonian syndromes in the anglo - saxon literature ( visser et al 2003 ) . finally , there are no epidemiological data on bd . thus , in this paper , we review bd , a postural disorder that has severe consequences and can compromise functional autonomy in elderly subjects . indeed , the autonomy and functional well - being of elderly subjects suffering from bd could be improved by early diagnosis and management using basic interventions ( manckoundia et al 2007b ) . cortical and subcortical lesions of the central nervous system are the main abnormalities leading to bd . there are reports of a positive correlation between cortical and subcortical cerebral lesions , identified on magnetic resonance imaging , and bd ( pfitzenmeyer et al 2002 ) . ischemic , degenerative and traumatic lesions of the cerebral trunk , the white matter , the frontal lobes , the gray matter and the cerebellum could induce bd in elderly subjects ( van wegen et al 2002 ) . ischemic and hemorrhagic strokes are the most frequent etiologies of bd as they are in nonidiopathic parkinsonian syndromes ( visser et al 2003 ) . in addition , the literature reports other subcortical abnormalities such as normal pressure hydrocephalus or amyotrophic lateral sclerosis , which could also be associated with bd ( desai and swash 1999 ; blomsterwall et al 2000 ) . backward disequilibrium could also be due to an imbalance between the ankle extensor muscles and the ankle flexor muscles , because of hypertonia of extensor muscles , which could be caused by a peripheral neurological lesion ( porter and lemon 1993 ) . the main psychosomatic disorder associated with bd is psychomotor disadaptation syndrome and its acute form , the post - fall syndrome ( pfitzenmeyer et al 2001 ) . psychomotor disadaptation syndrome , which was first described by gaudet and colleagues in 1986 , is characterized by postural impairments , including bd , neurologic signs and psychological disorders ( mourey et al 2004 ) . several chronic diseases such as parkinsonian syndromes , multiple system atrophy , progressive supranuclear palsy , lewy body disease , binswanger s disease , subcortical vascular lesions , normal pressure hydrocephalus , lymphomas of nervous system , meningoencephalitis , hiv infection , and depression may be associated with psychomotor disadaptation syndrome ( pfitzenmeyer 1999 ; mourey et al 2004 ) . finally , other situations such as confinement to bed and/ or depression could be associated with motor and postural disorders including bd ( mobily and skemp kelley 1991 ; mourey et al 2004 ; turcu et al 2004 ) . elderly subjects suffering from bd have a high risk of falling backwards ( pfitzenmeyer et al 1999 ) . falls are associated with an increase in morbi - mortality in the elderly ( wild et al 1981 ) . hip fracture is the more dreaded injury ( rose and maffulli 1999 ) , and there is a risk of posttraumatic subdural hematoma ( noltie and denham 1981 ) . fallers can also be affected by many other injuries ( tinetti et al 1995 ) . these traumatic consequences are associated with a high risk of mortality ( tinetti et al 1995 ) . among the psychological consequences of falls , there is the fear of standing , walking , and falling ( gomez and curcio 2007 ) , allof which can exacerbate bd behavior and result in a vicious circle . the social consequences of falls include the loss of autonomy , institutionalization and the risk of becoming bedridden ( tinetti and williams 1997 ) . in addition , the loss of autonomy is associated with a decrease in physical activity leading to reduced muscle mass and functional decline which predispose the elderly to falls . falls therefore have economic repercussions and represent a major public health problem ( tinetti et al 1994 ) . regarding the consequences of falls , it is necessary for all health workers , especially physicians , involved in the management of elderly subjects to know how to recognize and manage syndromes and afflictions that could cause falls . in this context , bd , which is one of main risk factors of falls , must be detected . however , elderly subjects suffering from mild form of bd show postural instability that does not necessarily result in falls . this , nevertheless , limits their daily activities , induces hypoactivity and may lead to the use of walking aids . backward disequilibrium could have severe psychological consequences in elderly subjects even in the absence of falls . indeed , bd is often associated with a loss of self - confidence during standing and walking . this could cause fear of standing , walking and falling as described in psychomotor disadaptation syndrome ( mourey et al 2004 ; manckoundia et al 2007b ) . this fear of standing , walking and falling can appear even in the absence of a history of falls ( legters 2002 ) . the progressive loss of autonomy is the logicaloutcome of both loss of self - confidence during standing / walking , and fear of standing . bd and its motor and psychological consequences lead to postural disadaptation which in turn leads to dependence . finally , bd can bring about depression mostly because of the fear of walking and falling , loss of independence and decreased of quality of life . indeed , elderly people who become aware of the deterioration in their physical capacities will lose self - confidence and self - esteem , develop a sense of insecurity and become withdrawn . as a result elderly subjects suffering from bd have a high risk of falling backwards ( pfitzenmeyer et al 1999 ) . falls are associated with an increase in morbi - mortality in the elderly ( wild et al 1981 ) . hip fracture is the more dreaded injury ( rose and maffulli 1999 ) , and there is a risk of posttraumatic subdural hematoma ( noltie and denham 1981 ) . fallers can also be affected by many other injuries ( tinetti et al 1995 ) . these traumatic consequences are associated with a high risk of mortality ( tinetti et al 1995 ) . among the psychological consequences of falls , there is the fear of standing , walking , and falling ( gomez and curcio 2007 ) , allof which can exacerbate bd behavior and result in a vicious circle . the social consequences of falls include the loss of autonomy , institutionalization and the risk of becoming bedridden ( tinetti and williams 1997 ) . in addition , the loss of autonomy is associated with a decrease in physical activity leading to reduced muscle mass and functional decline which predispose the elderly to falls . falls therefore have economic repercussions and represent a major public health problem ( tinetti et al 1994 ) . regarding the consequences of falls , it is necessary for all health workers , especially physicians , involved in the management of elderly subjects to know how to recognize and manage syndromes and afflictions that could cause falls . in this context , bd , which is one of main risk factors of falls , must be detected . however , elderly subjects suffering from mild form of bd show postural instability that does not necessarily result in falls . this , nevertheless , limits their daily activities , induces hypoactivity and may lead to the use of walking aids . backward disequilibrium could have severe psychological consequences in elderly subjects even in the absence of falls . indeed , bd is often associated with a loss of self - confidence during standing and walking . this could cause fear of standing , walking and falling as described in psychomotor disadaptation syndrome ( mourey et al 2004 ; manckoundia et al 2007b ) . this fear of standing , walking and falling can appear even in the absence of a history of falls ( legters 2002 ) . the progressive loss of autonomy is the logicaloutcome of both loss of self - confidence during standing / walking , and fear of standing . bd and its motor and psychological consequences lead to postural disadaptation which in turn leads to dependence . finally , bd can bring about depression mostly because of the fear of walking and falling , loss of independence and decreased of quality of life . indeed , elderly people who become aware of the deterioration in their physical capacities will lose self - confidence and self - esteem , develop a sense of insecurity and become withdrawn . as a result among the tests to assess balance and gait in elderly subjects , only the minimum motor test includes items for a qualitative analysis of bd ( mourey et al 2005 ) . the other usual tests , such as the tinettitest ( tinetti 1986 ) , the timed up and go test ( podsiadlo and richardson 1991 ) or the berg balance scale ( berg et al 1992 ) , are not able to diagnose bd . indeed , these tests are only suitable to evaluate the consequences of bd on certain daily tasks such as sit - to - stand , back - to - sit , maintaining a standing position or walking . given the absence of a specific tool to assess bd , diagnosis remains only qualitative , based on observations of elderly subjects in the sagittal plane , in the sitting position as well as in the static and dynamic standing position . however , a quality assessment of this kind of postural disorder requires a quantitative scale . the management of elderly subjects suffering from bd has to be multidisciplinary , associating different categories of health professionals including physicians , physiotherapists , psychologists , nurses , and nurse s aides . in addition , the management of bd must begin quickly , immediately after making the diagnosis , which is based on clinical observation . the medical management consists of the search for and the treatment of possible causes of the bd . the physician must therefore make a detailed examination , which could be completed by targeted biological analyses or radiological examinations . there are few reports concerning the rehabilitation of frail patients showing major limitations in motor performance . the association of strength training and progressive functional training improves the physical capacities in frail geriatric patients ( hauer et al 2001 ) . a recent review of the literature shows that progressive resistance training alone is not an effective therapy for balance control ( orr et al 2008 ) . improving the dynamic and static control of the centre of mass is the main objective of physiotherapy . practicing turning over in bed is introduced very early in the rehabilitation program in order to restore autonomy in bed and the capacity to achieve transfers . then , balance training is implemented in static and dynamic positions . it is advisable to start balance rehabilitation using intrinsic exercises first , followed by extrinsic exercises in the sitting position as well as in the standing position . the forward projection of the trunk and the stabilization of the head in space are developed in order to make the sit - to - stand easier . indeed , the sit - to - stand is a determinant stage of functional rehabilitation . in the standing position , the use of a removable heelpiece could help to reduce bd at the beginning of the physiotherapy . in addition , subjects suffering from bd have to be trained to rise from the floor . the physiotherapist , therefore has to control the heart rate and the breathing frequency ( brondel et al 2005 ) . the choice of exercise must be personalized according to the postural abilities of the subject . in parallel with rehabilitation by the physiotherapist , nurses and nurse s aides have to stimulate elderly patients suffering from bd through the activities of everyday life , but in doing so they must avoid pulling on the patient s upper limbs during transfers . finally , the psychologist must reassure and motivate elderly subjects suffering from bd and not overdramatize the situation to help them regain self - confidence . besides , the psychologist must help elderly subjects suffering from bd to overcome disafferentation , fear of standing and walking as well as fear of falling ( manckoundia et al 2007b ) . although it has been known by physicians for several decades , bd , which could have serious physical , psychologicalor social consequences , has rarely been studied in the literature . the causes of bd are often mixed and associate somatic and psychological pathologies among which there is post - fall syndrome . the management of bd must be interdisciplinary ( involving physicians , physiotherapists , psychologists , nurses , nurses aides ) . physiotherapy , which is a major component of the management strategy , must be started early and adjusted to allof the situations of everyday life .

we describe a rare clinical finding of conjunctival tick attachment in a child . a 10-year - old boy presented to the clinic with right - eye itch . he was found to have a live tick firmly attached to his right temporal conjunctiva . the tick was identified as the larval stage of the paralysis tick , ixodes holocyclus . the tick was removed completely by conjunctival excision . although various methods of removing a tick have been described in the literature , the goal of treatment is the safe and complete removal of the tick to prevent further transmission of pathogens , allergens , and toxins to the patient .

conjunctival tick attachment is a rare occurrence , with only 7 cases reported in the literature . the tick species ixodes holocyclus is found in australia and belongs to the family of hard ticks , ixodidae , which are a group of blood - sucking acarine ectoparasites [ 1 , 2 ] . it is also known as the paralysis tick because it produces a toxin that can cause motor paralysis , which is mainly a problem seen in veterinary medicine and rarely in humans , although children under 5 years of age are at greatest risk . ticks are known vectors of infectious diseases and are associated with a range of ocular and systemic conditions . we describe a rarely seen ophthalmic finding : conjunctival attachment of a live paralysis tick . we also discuss the literature regarding ocular tick attachment , clinical features , and management . a 10-year - old boy presented to the clinic with a 2-day history of right - eye redness and itch . the patient was visiting a property surrounded by bush land in the northern suburbs of sydney , n.s.w . his visual acuity was 6/6 in the right eye and 6/5 in the left eye . slit lamp examination revealed a live translucent - bodied organism with pulsatile fluid movement ( at a different rate to the patient 's heart ) , firmly attached to his right temporal bulbar conjunctiva ( fig . there was conjunctival vessel dilatation surrounding the organism , but otherwise , ocular examination was unremarkable . under general anaesthesia , the conjunctival tissue and the organism onto which it was attached ( see arrow in fig . 1b ) were elevated and excised en bloc . the excised area ( approximately 3 mm in diameter ) the patient was started on a 1-week course of chloramphenicol ophthalmic ointment and oral cephalexin 4 times a day . the recovery of our patient , after 3 weeks , has so far been uneventful . the organism was examined and identified as the larval stage of the tick species i. holocyclus from the family of hard ticks , ixodidae . histopathological examination revealed deep embedding of the larval tick 's mouthparts , with extension into the conjunctival stroma ( fig . conjunctival attachment of a live tick is a rare occurrence with only 7 cases ( 6 reports ) found in the literature [ 4 , 5 , 6 , 7 , 8 , 9 ] . the paralysis tick , i. holocyclus , is one of 7 species in the subgenus sternalixodes , all of which are exclusive to australia . the paralysis tick is distributed predominantly along the east coast from north queensland to southern victoria , and possibly into tasmania ( fig . hard ticks , which include the paralysis tick , go through 4 life stages from the egg to larva , to nymph , and finally to the adult stage . from the vegetation where they quest for a host , female ticks attach onto passing vertebrate hosts , including humans , and feed on the blood . paralysis ticks , including the larval form , have long mouthparts to enable a strong attachment to the host [ 1 , 10 ] . the paralysis tick tends to show seasonality in life stages , and the larval stage is most active at this time of the year ( summer ) in australia . during blood feeding , ticks transfer saliva , which contains a cocktail of chemicals including toxins , various allergy - inducing proteins , and potentially disease - causing pathogens [ 10 , 11 , 12 , 13 ] . tick bites to the eye may cause ocular irritation at the attachment site , conjunctivitis , uveitis , keratitis , and vasculitis . ticks can become embedded in the meibomian gland orifice and cause eyelid swelling and pain . although rarely reported in humans , toxins transmitted from the paralysis tick 's saliva have been associated with ascending motor paralysis , diplopia , blurred vision , and photophobia especially in children . the paralysis tick , however , needs several days of engorgement on its host before these signs of tick paralysis are manifested . this is because adult ticks go through various phases of feeding , including a slow phase , where the production of toxins in the tick 's salivary glands is very low on day 1 and 2 of the blood feed , and a rapid phase , where salivary production peaks on day 5 [ 3 , 13 ] . the timing of these phases varies according to life stage and , typically , larvae feed for a shorter duration . our patient had a larval tick attached to his conjunctiva for 2 days and has so far not developed any visual or neurological symptoms . paralysis ticks are vectors of infectious diseases such as queensland tick typhus and flinders island spotted fever . tick bites have been associated with lyme borreliosis , although more conclusive evidence is required to determine that the borrelia bacteria is present in ticks in australia . regarding the larval stage of hard ticks , the infectious diseases society of america ( idsa ) does not recommend routine antibiotic prophylaxis due to the low risk of disease transmission . this is because the larval form typically has not previously fed on another host from which they may have acquired a pathogen . however , antibiotic prophylaxis is often given when the tick bite has occurred in a location where infection would have more detrimental consequences , such as the eye . our patient was started on a 1-week course of oral cephalexin and chloramphenicol ophthalmic ointment . tick allergy has been well documented and ranges from mild urticaria , allergic conjunctivitis , to anaphylaxis . it was recently recognised that patients previously bitten by the paralysis tick may subsequently develop anaphylaxis from the consumption of mammalian meat and meat by - products such as gelatine , although this condition has not yet been associated with the larval stage . the mechanism of this is beyond the scope of this paper , but clinicians may consider close monitoring of the patient for hypersensitivity reactions . five out of the 7 cases of conjunctival tick attachment had the removal by conjunctival excision ( table 1 ) . the reasons for surgical removal were either a firmly attached tick [ 5 , 6 , 7 ] or simply that the conjunctiva was involved . one case report proposed that blunt , medium - tipped , angled forceps offered the best result for removing ticks not deeply seated in the conjunctiva . another case successfully used blunt force with a cotton applicator but admitted difficulty with this approach for embedded ticks . in all 7 cases , recovery of the patient was uneventful , and no parts of the tick remained in the conjunctiva . the australasian society of clinical immunology and allergy ( ascia ) recommends first killing an embedded tick with ether - containing agents or permethrin - based products and then letting it drop off the host or removing it with fine - tipped forceps to avoid squeezing saliva into the host 's vasculature . however , ascia recognises that this method is not suitable for all patients , especially when the tick is embedded in sensitive areas of the body such as the eye . despite the different tick removal methods postulated , there is consensus in the literature that the goal of treatment should be complete removal of the tick from the host to prevent further transmission of pathogens , allergens , and toxins [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ] . in our patient , the larval tick was too firmly attached to the bulbar conjunctiva , so complete and safe removal of it using forceps was not possible in a conscious child . it is not uncommon for ticks to inject further toxins into the host if disturbed . the tick was removed completely and our patient has so far not experienced any post - operative complications or systemic illness . the goal of treatment for conjunctival tick attachment should be to completely and safely remove the tick as soon as possible to prevent further transmission of pathogens , toxins , and allergens to the patient . surgical removal of the tick and surrounding conjunctiva ( with general anaesthetic if necessary ) proved simple and effective with no adverse sequelae , a decision supported by the histopathological examination revealing deep embedding of the tick 's mouthparts into the conjunctiva .

negative affect in healthy populations regulates the appraisal of demanding situations , which tunes subsequent effort mobilization and adjustments in cognitive control . in the present study , we hypothesized that dysphoria in depressed individuals similarly modulates this adaptation , possibly through a neural mechanism involving serotonergic regulation . we tested the effect of dysphoria induced by acute tryptophan depletion ( atd ) in remitted depressed patients on conflict adaptation in a simon task . atd temporarily lowers the availability of the serotonin precursor l - tryptophan and is known to increase depressive symptoms in approximately half of remitted depressed participants . we found that depressive symptoms induced by atd were associated with increased conflict adaptation . our finding extends recent observations implying an important role of affect in regulating conflict - driven cognitive control .

twenty - three patients were administered a high - dose and low - dose atd mixture ( 100 vs. 25 g amino acids ) in a double - blind randomized crossover design with two sessions , separated by at least 4 days ( booij et al . the 100 g and 25 g atd mixtures have previously been shown to lower plasma trp levels by approximately 90% and 50% , respectively , in this sample ( booij et al . , 2005b ) as well as in other samples ( e.g. , booij , van der does , haffmans , & riedel , 2005a ) . the study was approved by an independent medical ethics committee ( metigg , utrecht ) and was performed according to their guidelines and regulations . all patients were informed about the study by their clinician and in detail by one of the investigators ( l.b . ) , and provided written informed consent . the sample has been described in detail previously ( booij et al . , 2005b ) . inclusion criteria were : age between 18 and 65 years ; ongoing treatment with an ssri or a serotonin noradrenaline reuptake inhibitor for at least 4 weeks , meeting dsm - iv criteria for depression in full or partial remission , and a hamilton depression rating scale ( hrsd , 17 items ) ( hamilton , 1960 ) lower than 15 ( frank et al . , 1991 ) . exclusion criteria were : substance abuse within the past 3 months , psychosis ( lifetime ) , major physical illness , lactation , and pregnancy . after excluding two drop - outs and two statistical outliers , 19 participants remained for statistical analyses ( cf . booij et al . , 2005b ) . as described in the original article ( booij et al . , 2005b ) , depressive symptoms were assessed with the 10-item montgomery asberg depression rating scale ( madrs ) ( montgomery & asberg , 1979 ) . the sleep items were omitted , since this could not change within an atd session . diagnoses , and demographic and clinical background variables were verified with the structured clinical interview for dsm - iv ( scid - i ) ( first , spitzer , gibbon , & williams , 2005 ) . the simon task consisted of 64 trials presented in two consecutive blocks in which the stimulus interval differed ( 2,250 ms fixed vs. 2,2505,500 ms variable ; originally included to investigate a possible effect of task complexity ) right was presented in randomized order either at the left or the right side of the screen . participants were instructed to respond to the meaning of the word ( target)that is , making a left - hand response to the word left and a right - hand response to the word the same number of compatible ( distracter location matches the target word ) and incompatible ( distracter location conflicts with the target word ) stimuli was used . 1 venous blood ( 10 ml ) was taken in the morning , 6 hr after atd and the next day ( t + 24 ) and was analyzed for both total plasma trp and the other large neutral amino acids ( fekkes , vandalen , edelman , & voskuilen , 1995 ) . mood was assessed 1 hr before atd ( t 1 ) , 6.5 hr later ( t + 6.5 ) , and the next morning ( t + 24 ) . the simon task ( left / right task ) was administered ( booij et al . , 2005b ) approximately 5.5 hr after administration of the atd mixture . the order of the low - dose session and the high - dose session was counterbalanced . cognitive performance and mood repeated measures anovas were used for sequential analysis of simon performance , separately for correct reaction time ( rt ) and percent accuracy . in order to measure sequential adjustments in simon task performance , we not only included the factor compatibility of the current trial as is usually done , but we also added the factor compatibility of the previous trial . standard conflict adaptation that is , the modulation of the compatibility effect as a function of previous - trial compatibility ( cf . 1a)should yield an interaction effect between current and previous trial compatibility ( egner , 2007 ; gratton et al . , 1992 ) . to analyze direct effects of the atd manipulation on conflict adaptation , intervention ( baseline vs. low - dose vs. high - dose atd ) the effect of mood state on conflict adaptation , for the low - dose atd and high - dose atd sessions separately , was analyzed by using mood scores ( measured at + 6.5 hr ) as a covariate . additionally , we also ran separate anovas in which we included the binary between - subjects factors ongoing treatment ( ssri vs. snri ) and mood response ( absence vs. presence of depressed mood increase from t-1 to t + 6.5 in the high - dose atd session).fig . 1a conflict adaptation in the simon task as evidenced by a reduced compatibility effect after conflict trials versus nonconflict trials . b more depressive symptoms ( madrs score ) after the high - dose atd intervention are associated with increased conflict adaptation a conflict adaptation in the simon task as evidenced by a reduced compatibility effect after conflict trials versus nonconflict trials . b more depressive symptoms ( madrs score ) after the high - dose atd intervention are associated with increased conflict adaptation to visualize the hypothesized association between mood and conflict adaptation , we calculated standard individual conflict - adaptation scores by subtracting the interference effect following a correct conflict ( incompatible ) trial from the interference effect following a correct nonconflict ( compatible ) trial ( cf . 1a ) . before averaging sequential conditions for each individual , the first trial of each block , trials following an error , and trials with rts not fitting the outlier criterion ( deviating more than 2.5 sds from the individual condition - specific mean ) were excluded from analyses . the sample has been described in detail previously ( booij et al . , 2005b ) . inclusion criteria were : age between 18 and 65 years ; ongoing treatment with an ssri or a serotonin noradrenaline reuptake inhibitor for at least 4 weeks , meeting dsm - iv criteria for depression in full or partial remission , and a hamilton depression rating scale ( hrsd , 17 items ) ( hamilton , 1960 ) lower than 15 ( frank et al . , 1991 ) . exclusion criteria were : substance abuse within the past 3 months , psychosis ( lifetime ) , major physical illness , lactation , and pregnancy . after excluding two drop - outs and two statistical outliers , 19 participants remained for statistical analyses ( cf . as described in the original article ( booij et al . , 2005b ) , depressive symptoms were assessed with the 10-item montgomery asberg depression rating scale ( madrs ) ( montgomery & asberg , 1979 ) . the sleep items were omitted , since this could not change within an atd session . diagnoses , and demographic and clinical background variables were verified with the structured clinical interview for dsm - iv ( scid - i ) ( first , spitzer , gibbon , & williams , 2005 ) . the simon task consisted of 64 trials presented in two consecutive blocks in which the stimulus interval differed ( 2,250 ms fixed vs. 2,2505,500 ms variable ; originally included to investigate a possible effect of task complexity ) . the word left or right was presented in randomized order either at the left or the right side of the screen . participants were instructed to respond to the meaning of the word ( target)that is , making a left - hand response to the word left and a right - hand response to the word right,and to ignore its location ( distracter ) , as fast as possible . the same number of compatible ( distracter location matches the target word ) and incompatible ( distracter location conflicts with the target word ) stimuli was used . 1 venous blood ( 10 ml ) was taken in the morning , 6 hr after atd and the next day ( t + 24 ) and was analyzed for both total plasma trp and the other large neutral amino acids ( fekkes , vandalen , edelman , & voskuilen , 1995 ) . mood was assessed 1 hr before atd ( t 1 ) , 6.5 hr later ( t + 6.5 ) , and the next morning ( t + 24 ) . the simon task ( left / right task ) was administered ( booij et al . , 2005b ) approximately 5.5 hr after administration of the atd mixture . the order of the low - dose session and the high - dose session was counterbalanced . cognitive performance and mood repeated measures anovas were used for sequential analysis of simon performance , separately for correct reaction time ( rt ) and percent accuracy . in order to measure sequential adjustments in simon task performance , we not only included the factor compatibility of the current trial as is usually done , but we also added the factor compatibility of the previous trial . standard conflict adaptation that is , the modulation of the compatibility effect as a function of previous - trial compatibility ( cf . 1a)should yield an interaction effect between current and previous trial compatibility ( egner , 2007 ; gratton et al . , 1992 ) . to analyze direct effects of the atd manipulation on conflict adaptation , intervention ( baseline vs. low - dose vs. high - dose atd ) the effect of mood state on conflict adaptation , for the low - dose atd and high - dose atd sessions separately , was analyzed by using mood scores ( measured at + 6.5 hr ) as a covariate . additionally , we also ran separate anovas in which we included the binary between - subjects factors ongoing treatment ( ssri vs. snri ) and mood response ( absence vs. presence of depressed mood increase from t-1 to t + 6.5 in the high - dose atd session).fig . 1a conflict adaptation in the simon task as evidenced by a reduced compatibility effect after conflict trials versus nonconflict trials . b more depressive symptoms ( madrs score ) after the high - dose atd intervention are associated with increased conflict adaptation a conflict adaptation in the simon task as evidenced by a reduced compatibility effect after conflict trials versus nonconflict trials . b more depressive symptoms ( madrs score ) after the high - dose atd intervention are associated with increased conflict adaptation to visualize the hypothesized association between mood and conflict adaptation , we calculated standard individual conflict - adaptation scores by subtracting the interference effect following a correct conflict ( incompatible ) trial from the interference effect following a correct nonconflict ( compatible ) trial ( cf . 1a ) . before averaging sequential conditions for each individual , the first trial of each block , trials following an error , and trials with rts not fitting the outlier criterion ( deviating more than 2.5 sds from the individual condition - specific mean ) were excluded from analyses . as described in detail elsewhere ( booij et al . , 2005b ) , high - dose atd but not low - dose atd led to a both statistically and clinically significant induction of depressive symptoms as measured by madrs scores 6.5 hr after depletion [ 7.9 7.8 vs. 3.7 3.8 , mean standard deviation ; t(18 ) = 3.34 , p < .005 ] . the simon task produced a standard interference effect : incompatible trials produced longer rts than compatible trials , f(1 , 18 ) = 23.47 , p < .001 , mse = 1933.65 . the analysis also revealed a robust conflict - adaptation effect as indicated by an interaction between current- and previous - trial compatibility , f(1 , 18 ) = 38.27 , p < .001 , mse = 1143.45 . 1a shows , the interference effect was eliminated after conflict ( incompatible ) trials but not after nonconflict ( compatible ) trials , indicating standard conflict adaptation that is , reduced interference after conflict trials . this reduction in interference was driven by both postconflict speeding of incompatible trials [ illustrating that increased conflict - driven control reduces interference ; t(18 ) = -4.2 , p < .001 ] and by postconflict slowing of compatible trials [ illustrating that increased conflict - driven control reduces facilitation ; t(18 ) = 4.5 , p < .001 ] . although participants made only a few errors in general , error rate analyses still showed standard interference , f(1 , 18 ) = 5.38 , p < .05 , mse = 0.001 , and conflict adaptation , f(1 , 18 ) = 6.30 , p < .05 , mse = 0.001 , effects . in addition , they revealed a main effect of previous compatibility , f(1 , 18 ) = 4.46 , p < .05 , mse = .002 , indicating improved accuracy after conflict . notably , no main effects or interactions with congruency or conflict adaptation in rt or accuracy were observed for atd intervention . thus , atd did not have an effect on interference ( as reported earlier by booij et al . ongoing treatment did not have main or interactive effects.table 1behavioral data for all sessions and conditionsreaction time ( in milliseconds)error rate ( in percentages)msemsebaselinecompatible trial following a compatible trial65514.40.60.3incompatible trial following a compatible trial70512.33.61.2compatible trial following an incompatible trial67612.10.90.4incompatible trial following an incompatible trial67511.61.00.5low - dose sessioncompatible trial following a compatible trial65016.81.40.9incompatible trial following a compatible trial70618.22.91.0compatible trial following an incompatible trial68816.70.90.9incompatible trial following an incompatible trial68014.40.80.8high - dose sessioncompatible trial following a compatible trial64918.31.40.8incompatible trial following a compatible trial71119.92.30.9compatible trial following an incompatible trial66717.30.90.5incompatible trial following an incompatible trial67816.41.10.8 behavioral data for all sessions and conditions an ancova using mood score as a covariate confirmed our hypothesis : depressed - mood scores during the high - dose atd condition predicted increased conflict adaptation in rt as indicated by a significant three - way interaction between mood , current - trial compatibility , and previous - trial compatibility , f(1 , 17 ) = 5.30 , p < .05 , mse = 396.75 . as fig . 1 shows , individuals with more depressive symptoms after the atd intervention showed more conflict adaptation in the simon task . as is typically observed ( chepenik et al . , 2007 ; van steenbergen et al . , 2010 2 moreover , no mood effects were found for accuracy , thus showing that the effect on conflict adaptation could not be attributed to a speed accuracy tradeoff . because the low - dose atd session did not lead to any mood changes ( booij et al . , 2005b ) , data from this session were used for a control analysis : no association between mood and performance emerged.3 we report the first evidence for a link between low tryptophan concentrations , depressed mood , and conflict adaptation in remitted depressed patients : individuals with higher levels of depressive symptoms following high - dose atd showed increased conflict adaptation . this finding is in line with predictions derived from mbm theory ( gendolla , 2000 ) , with earlier behavioral and physiological observations from mood - induction studies in healthy populations ( e.g. , gendolla , abele , & krusken , 2001 ; gendolla & krusken , 2002 ; van steenbergen et al . , 2010 ) , and with neural evidence ( e.g. , evers et al . , 2009 ) our present study has important implications for understanding how depressive affect regulates cognitive control . in line with mbm theory ( gendolla , 2000 ) , our data illustrate that depressed mood per se does not have motivational implications ( as would be indicated by a modulation in attentional interference effects ) , but may facilitate increased cognitive control after a behavioral challenge . interestingly , this effect was observed in a relatively low - demanding simon task in which people were merely instructed to do their best ( see also brinkmann & gendolla , 2007 ) , and in the context of depression scores that were mainly below the cut - off value for a depression diagnosis , but were still clinically relevant and much larger than the effect of mood inductions in healthy participants . note that our findings suggest that depressive symptoms as induced by atd did not simply mimic the effect of depressive symptoms in currently depressed patients . that is , whereas mdd has been associated with reduced postconflict adaptation ( meiran et al . , 2011 ; pizzagalli , 2011 ) mbm theory ( gendolla , 2000 ) has proposed that the association between mood and effort mobilization is nonlinear , because it is mediated by task difficulty appraisal . accordingly , negative mood will facilitate demand - driven adjustments in effort only up to a certain level : if demands become perceived as too high to actively cope with , negative mood may actually trigger demand - driven disengagement ( cf . evidence for this effect has been reported in mood - induction studies and can also be shown in dysphoric participants when they perform tasks with extremely high fixed demands ( brinkmann & gendolla , 2008 ) . taken collectively , these observations suggest an inverted - u relationship between depressive symptoms and conflict adaptation ( see brehm & self , 1989 ) . it is an important aim for future studies to understand the generalizability of these findings and to disentangle the effects of increased negative affect and putative reduced availability of resources ( e.g. , due to rumination ) in mdd ( cf . mbm theory assumes that the interaction between both factors determines the actual appraisal of the demand , which in turn modulates effort mobilization . this hypothesis is now ripe for further testing in other studies using sequential analyses of conflict - task performance . at the neural level , the joint impact of depressed mood and demand evaluation on subsequent effort mobilization and cognitive control may be associated with ( hyper)activation of the anterior cingulate cortex ( acc ) , a region important for signaling the need for more cognitive effort to the dorsolateral prefrontal cortex ( dlpfc ) ( botvinick et al . , 2001 ; davidson , pizzagalli , nitschke , & putnam , 2002 ; olvet & hajcak , 2008 ; pizzagalli et al . , dlpfc circuit in unipolar depression also produces catastrophic reactions to errors ( for a review , see pizzagalli , 2011 ) . further study is needed to understand the exact neuromodulating role that 5-ht may play in this affective regulation ( cf . future studies that combine neuroimaging methods with effort - related physiological and behavioral measures will advance our understanding of the functional role of the acc in the affective ( dys)regulation of adaptive control to fluctuating task demands .

objectives : masking ability of a restorative material plays a role in hiding colored substructures ; however , the masking ability of zirconia ceramic ( zrc ) has not yet been clearly understood in zirconia - based restorations . this study evaluated the effect of three different core materials on masking ability of a zrc.materials and methods : ten zirconia disc samples , 0.5 mm in thickness and 10 mm in diameter , were fabricated . a white ( w ) substrate ( control ) and three substrates of nickel - chromium alloy ( nca ) , non - precious gold alloy ( npga ) , and zrc were prepared . the zirconia discs were placed on the four types of substrates for spectrophotometry . the l * , a * , and b * values of the specimens were measured by a spectrophotometer and color change ( e ) values were calculated to determine color differences between the test and control groups and were then compared with the perceptual threshold . randomized block anova and bonferroni test analyzed the data . a significance level of 0.05 was considered.results:the mean and standard deviation values of e for nca , npga , and zrc groups were 10.262.43 , 9.451.74 , and 6.701.91 units , respectively . significant differences were found in the e values between zrc and the other two experimental groups ( nca and npga ; p<0.0001 and p=0.001 , respectively ) . the e values for the groups were more than the predetermined perceptual threshold.conclusions:within the limitations of this study , it was concluded that the tested zrc could not well mask the examined core materials .

metal - ceramic restorations have been successfully applied in restorative dentistry due to high fracture strength . however , achieving a natural translucency is more difficult with metal - ceramic restorations than all - ceramic restorations because metal substructures prevent light transmission in metal - ceramic restorations . this has led to an increase in use of all - ceramic restorations . among all - ceramic restorations , however , high translucency of a restoration is not always an advantage , for instance in cases with discolored teeth , colored core materials and cast metal post and cores . in these clinical conditions , a restorative material with optimal masking ability of the underlying substructures a method to evaluate the masking ability of restorative materials is to measure color differences in cie lab color system . in this color system , the l * , a * and b * color parameters and e refer to lightness , red - green value , yellow - blue value , and color difference , respectively , which can be measured by a spectrophotometer . the e color difference is calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 which is the most commonly used formula for e . this formula can detect even a small amount of color difference between two objects [ 9 , 10 ] . as spectrophotometers can detect even the slightest amount of color difference not perceptible by the human eye , perceptual and clinically acceptable thresholds have been defined based on e values . it has been reported that the clinically acceptable threshold is more than the perceptual threshold , because of optical conditions of oral environment . if the e ( color difference ) between two objects is more than the perceptual threshold , a color mismatch will be detected by the human eye . the perceptual threshold has been reported from e=1 to e=5.5 in different studies [ 1214 ] . if a restorative material has an ideal masking ability , e will be equal to zero when placing the material on white and black substrates , namely that the material has a similar color on different substrates , and the substrates can not affect the color of the material . from a clinical point of view , if e of a material on different backgrounds is less than the threshold , it can thoroughly mask the background color . several studies have surveyed the optical properties of different ceramic materials [ 8 , 1518 ] . some studies have evaluated various factors affecting the final color of zirconia restorations including dental substrates [ 1922 ] , luting agents [ 21 , 23 ] , ceramic veneers [ 2426 ] , glaze , and firing process . suputtamongkol et al , reported that the background color could affect the overall color of posterior zirconia restorations on a metal post and core or a prefabricated post and a composite core ; however , color changes did not exceed the perceptual threshold . choi and razzoog assessed the masking ability of a zirconia ceramic ( zrc ) with and without the veneering porcelain and revealed that the zirconium oxide coping material alone had some degree of masking ability . oh and kim disclosed that abutment shade , ceramic thickness and coping type affected the resulting color of zirconia restorations . vichi et al , evaluated the effects of different substrates with different colors on the final color of all - ceramic ips - empress glass - ceramic restorations , and advised to take the color of substrate into consideration in cases with ceramic thickness of less than 1 mm . masking ability of a zrc is related to its color coverage over underlying substructures including cement and dental substrates . it may not be feasible to mask a substrate with cements , because different shades do not exist for all cements . thus , it seems that the color masking ability of a zrc on different substrates may be an important factor affecting the esthetic results of zirconia - based restorations . on the other hand , posts and cores , which are widely applied in restorative dentistry to restore severely damaged teeth the effects of these core materials ( as substrates ) on masking ability of zrcs ( as substructures in zirconia - based restorations ) have to be clearly determined . therefore , this in vitro study aimed to evaluate the effect of three different core materials including a nickel - chromium alloy ( nca ) , a non - precious gold alloy ( npga ) , and a zrc on masking ability of a zrc . the null hypothesis was that the zrc would mask the three aforementioned core materials with no significant difference . the discs were placed over four substrates including a white ( w ) substrate ( as the control ) , nca , npga and zrc . the procedure was conducted as follows : specimen preparation : a computer - aided design / computer - aided manufacturing system ( coritec 250i , imes - icore gmbh , eiterfeld , germany ) milled zirconia blanks ( luminesse high strength / low translucency , 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate zirconia discs . the zirconia blanks had 37% translucency and were used to fabricate zirconia frameworks in zirconia - based restorations . the discs had 0.5 mm thickness and 10 mm diameter [ 20 , 23 ] . all the zirconia discs were sintered at 1520c for a 12-hour process in a sintering furnace ( isint ht , imes - icore gmbh , eiterfeld , germany ) . a digital micrometer ( 293 mdc - mx lite ; mitutoyo corporation , tokyo , japan ) with the accuracy of 0.002 mm an adjustment / polishing kit ( bruxzir ; glidewell direct , irvine , ca , usa ) was used to reduce the thicknesses according to the afore - mentioned acceptable range . in case of lack of acceptable thickness the zirconia discs were polished , cleaned in an ultrasonic bath ( elmasonic s-30 ; dentec , north shore , australia ) containing 98% ethanol for 15 minutes , and were finally dried . the zirconia disc specimens were prepared as such , and had natural white color of zirconia ( fig . 1).substrate preparation : in order to fabricate the w substrate with the dimensions of 10 mm in diameter and 10 mm in height , a white teflon material ( ptfe , omnia plastica spa , busto arsizio , italy ) was milled . then , two cylindrical acrylic resin ( duralay , reliance dental mgf co. , worth , il , usa ) patterns were formed according to the afore - mentioned dimensions . the patterns were cast by a nca ( verabond v ; alba dent , fairfield , ca , usa ) and a npga ( alba dent , fairfield , ca , usa ) to fabricate nca and npga substrates , respectively . an adjusting / polishing kit ( np alloy adjustment kit ; shofu inc . , kyoto , japan ) was employed to polish the alloy substrates . in order to fabricate the zrc substrate , a cylindrical pattern was virtually designed by a software program ( solidworks 2015 , solidworks corp . , the same computer - aided design / computer - aided manufacturing system milled a zirconia blank ( luminesse high strength 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate a zirconia substrate based on the above - mentioned virtual design . the zirconia substrate was dipped in a2 shade color liquid ( dd bio zx2 monolithic zero lzdd ; dental direkt gmbh , spenge , germany ) for 10 seconds . the zirconia substrate was sintered at 1520c for a 12-hour process in the same sintering furnace , and was then polished by the same polishing kit . the four substrates of w , nca , npga , and zrc were prepared as such . all the substrates were cleaned in the same ultrasonic bath containing 98% ethanol for 15 minutes ( fig . 2 ) color measurement : a spectrophotometer ( spectroshade micro , mht , verona , italy ) was employed for spectrometric measurements . a putty silicone material ( speedex ; coltene , altstatten , switzerland ) was adapted to the mouth piece of the spectrophotometer to match the conditions of spectrophotometry for all specimens and to prevent external lights . the specimens were located at the center of this putty mold . before each measurement , the spectro - photometer was calibrated by the white and green calibration plates , respectively . the discs were placed over the substrates with a water drop in - between to prevent the refraction of light . each disc was placed on each of the substrates , and the color measurements were made . all the color measurements were made at the center of specimens marked by a pen on the monitor screen of spectrophotometer . the color parameters of l * , a * , and b * were recorded for each specimen . additionally , e values were calculated to determine the e of disc on the substrates . in order to compare the color of specimens on the nca , npga , and zrc substrates with their color on the w substrate , the e values were measured in three situations including : w - nca , w - npga , and w - zrc . the e w - nca e w - npga , and e w - zrc values were calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 specimen preparation : a computer - aided design / computer - aided manufacturing system ( coritec 250i , imes - icore gmbh , eiterfeld , germany ) milled zirconia blanks ( luminesse high strength / low translucency , 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate zirconia discs . the zirconia blanks had 37% translucency and were used to fabricate zirconia frameworks in zirconia - based restorations . the discs had 0.5 mm thickness and 10 mm diameter [ 20 , 23 ] . all the zirconia discs were sintered at 1520c for a 12-hour process in a sintering furnace ( isint ht , imes - icore gmbh , eiterfeld , germany ) . a digital micrometer ( 293 mdc - mx lite ; mitutoyo corporation , tokyo , japan ) with the accuracy of 0.002 mm an adjustment / polishing kit ( bruxzir ; glidewell direct , irvine , ca , usa ) was used to reduce the thicknesses according to the afore - mentioned acceptable range . in case of lack of acceptable thickness the zirconia discs were polished , cleaned in an ultrasonic bath ( elmasonic s-30 ; dentec , north shore , australia ) containing 98% ethanol for 15 minutes , and were finally dried . the zirconia disc specimens were prepared as such , and had natural white color of zirconia ( fig . substrate preparation : in order to fabricate the w substrate with the dimensions of 10 mm in diameter and 10 mm in height , a white teflon material ( ptfe , omnia plastica spa , busto arsizio , italy ) was milled . then , two cylindrical acrylic resin ( duralay , reliance dental mgf co. , worth , il , usa ) patterns were formed according to the afore - mentioned dimensions . the patterns were cast by a nca ( verabond v ; alba dent , fairfield , ca , usa ) and a npga ( alba dent , fairfield , ca , usa ) to fabricate nca and npga substrates , respectively . an adjusting / polishing kit ( np alloy adjustment kit ; shofu inc . , kyoto , japan ) was employed to polish the alloy substrates . in order to fabricate the zrc substrate , a cylindrical pattern was virtually designed by a software program ( solidworks 2015 , solidworks corp . , the same computer - aided design / computer - aided manufacturing system milled a zirconia blank ( luminesse high strength 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate a zirconia substrate based on the above - mentioned virtual design . the zirconia substrate was dipped in a2 shade color liquid ( dd bio zx2 monolithic zero lzdd ; dental direkt gmbh , spenge , germany ) for 10 seconds . the zirconia substrate was sintered at 1520c for a 12-hour process in the same sintering furnace , and was then polished by the same polishing kit . the four substrates of w , nca , npga , and zrc were prepared as such . all the substrates were cleaned in the same ultrasonic bath containing 98% ethanol for 15 minutes ( fig . 2 ) color measurement : a spectrophotometer ( spectroshade micro , mht , verona , italy ) was employed for spectrometric measurements . a putty silicone material ( speedex ; coltene , altstatten , switzerland ) was adapted to the mouth piece of the spectrophotometer to match the conditions of spectrophotometry for all specimens and to prevent external lights . the specimens were located at the center of this putty mold . before each measurement , the spectro - photometer was calibrated by the white and green calibration plates , respectively . the discs were placed over the substrates with a water drop in - between to prevent the refraction of light . each disc was placed on each of the substrates , and the color measurements were made . all the color measurements were made at the center of specimens marked by a pen on the monitor screen of spectrophotometer . the color parameters of l * , a * , and b * were recorded for each specimen . additionally , e values were calculated to determine the e of disc on the substrates . in order to compare the color of specimens on the nca , npga , and zrc substrates with their color on the w substrate , the e values were measured in three situations including : w - nca , w - npga , and w - zrc . the e w - nca e w - npga , and e w - zrc values were calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 zirconia disc specimens tested substrates ( from left to right : zirconia ceramic , non - precious gold alloy , nickel - chromium alloy and white substrate ) the perceptual threshold of e=2.6 was considered in this study [ 1214 ] . normal distribution of the data was accepted in all groups by the kolmogorov - smirnov test ( p>0.05 ) . the values of l * , a*and b * for each disc in npga , nca and zrc groups were subtracted from l*white , a*white and b*white values , which were the corresponding values for w substrate . in order to compare the standard l * , a * , b * ( from which , the values of w substrate were subtracted ) and e values among the groups , randomized block anova was employed . stata software ( statacorp lp , lakeway , tx , usa ) compared the e values with the predetermined perceptual threshold of e=2.6 using one - sample t - test . the results were explained according to the measured variables of l * , a * , b * , and e in four sections . l * parameter ( lightness ) : the mean and standard deviation of the l * values for the w , nca , npga and zrc groups were 88.351.46 , 78.241.31 , 78.981.26 , and 82.161.16 units , respectively ( table 1 , fig . 3 ) . statistical analysis showed a significant difference among the groups in this regard ( p<0.0001 ) . pairwise comparisons of the groups using bonferroni test revealed significant differences between the nca and zrc ( p<0.0001 ) , and npga and zrc ( p<0.0001 ) . the color parameter values in the four groups of white substrate ( w ) , nickel - chromium alloy ( nca ) , non - precious gold alloy ( npga ) and zirconia ceramic ( zrc ) the mean l * values of the groups a * parameter ( red - green value ) : the mean and standard deviation of the a * values for the w , nca , npga and zrc groups were 0.400.42 , 0.040.52 , 0.600.42 , and 1.520.38 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test revealed significant differences between nca and npga ( p<0.0001 ) , nca and zrc ( p<0.0001 ) and npga and zrc ( p<0.0001 ) . the a * values increased in all the groups compared to the control . the increase in the a the mean a * values of the groups b * parameter ( yellow - blue value ) : the mean and standard deviation of the b * values for the w , nca , npga , and zrc groups were 3.380.36 , 1.921.01 , 3.860.49 , and 4.880.57 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test showed significant differences between nca and npga ( p<0.0001 ) , nca and zrc ( p<0.0001 ) , and npga and zrc ( p<0.001 ) . zrc had the highest b * value , and nca had the lowest b * value among the groups . the mean b * values of the groups e ( color difference ) : the mean and standard deviation of the e values for the nca , npga and zrc groups were 10.262.43 , 9.451.74 , and 6.701.91 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test showed significant differences between nca and zrc ( p<0.0001 ) , and npga and zrc ( p=0.001 ) . the difference between nca and npga was not statistically significant ( p=0.65 ) . in order to compare the e values of the groups with the predetermined perceptual threshold of e=2.6 , the null hypothesis of e 2.6 was rejected for all the groups ( p<0.0001 ) . the present study evaluated the color parameters of l * , a * , b * , and e for zirconia disc specimens on four different substrates including w , nca , npga and zrc . statistical analysis indicated significant differences among the groups in the l * , a * , b * and e values . the examined zrc on the tested substrates showed perceptible color change namely that the examined zrc could not thoroughly mask the tested substrates . the l * ( lightness ) values decreased in all the groups compared with the control . the change in l * value shows the impact of the substrates on the zirconia specimens . this can be explained by the optical properties of zrc , which is a semi - translucent material . according to the results , metal core materials such as ncas and npgas can decrease the lightness of zrc more than a zirconia core material the a * ( red - green ) values increased in all the groups compared to the control . a reason for this may be the natural a * value of the zrc as a substrate . the tested zirconia substrate ( a2 shade ) positively shifted the a * value towards red . the b * ( yellow - blue ) values changed in the groups depending on the substrates . the nickel - chromium substrate decreased the b * value while the non - precious gold and zirconia substrates increased the b * value compared to the control . this may be due to the yellow color tendency of a2 shade zirconia substrate and npga , which impact on the color of zrc and shift it towards yellow . on the contrary , nca shifted the color of zrc towards blue , and negatively affected the b * value . the e values of all groups were more than the predetermined perceptual threshold of e=2.6 . assessment of the l * , a * and b * values indicated their changes in the substrate groups . thus , color changes were derived from all three color parameters . the l * , a * , and b * values manifested significant differences between the substrate groups . this demonstrated that the quality of the color change was different depending on the substrate . the e values manifested no significant difference between the metal substrates ; however , significant differences existed between the zirconia substrate and the metal substrates , and the least color change belonged to the zirconia substrate . this demonstrated that the quantity of the color change was different depending on the substrate as well . hence , both quality and quantity of the color of zrc were altered by the substrates . the used metal substrates did not differ in terms of the quantity of color change , and both affected the quantity of the color change more than the zirconia substrate . suputtamongkol et al , reported that the color of an underlying substructure could affect the overall color of posterior zirconia - based restorations on a metal post and core or a prefabricated post and a composite core , ranging from e = 1.2 to 3.1 . additionally , slight color changes of zirconia crowns were detected by measuring the e . although some differences exist between the afore - mentioned study and the current research such as zrc brands , thicknesses of ceramics , veneered versus non - veneered zrcs , hypothesized perceptual thresholds and substrate types , both studies showed that zrc could not thoroughly mask the underlying materials . oh and kim in an in vitro study of color masking ability assessed the effects of abutment shade , ceramic thickness and coping type on the final color of zirconia restorations . the abutments were fabricated of gold alloy , base metal ( nickel - chromium ) alloy , and four different shades of composite resins . in their study , the average e value of lava specimens between the a2 shade composite resin and gold alloy abutments was higher than that between the a2 shade composite resin and other abutments , and was close to e=5.5 . a similar result was obtained in the current study , although the substrates and zrcs used in the two studies were different . according to the study by oh and kim , gold alloy substrate yielded the highest e among the tested substrates ; however , the npga and the nca yielded close e values in the current study . this dissimilarity in the results may be caused by the color difference of the precious gold alloy used by oh and kim and the npga used in the current study . choi and razzoog evaluated the masking ability of zrc with and without a veneering porcelain . in their study , color differences caused by zrc and the veneering porcelain were compared with substrates alone . they concluded that the non - veneered zrc had some degree of masking ability over different tested substrates ( white , black , gray , and a3 shade tooth - colored substrates ) . however , the current study compared substrate - induced color differences with a zrc over w substrate and concluded that the tested zrc could not mask the examined substrates . choi and razzoog measured the e values between the substrate alone ( as control ) and the zrc over the substrate , while the current study measured the e values between the zrc over w substrate ( as control ) and zrc over the tested substrates . based on the results of our study , the three core materials including nca , npga and zrc can change the color of tested zrc . the color of zrc may be closer to the final color of a restoration than the metal cores . therefore , it is esthetically advised to preferably choose a zrc post and core instead of nca or npga post and cores in zirconia - based restorations and to reduce the core material - induced color change by further core reduction , applying sufficient thickness of the veneering porcelain , and use of proper luting agents in zirconia - based restorations . it should be noted that given that the physical and mechanical properties of zirconia post and core are confirmed in further studies , zirconia post and core should be clinically recommended . this needs future investigations . considering the optical properties of zrc and the thickness of zirconia sub - structures , which is approximately 0.5 mm in a normal case , light transmission through the zirconia structure can be expected in zirconia - based restorations . the present study evaluated the effects of three core materials in this respect . on the other hand , the zirconia core overlaying materials such as porcelain veneers and glaze may influence the final color of zirconia - based restorations , which were not assessed in this study . therefore , evaluation of the effect of other factors in this respect is recommended in future studies . the present study had some limitations such as using a specific brand of zrc , an uncolored zrc , and a specific brand of composite resin . within the limitations of this study , it was concluded that the tested zrc could not thoroughly mask the three core materials of nca , npga , and zrc .

a 47-year - old indian woman with dark hair developed dramatic alopecia of the occipito - vertical area two weeks after a fluoroscopically - guided endovascular procedure for treatment of cerebral aneurysm . there was spontaneous repopulation of hair in 14 weeks . neuro - radiological intervention procedures are becoming commoner by the day in india , and the rare but documented possibility of such reactions occurring in patients should be kept in mind by the treating surgeon as well as dermatologist . necessary counseling regarding this uncommon side - effect is of essence , especially when the radiation dose exceeds 3 gy . we believe this is the first case ever reported in indian dermatology literature .

however , it has not been reported with fluoroscopically - guided endovascular procedures.the rapidly proliferating keratinocytes in the hair matrix are most sensitive to radiation . the insult damages the keratinocytes , which results in thin , weakened hair shaft that is susceptible to trauma.complete hair re - growth occurs in a time period between two to four months following the insult . the insult damages the keratinocytes , which results in thin , weakened hair shaft that is susceptible to trauma . complete hair re - growth occurs in a time period between two to four months following the insult . fluoroscopy - guided neuro - radiological endovascular procedures for treatment of arteriovenous malformations are getting commonplace in modern health care facilities of india . temporary alopecia of the scalp is an uncommon but documented side - effect of these procedures , especially when a certain threshold of radiation is exceeded . to the best of our knowledge , this is the first case of temporary alopecia after fluoroscopically - guided coiling procedure for a ruptured cerebral aneurysm being described in a dark haired woman from india . a 47-year - old apparently healthy woman was referred to this clinic ( sv ) for a dramatic non - scarring hair loss of occipito - vertical region since a month . it became known upon eliciting history that three weeks ago , she developed acute excruciating headache of the nape of the neck , profuse perspiration , vomiting , and lack of consciousness with incontinence . an urgent ct scan showed a ruptured anterior communicating artery aneurysm . a fluoroscopically - guided guglielmi detachable coil ( gdc ) coiling procedure as a non - invasive treatment was suggested . a preliminary angiography lasting for two and half hours was done followed by an attempt to perform coiling that lasted unsuccessfully for four hours . shoots had to be taken due to severe vasospasm . after waiting for two days to let the dye drain out , the procedure was attempted again , and coiling was done successfully in two and a half hours . a week later , the patient developed headache , for which another ct scan was done . the headache was attributed to cerbritis , and 24 mg of methylprednisolone was instituted on a slow taper . two weeks later , she noticed clumps of hair falling out initially during combing and then even while spontaneously running her fingers through her hair . there was a 6 4 inch - sized , asymptomatic , skin - colored alopecic patch in the occipital area with no erythema , scarring or scaling [ figure 1 ] . hair was sent for trichogram to an academic hospital in coimbatore where they demonstrated 80% anagen hair showing dystrophy , the so called bayonet hair. [ figure 2 ] . she had no exclamation mark hairs , poliosis , hair shaft disorders or positive history of similar affliction or atopy in her family . a vascular cause for the hair loss was ruled out by loud arterial pulse palpable in the temporal and occipital arteries . pressure alopecia due to prolonged supine position was ruled out by absence of fractured hair shafts . occipito - vertical alopecia after radiation exposure anagen hair with the pointed distal end to the left and telogen hair to the right diagnosis of fluoroscopic endovascular catheterization - induced alopecia was made . after a period of 14 weeks of observation and no active treatment , she showed significant re - growth of dark hair [ figure 3 ] . flouroscopically - guided endovascular procedures are becoming increasingly common all over the globe . while dermatologists have since long been consulted for hair loss following radiotherapy of tumors , there are only few reports of temporary hair loss following fluoroscopic endovascular procedures in dermatologic literature . other than radiation oncology , the maximum radiation exposure comes from equipment used for non - invasive interventions such as these . the two commonest temporary side - effects of such procedures , erythema , and temporary hair loss are termed deterministic and are indicative of a certain threshold of irradiation , below which the phenomenon would not be seen . the probability of encountering such a side - effect would increase as the dose exceeds the threshold . the time between the exposure and appearance of clinical symptoms depend on the cell turnover rate . in tissues such as hair follicle with its rapid turnover rate , it is thought that irradiation doses exceeding 3 gy are followed by such a hair loss during fluoroscopic procedures . an estimated 7% of such radiological interventions are said to reach the threshold set for temporary hair loss by the international commission on radiological protection . according to the interventionalist , it would have grossly exceeded 3 gy because the procedure turned out to be a very long one with prolonged exposure to the radiation owing to the difficulty in negotiating the loop through the site of pathology . it is not unusual for the patient to skip the history of radiation exposure because they are unaware of the fact that the procedure they have undergone involves radiation exposure and have not been forewarned by the interventionalist . severity of radiation - induced temporary alopecia is largely dependent on the dose , total duration of irradiation , interval between irradiations ( dose fractionation where applicable ) , the size of the skin area irradiated , the angle of irradiation , all affect the expression and severity of the injury . one of the commonest working positions during gdc coiling procedure is posteroanterior , which explains the occipito - vertical hair loss in this patient . loss of scalp hair can occur at relatively lower doses when compared with doses at which other body hair is lost . our patient with jet black hair developing this dramatic hair loss is suggestive of gross excess of radiation . the radiation insult damages these keratinocytes and diminishes the growth of the growing hair shaft . this results in thin , weakened hair shaft that is susceptible to trauma when it reaches the surface of scalp . hair breakage in anagen effluvium occurs within days to weeks ( 1 - 3 weeks ) following insult to the follicle . microscopic hair examination shows that the distal hair shaft is dystrophic with a rapidly tapering configuration called the bayonet hair. tapered hair can arise in any process that inhibits the cell division in the hair matrix . since 90% of hair on the scalp is in growing phase , smoking , malnutrition , pre - existing autoimmune and connective tissue disorders like diabetes , hyperthyroidism , scleroderma , rheumatoid arthritis , and systemic lupus erythematosus predispose patients to the development of severe radiation effects in an unpredictable manner . drugs that increase radiosensitivity include actinomycin d , doxorubicin , bleomycin , 5-fluorouracil , and methotrexate , mitoxantrone , 5-fluorourcil , cyclophosphamide , paclitaxel , docetaxel , and possibly tamoxifen . , it was literally adding insult to injury considering she had just recovered from a life - threatening situation . fortunately , unassisted re - population of hair in 2 - 4 months is the most likely outcome and , therefore , a gentle handholding and confidence building are all that is required . interventionalists should explain this possibility to patients in whom such a deterministic dose has been exceeded to avoid distress . fortunately , at such doses , there are very few chances of a serious or irreversible long - term consequence . however , all dermatologists should be aware of the association of hair loss with prolonged fluoroscopic endovascular procedures where the deterministic dose has been exceeded . the absorbed skin dose should be recorded on the patient 's charts or medical record . it is recommended that this dose be taken into account if the patient requires a future procedure involving exposure to ionizing radiation . minimizing the effective dose to achieve the therapeutic goal is the aim , and efforts are on to reach that goal . fluoroscopically - guided interventional procedures are now performed fairly commonly in advanced tertiary care centers in india . dermatologists who saw radiation - induced dermatoses until recently will now increasingly start seeing side - effects to the skin and hair due to these procedures , which is the second commonest cause of radiation exposure after radiotherapy.temporary alopecia of the scalp is a well - known early side - effect of ionizing radiation , which is emitted during the fluoroscopic - guided interventional procedures and is seen when a dose of 3 gy is exceeded . other factors that play a role include total duration of irradiation , interval between irradiations , size of the skin area irradiated , and the angle of irradiation.the resultant alopecia is due to anagen effluvium , and there is complete spontaneous re - growth in about 2 - 4 months . fluoroscopically - guided interventional procedures are now performed fairly commonly in advanced tertiary care centers in india . dermatologists who saw radiation - induced dermatoses until recently will now increasingly start seeing side - effects to the skin and hair due to these procedures , which is the second commonest cause of radiation exposure after radiotherapy . temporary alopecia of the scalp is a well - known early side - effect of ionizing radiation , which is emitted during the fluoroscopic - guided interventional procedures and is seen when a dose of 3 gy is exceeded . other factors that play a role include total duration of irradiation , interval between irradiations , size of the skin area irradiated , and the angle of irradiation . the resultant alopecia is due to anagen effluvium , and there is complete spontaneous re - growth in about 2 - 4 months .

the objective of this study is to examine the efficacy of gabapentin for the treatment of hemicrania continua ( hc ) in cases where patients had difficulty tolerating indomethacin due to adverse effects . a retrospective chart review of nine patients with hc between october 2006 and february 2008 . inclusion criteria included men and women age 18 or above presenting to the headache center with a headache that meets international headache society criteria for hc including a response to indomethacin , but were not able to continue on indomethacin secondary to adverse effects . four patients report being pain free , three patients report a 5080% reduction of pain , one patient reports a 10% reduction of pain , and one patient reports no change in pain . seven out of nine patients demonstrated at least a 50% reduction of pain on gabapentin , four patients becoming completely pain free .

hemicrania continua ( hc ) is an uncommon primary headache disorder characterized by a continuous , mild to moderate intensity , unilateral headache [ 14 ] . most patients will experience superimposed exacerbations of more severe pain , often associated with ipsilateral autonomic symptoms . headache for greater than 3 months fulfilling the following criteria : all of the following : unilateral pain without side shift , daily and continuous , without pain - free periods , moderate intensity , but with exacerbations of severe pain.at least one of the following autonomic features occurs during exacerbations and ipsilateral to the side of pain : conjunctival injection and/or lacrimation , nasal congestion and/or rhinorrhea , ptosis and/or miosis.a complete response to therapeutic doses of indomethacin.finally , not attributed to another disorder . unilateral pain without side shift , daily and continuous , without pain - free periods , moderate intensity , but with exacerbations of severe pain . at least one of the following autonomic features occurs during exacerbations and ipsilateral to the side of pain : conjunctival injection and/or lacrimation , nasal congestion and/or rhinorrhea , ptosis and/or miosis . a complete response to therapeutic doses of indomethacin . finally , not attributed to another disorder . there is a group of patients that meets the above criteria for hc , but has to discontinue indomethacin secondary to adverse effects , primarily involving the gastrointestinal ( gi ) tract . the side effects can range from gastritis to severe bleeding , patients may also develop an allergy to indomethacin , see an elevation of blood pressure , or an exacerbation of asthma symptoms . gabapentin is often used in an off label fashion for the treatment of neuropathic pain and is well tolerated from a side effect profile . marinano da silva , alcantara , bordini , and speciali reported a case of a unilateral headache similar to hc that was responsive to gabapentin . institutional review board approval was obtained for the study . a retrospective chart review of nine patients with hc between october 2006 and february 1 , 2008 . inclusion criteria included men and women age 18 or above presenting to the headache center with a headache that meets ihs criteria for hc , including a response to indomethacin , but were unable to continue on indomethacin secondary to adverse side effects . exclusion criteria included anyone under the age of 18 and anyone not meeting ihs criteria for hc including an adequate trial at therapeutic doses of indomethacin and response to indomethacin . patients with concomitant disease , specifically involving pain were not excluded , although every effort was made to enroll patients where hc was their primary complaint . this was decided since the purpose of this study was to examine gabapentin as an alternative therapy , indomethacin remains the gold standard . points of measurement included the duration of disease which is considered to be from onset to completion of the study , temporal profile , duration of therapy , range of dose , and concurrent use of other medications for pain , concomitant disease , especially related to pain , reduction of pain , and the side effects of gabapentin . the duration of therapy on gabapentin ranged from 3 months to 13 months with eight out of nine patients remaining on gabapentin at the end of the study . one patient discontinued after reaching 3,600 mg a day due to lack of efficacy . the other doses ranged from 600 mg to 3,600 mg per day of gabapentin . one patient also on fentanyl patch 50 mcg every 72 h reported an 80% reduction in pain after reaching 2,400 mg of gabapentin . the second patient was taking topiramate and became pain free upon reaching 600 mg of gabapentin.table 1treatment of hc with gabapentinpgardi / day ( mg)arirdg dose / day ( mg)rpdd ( months)tpdt ( months)cdcmse#1f41150gastritis1,5008/100/1013c5nonenonenone#2f5575gastritis2,4006/101/106c4lumbar radiculopathyfentanyl patch 50 mcg/72 hsedation#3f44300g.i . bleed 2 episodes3,6009/109/1025c6nonenonecognitive impairment#4f25150fatique9004/101/1036c12nonenonenone#5f46150gastritis9005/104/107c4nonenonenone#6f33150gastritis1,8008/100/1034c7nonenonevertigo#7m6375rectal bleed3,60010/104/1017c13nonenonenone#8f45300excerbation of asthma60010/100/108c5migrainetopiramate 50 mg / daynone#9f42250gastritis9005/100/103ep3atypical facial painnoneweight gainp patient , g gender , f female , m male , a age , rdi responsive dose of indomethacin , ari adverse reaction to indomethacin , rdg responsive dose of gabapentin , rp reduction of pain , dd disease duration , tp temporal profile , c chronic , ep episodic , dt duration of treatment , cd concomitant disease , cm concomitant medication , se side effects on gabapentin treatment of hc with gabapentin p patient , g gender , f female , m male , a age , rdi responsive dose of indomethacin , ari adverse reaction to indomethacin , rdg responsive dose of gabapentin , rp reduction of pain , dd disease duration , tp temporal profile , c chronic , ep episodic , dt duration of treatment , cd concomitant disease , cm concomitant medication , se side effects on gabapentin the seven patients that achieved the primary end point and reported a 5080% reduction of pain had doses ranging from 900 to 3,600 mg per day . the one man reported a 50% reduction of pain and reached the maximum dose of 3,600 mg of gabapentin per day . four patients achieved the secondary end point and reported being completely pain free on gabapentin . the dose ranges for these patients were , 600 mg , 900 mg , 1,500 mg , and 1,800 mg / day respectively . it should be noted that one of the four patients was also taking topiramate , which has been reported to be efficacious for hc [ 7 , 8 ] . the patient , who reported no benefit , responded to indomethacin on two separate occasions but developed gi bleeding as a result of indomethacin . the major side effects reported on gabapentin include sedation , cognitive impairment , depression , and weight gain . none of the patients chose to discontinue gabapentin as a result of the above side effects . hc was first described in 1981 and officially named by sjaastad and spierings in 1984 [ 9 , 10 ] . hc was originally believed to be a rare primary headache disorder but more recent evidence suggests that hc is not rare , just under recognized . in the clinical setting , hc is characterized by a unilateral headache that is continuous and of mild to moderate severity . the pain is usually reported as dull , aching , or pressure - like pain without associated symptoms . exacerbations or more severe pain , lasting anywhere from 20 min to several days , are experienced in the majority of those with hc , and when present are associated with one or more autonomic symptoms on the ipsilateral side . migrainous features , such as photophobia , phonophobia , nausea , and vomiting commonly occur during exacerbations of pain . many patients will experience a sensation of a foreign body in their eye similar to sand or an eyelash on the affected side . the pathophysiology of hc is not known , some scientist believe it is a subtype of migraine , while others believe it is more closely related to the trigeminal autonomic cephalalgias ( tac ) . it was hoped that functional brain imaging would help to clarify our understanding of hc , but it revealed that hc is a distinct headache syndrome , not belonging to the migraine or tac group . the scans revealed activation of the contralateral posterior hypothalamus and ipsilateral dorsal rostral pons , as well as activation of the ipsilateral ventrolateral midbrain , extending over the red nucleus and substania nigra and the bilateral pontomedullary junction . these areas have been previously demonstrated to be the sites of activation in the tacs and migraine , ipsilateral activation hypothalamic activation in short - lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing ( sunct ) and cluster ; in hc the findings are reversed . hc was not included in the first edition of the ichd - i , but is included within the other primary headaches section of the second edition of the ichd - ii . hemicrania continua presents as two temporal profiles : an episodic form with distinct headache phases separated by periods of pain - free remissions , and a chronic form in which the headache persists without remission for years . hc is chronic from onset in 53% of patients ; the disorder began in the episodic form and evolved into the chronic form in 35% , and it begins and remains episodic in 12% of patients . this can occur by focusing exclusively on the ipsilateral autonomic features that accompany the painful exacerbations of hc . similarly , focusing on the associated photophobia , phonophobia , nausea , and vomiting that may occur during exacerbations of hc may lead to misdiagnosing hc as migraine . autonomic features on the ipsilateral side , which are present during exacerbations of hc , are at times also present during acute attacks of migraine . hc is also distinguished from cluster headache and migraine by the presence of a continuous baseline headache that is usually unilateral and mild to moderate in severity . the associated features of photophobia , phonophobia , nausea , and vomiting , as well as the ipsilateral autonomic features of cluster are absent with the continuous baseline pain of hc . some patients are unable to tolerate indomethacin due to gi distress , renal disease , or concurrent anticoagulation therapy , and alternative types of therapy must be considered . peres and silberstein demonstrated in a study using cyclooxygenase-2 inhibitors ( cox-2 ) , efficacy of celecoxib and rofecoxib , the latter of which has since been removed from the market . they found 60% of patients who received celecoxib and 33% who received rofecoxib experienced a complete response . there is one case in the literature of a unilateral headache similar to hc that did not respond to indomethacin , and many other standard headache agents , but was responsive to gabapentin at 1,200 mg per day . there have also been two separate reports of melatonin being efficacious for hc , one case reported by spears , and three cases by rozen . there have also been four cases in the literature of patients responding to topiramate , one by camarda et al . , one by matharu et al . , and two cases by brighina et al . . gabapentin is an antiepileptic drug that is approved for use in the adjunctive treatment of partial seizures with and without secondary generalization in patients over 12 years of age . gabapentin is also indicated as adjunctive therapy in the treatment of partial seizures in pediatric patients age 312 years of age . benefit has been reported in gabapentin treatment for hypobaric hypoxia - induced headache , high altitude headache , nummular headache , sunct syndrome , chronic cluster headache , post - dural puncture headache , and primary thunderclap headache just to mention headaches disorders [ 1826 ] . the mechanism by which gabapentin exerts its analgesic action is unknown , but in animal models of analgesia , gabapentin prevents allodynia ( pain related behavior in response to a normally innocuous stimulus ) and hyperalgesia ( exaggerated response to painful stimuli ) . the mechanism by which gabapentin exerts its anticonvulsant action is unknown , but in animal test systems designed to detect anticonvulsant activity , gabapentin prevents seizures . gabapentin is structurally related to the neurotransmitter gamma - aminobutyric acid ( gaba ) but does not modify gaba a or gaba b radioligand binding , it is not converted metabolically into gaba or a gaba agonist , and it is not an inhibitor of gaba uptake or degradation . gabapentin was tested in radioligand binding assays and did not exhibit affinity for a number of other common receptor sites including benzodiazepine , glutamate , n - methyl - d - aspartate ( nmda ) , quisqualate , kainate , strychnin - insensitive glycine , alpha 1 , alpha 2 , or beta adrenergic , adenosine a1 or a2 , cholinergic muscarinic or nicotinic , dopamine d1 or d2 , histamine h1 , serotonin s1 or s2 , opiate mu , delta or kappa , cannabinoid 1 , voltage sensitive calcium channel sites labeled with nitrendipine or diltiazem , or at voltage sensitive sodium channel sites labeled with batrachotoxinin a 20 alpha benzoate . one patient reported an 80% reduction of pain on 2,400 mg of gabapentin but was also on the fentanyl patch . there have been reports in the literature of enhancement of pain control on the combination of opioids and gabapentin [ 28 , 29 ] . this patient met the primary endpoint , but the concomitant use of an opioid is a confounding factor . this study demonstrates that gabapentin is a good choice for alternative therapy in the treatment of hc . seven out of nine patients demonstrated at least a 50% reduction of pain on gabapentin , four patients becoming completely pain free . the results of the study are confounded in two patients that were taking concomitant medications specifically fentanyl patch and topiramate . another potential confounding factor is that a proton pump inhibitor was not initiated when patients had difficulty tolerating indomethacin . clinically it may be prudent to first try initiating therapy with a proton pump inhibitor while continuing indomethacin prior to changing to gabapentin . hc is a particularly difficult headache disorder to treat if the patient is not able to tolerate indomethacin , therefore alternative therapies are being sought to provide relief to this patient population . the results of this study should be validated in a double - blinded randomized control trial .

objectiveserine - threonine kinase stk11 catalyzes the amp - activated protein kinase complex . we tested the hypothesis that a gene variant in stk11 contributes to variation in insulin sensitivity and metformin efficacy.research design and methodswe studied the effects of a single nucleotide polymorphism ( snp ) ( rs8111699 ) in stk11 on endocrine - metabolic and body composition indexes before and after 1 year of metformin in 85 hyperinsulinemic girls with androgen excess , representing a continuum from prepuberal girls with a combined history of low birth weight and precocious pubarche over to postmenarchial girls with hyperinsulinemic ovarian hyperandrogenism . metformin was dosed at 425 mg / day in younger girls and 850 mg / day in older girls . stk11 rs8111699 was genotyped . endocrine - metabolic features were assessed in the fasting state ; body composition was estimated by absorptiometry.resultsgenotype effects were similar in younger and older girls . at baseline , the mutated g allele in stk11 rs8111699 was associated with higher insulin and igf - i levels ( both p < 0.005 ) . the response to metformin differed by stk11 genotype : gg homozygotes ( n = 24 ) had robust metabolic improvements , gc heterozygotes ( n = 38 ) had intermediate responses , and cc homozygotes ( n = 23 ) had almost no response . such differences were found for 1-year changes in body composition , circulating insulin , igf - i , free androgen index , and lipids ( all p < 0.005).conclusionsin hyperinsulinemic girls with androgen excess , the stk11 rs8111699 snp influences insulin sensitivity and metformin efficacy , so that the girls with the least favorable endocrine - metabolic profile improve most with metformin therapy .

the study population consisted of 85 caucasian , northern spanish girls representing a continuum of female subjects with hyperinsulinemic androgen excess that commences in prepuberty in those girls with a combined history of lbw and precocious pubarche , is followed by a preclinical phase starting around menarche of biochemical ovarian hyperandrogenism and yields to an overt clinical period of ovarian hyperandrogenism 23 years after menarche . all subjects were included in similar clinical trials investigating the safety and efficacy of metformin treatment on their hyperinsulinemic androgen excess . the prepubertal girls ( n = 18 ; tanner stage i ; aged 7.9 0.2 years ; bmi 19.3 0.6 kg / m ) had a combined a history of lbw ( 2.3 0.1 kg at term birth ) and precocious pubarche ( appearance of pubic hair before age 8 years ) attributable to an exaggerated adrenarche , based on high serum levels of dehydroepiandrosterone sulfate or androstenedione at diagnosis of precocious pubarche ( 8) . the girls were , in addition , hyperinsulinemic , as judged by the insulin response after an oral glucose tolerance test ( 16 ) . all of these subjects participated in a controlled long - term study exploring the effects of early metformin treatment ( isrctn84749320 ) ; more specifically , they form together the study subgroup that was initially randomly assigned to receive metformin for at least 1 year ( 425 mg , daily at dinner time ) ( 9 ) . the early postmenarcheal girls ( n = 31 ; aged 12.2 0.1 years ; bmi 21.5 0.5 kg / m ) manifested biochemical androgen excess characterized as the ensemble of 1 ) absence of clinical signs or symptoms of androgen excess ( 17 ) and 2 ) hyperinsulinemia ( oral glucose tolerance test ) and 3 ) ovarian androgen excess suggested by increased testosterone levels and confirmed by 17-hydroxyprogesterone hyperresponse to gonadotropin - releasing hormone agonist ( 17,18 ) . the girls in the present analysis were enrolled in clinical studies that were initiated before 2003 , thus before compulsory trial registration ; as described , all these girls had a history of lbw - precocious pubarche and were already in the early postmenarche phase ( 612 months beyond menarche ) at start of metformin intervention ( 8,19 ) . the adolescent girls ( n = 36 ; aged 15.5 0.3 years ; bmi 21.9 0.5 kg / m ) were consecutive patients with pcos who were at least 2 years beyond menarche and who were followed in the outpatient clinic according to standard procedures , including yearly blood sampling in the fasting state ( once with dna extraction ) and yearly assessment of body composition . the inclusion criteria were the same as those for the early postmenarcheal girls , except that the presence of clinical markers of androgen excess was now required : hirsutism ( ferriman - gallwey score > 8) plus either amenorrhea ( menses absent for > 3 months ) or oligomenorrhea ( menstrual cycle > 45 days ) . in all postmenarcheal girls , baseline assessments were performed in the follicular phase ( day 37 ) or after 2 months of amenorrhea . all postmenarcheal girls received metformin for at least 1 year in monotherapy ( 850 mg , daily at dinner time ) . at the start of metformin treatment , none of the 85 girls presented evidence for thyroid dysfunction , cushing syndrome , hyperprolactinemia , glucose intolerance , or late - onset adrenal hyperplasia ; for at least 6 months before the start of metformin start , none of the girls received an estro - progestagen or other medication known to affect gonadal function or carbohydrate metabolism . height and weight were measured , and bmi was calculated ( weight in kilograms divided by the square of height in meters ) . bmi standard deviation scores ( sdss ) were derived from regional normative data ( 20 ) . fasting blood glucose , serum insulin , igf - i , sex hormone binding globulin ( shbg ) , and testosterone were measured as described previously ( 9 ) . genomic dna was purified from whole blood samples using commercial reagents ( gentra puragene cell kit ; qiagen iberia , madrid , spain ) . the stk11 rs8111699 snp was genotyped by a fluorescent genotyping system ( kaspar snp genotyping system ; kbiosciences , hoddesdon , u.k . ) . in brief , 10 pg genomic dna was incubated in a 8-l solution of a combined mix containing 100 mol / l allele specific primer 1 ( fam , x - axis , allele g ) , 5-gaaggtgaccaagttcatgctccactgactgttcaggaaagcac-3 , 100 mol / l allele - specific primer 2 ( vic , y - axis , allele c ) , 5-gaaggtcggagtcaacggattccactgactgttcaggaaagcag-3 , 100 mol / l common reverse primer 5-ggtggaagcctgactgtgagagt-3 , 10 mol / l tris - hcl , ph 8.3 , ktaq polymerase , 1.8 mmol / l mgcl2 , and rox as a passive reference . the pcr mix was incubated at 94c for 15 min , followed by 20 cycles of 94c for 10 s , 57c for 5 s , and 72c for 10 s. thereafter , the pcr mix was incubated for 18 cycles at 94c for 10 s , 57c for 20 s , and 72c for 40 s. samples were then read in a fluorescence plate reader . the kaspar system uses the fluors fam and vic for distinguishing among genotypes and rox as a passive reference . the corresponding genotypes for this snp ( cc vs. cg vs. gg ) did not differ among prepuberty ( 33% vs. 44% vs. 22% ) , early postmenarche ( 26% vs. 49% vs. 26% ) , and pcos ( 25% vs. 42% vs. 33% ) subjects in our study ( p = 0.89 ) . the distributions were also tested for hardy - weinberg equilibrium , and none deviated significantly . body composition was assessed by dual - energy x - ray absorptiometry with a lunar prodigy and lunar software ( version 3.4/3.5 ; lunar , madison , wi ) ( 9 ) . total irradiation dose per assessment was 0.1 msevert ; coefficients of variation for scanning precision are 2.2 and 2.6% for fat and lean mass ( 8,9,19 ) . the study protocol was approved by the institutional review board of hospital of sant joan de du . informed consent was obtained from the patients and/or from parents , and assent was obtained from girls , as appropriate . the clinical , biochemical , and body composition data from prepubertal and early postmenarcheal subjects have been reported separately ( 8,9,19 ) . statistical analyses were performed using spss ( version 12.0 ; spss , chicago , il ) . quantitative phenotypic data were compared across genotypes by a repeated - measures general linear model , computing the genotype as the between - subjects effect and both baseline and 1-year values as the within - subjects effect . differences in 1-year changes across genotypes were tested by the interaction term among the between- and within - subjects effects . height and weight were measured , and bmi was calculated ( weight in kilograms divided by the square of height in meters ) . bmi standard deviation scores ( sdss ) were derived from regional normative data ( 20 ) . fasting blood glucose , serum insulin , igf - i , sex hormone binding globulin ( shbg ) , and testosterone were measured as described previously ( 9 ) . genomic dna was purified from whole blood samples using commercial reagents ( gentra puragene cell kit ; qiagen iberia , madrid , spain ) . the stk11 rs8111699 snp was genotyped by a fluorescent genotyping system ( kaspar snp genotyping system ; kbiosciences , hoddesdon , u.k . ) . in brief , 10 pg genomic dna was incubated in a 8-l solution of a combined mix containing 100 mol / l allele specific primer 1 ( fam , x - axis , allele g ) , 5-gaaggtgaccaagttcatgctccactgactgttcaggaaagcac-3 , 100 mol / l allele - specific primer 2 ( vic , y - axis , allele c ) , 5-gaaggtcggagtcaacggattccactgactgttcaggaaagcag-3 , 100 mol / l common reverse primer 5-ggtggaagcctgactgtgagagt-3 , 10 mol / l tris - hcl , ph 8.3 , ktaq polymerase , 1.8 mmol / l mgcl2 , and rox as a passive reference . the pcr mix was incubated at 94c for 15 min , followed by 20 cycles of 94c for 10 s , 57c for 5 s , and 72c for 10 s. thereafter , the pcr mix was incubated for 18 cycles at 94c for 10 s , 57c for 20 s , and 72c for 40 s. samples were then read in a fluorescence plate reader . the kaspar system uses the fluors fam and vic for distinguishing among genotypes and rox as a passive reference . the corresponding genotypes for this snp ( cc vs. cg vs. gg ) did not differ among prepuberty ( 33% vs. 44% vs. 22% ) , early postmenarche ( 26% vs. 49% vs. 26% ) , and pcos ( 25% vs. 42% vs. 33% ) subjects in our study ( p = 0.89 ) . the distributions were also tested for hardy - weinberg equilibrium , and none deviated significantly . body composition was assessed by dual - energy x - ray absorptiometry with a lunar prodigy and lunar software ( version 3.4/3.5 ; lunar , madison , wi ) ( 9 ) . total irradiation dose per assessment was 0.1 msevert ; coefficients of variation for scanning precision are 2.2 and 2.6% for fat and lean mass ( 8,9,19 ) . the study protocol was approved by the institutional review board of hospital of sant joan de du . informed consent was obtained from the patients and/or from parents , and assent was obtained from girls , as appropriate . the clinical , biochemical , and body composition data from prepubertal and early postmenarcheal subjects have been reported separately ( 8,9,19 ) . statistical analyses were performed using spss ( version 12.0 ; spss , chicago , il ) . quantitative phenotypic data were compared across genotypes by a repeated - measures general linear model , computing the genotype as the between - subjects effect and both baseline and 1-year values as the within - subjects effect . differences in 1-year changes across genotypes were tested by the interaction term among the between- and within - subjects effects . genotype effects were similar in prepubertal , early postmenarcheal , and pcos subjects , and therefore genotype - phenotype associations are presented for all 85 girls studied as a single group ( adjusted for group allocation ) to maximize the statistical power . clinical , biochemical , and body composition variables before and after 1 year of metformin in the study subjects values are means sem . values in healthy girls matched for bmi : prepubertal ( n = 24 ; aged 8.3 0.3 years ) : insulin 6.5 0.5 miu / l , igf - i 200 15 ng / ml , shbg 3.6 0.1 g / dl , testosterone 16 2 ng / dl ; pubertal ( n = 24 ; age 15.3 0.2 years ) : insulin 8.8 0.4 miu / l , igf - i 384 26 ng / ml , shbg 1.9 0.1 g / dl , testosterone 31 3 ng / dl . lipid profile : triglycerides 55 4 mg / dl ; ldl 83 3 mg / dl ; hdl 65 2 mg / dl ( 8,23 and ibez et al . ; jcem 2004 ; 89:4,331337 ) . the g allele in stk11 rs8111699 was associated with a poorer metabolic profile , specifically with higher insulin and igf - i ( both p < 0.005 ) ( table 2 ) . the presence of this allele was at baseline unrelated to age , weight , body composition parameters , and serum androgens and lipids ( table 2 ) . clinical , biochemical , and body composition variables before and after 1 year on metformin according to stk11 rs8111699 genotype in the study subjects values are means sem . p values for comparisons of baseline , 1-year data , and 1 year changes across genotypes are from a repeated - measures general linear model . results are adjusted for group allocation ( i.e. , prepubertal , early postmenarcheal , or pcos ) ; those for 1 year of metformin and for 01 year are also adjusted for baseline insulin levels . p 0.005 is considered the acceptable threshold of significance in these data because of the multiple tests performed ; these values appear in bold in the table . values in healthy girls matched for bmi : prepubertal ( n = 24 ; aged 8.3 0.3 years ) : insulin 6.5 0.5 miu / l ; igf - i 200 15 ng / ml ; shbg 3.6 0.1 g / dl ; testosterone 16 2 ng / dl ; pubertal ( n = 24 ; aged 15.3 0.2 years ) : insulin 8.8 0.4 miu / l ; igf - i 384 26 ng / ml ; shbg 1.9 0.1 g / dl ; testosterone 31 3 ng / dl . lipid profile : triglycerides 55 4 mg / dl ; ldl 83 3 mg / dl ; hdl 65 2 mg / dl ( 8,23 ) . after 1 year of therapy , there were pronounced differences in the response to metformin according to the stk11 rs8111699 genotype , as reflected by a robust response in the main endocrine - metabolic and body composition parameters in gg homozygotes , with an intermediate response in cg heterozygotes and almost no response in cc homozygotes ( table 2 ) . such differences were found for 1-year changes in body composition , circulating insulin , igf - i , free androgen index , and lipids ( all p < 0.005 ; table 2 ) . our results indicate that the stk11 rs8111699 snp influences both insulin sensitivity and metformin efficacy in hyperinsulinemic girls with androgen excess . despite the fact that neither stk11 nor ampk is a direct target of metformin ( 14 ) , both are necessary for metformin actions ( 14 ) . it has been proposed that stk11 is constitutively active and that metformin exerts its hypoglycemic effects through a modification in ampk , rendering it a better substrate for stk11 ( 10 ) . although the role of the studied intronic snp in stk11 has yet to be defined , it is known that intronic snps may regulate gene expression and alternative splicing ( 21 ) . the stk11 rs8111699 snp may also be in linkage disequilibrium with an active snp in the kinase gene region . the stk11 rs8111699 snp was associated with baseline insulin sensitivity in girls with hyperinsulinemic androgen excess . bearing in mind that stk11 may be constitutively active , we postulate that carrying the g allele in the stk11 rs8111699 snp can result in lower stk11 activity and therefore lower efficiency of ampk phosphorylation by stk11 . the same g allele in the stk11 rs8111699 snp was associated with pronounced changes in endocrine - metabolic and body composition parameters upon metformin treatment , effects that were nearly absent in c homozygous subjects . we suggest that metformin restores the efficiency of ampk phosphorylation by stk11 in carriers of the g allele and has less or no effect in the absence of such allele . ( 15 ) , who found a stepwise increase in ovulation rate from c / c to c / g to g / g genotype in women with pcos treated with metformin . the stk11 rs8111699 snp may thus play a broader role in metabolic and cardiovascular diseases by modulating the baseline condition as well as the response to treatment with the above - mentioned pharmacological agents . a limitation of our study is the insufficient power to analyze the effect of the stk11 snp in each of the three subgroups within the total study population . such an analysis would have been of interest because each of the consecutive subgroups is thought to represent a developmental stage of hyperinsulinemic androgen excess . it should be noted , however , that the results remained essentially the same with and without adjustment for subgroup allocation in the multivariate model . the identification of genetic polymorphisms that contribute to predict the response to commonly prescribed medications should assist clinicians in their selection of available drugs to individualize treatment , thereby improving the therapeutic response , both on the efficacy and on the safety side .

focus area : pediatricsintroduction : the centers for disease control and prevention report that autism spectrum disorder ( asd ) now affects 1 in 88 children in the usa and 1 in 47 in utah . asd is 5 times more likely in boys with 1 in 54 boys identified nationally . more children will be diagnosed with asd this year than with aids , diabetes , and cancer combined . a diagnosis of autism is based on observed behavior and educational and psychological testing . currently , there is no single definitive treatment for autism , nor is there presently a cure . traditional treatments may be biomedical , sensory , behavioral , developmental , sports - related , or arts-based.objectives:the objective was to conduct qualitative research using light hands - on touch energy work with children diagnosed with asd with focus on the following behaviors : eye contact , appropriate social behavior , verbal tics , motor control and coordination , neural impulses related to the sensory organs , immune system , and ability to learn.methods or target groups : six participants between 9 and 16 years of age were treated by teams of two practitioners in 10 sessions using light touch energy work protocol focusing on boosting the immune system and strengthening organs of the body and brain functioning . assessments included parental and practitioner observations using verified and unverified research instruments.results or activities : over a 15-month period , participants had an overall improvement of 70% in 5 categories evaluated . specifically , there was an improvement of 81% in eye contact , 72% in social behavior , 61% in motor control / coordination , 75% in sensory neural reorganization , and 65% in learning evolution.conclusion or deliverables : energy work may positively impact people living with asd and can be safely integrated with other healthcare modalities . further research needs to be conducted , and more reliable research instruments for complementary and alternative healthcare need to be developed .

the centers for disease control and prevention report that autism spectrum disorder ( asd ) now affects 1 in 88 children in the usa and 1 in 47 in utah . asd is 5 times more likely in boys with 1 in 54 boys identified nationally . more children will be diagnosed with asd this year than with aids , diabetes , and cancer combined . a diagnosis of autism is based on observed behavior and educational and psychological testing . currently , there is no single definitive treatment for autism , nor is there presently a cure . traditional treatments may be biomedical , sensory , behavioral , developmental , sports - related , or arts - based . the objective was to conduct qualitative research using light hands - on touch energy work with children diagnosed with asd with focus on the following behaviors : eye contact , appropriate social behavior , verbal tics , motor control and coordination , neural impulses related to the sensory organs , immune system , and ability to learn . six participants between 9 and 16 years of age were treated by teams of two practitioners in 10 sessions using light touch energy work protocol focusing on boosting the immune system and strengthening organs of the body and brain functioning . over a 15-month period , participants had an overall improvement of 70% in 5 categories evaluated . specifically , there was an improvement of 81% in eye contact , 72% in social behavior , 61% in motor control / coordination , 75% in sensory neural reorganization , and 65% in learning evolution . energy work may positively impact people living with asd and can be safely integrated with other healthcare modalities . further research needs to be conducted , and more reliable research instruments for complementary and alternative healthcare need to be developed .

introduction : with the improvement in anastomotic technique , it is rare to find anastomotic site leak after robot - assisted radical prostatectomy ( rarp ) . it may not always be necessary to do regular check cystogram before catheter removal . we evaluated our 230 consecutive rarp patients and their cystograms to determine the indications for selective use of cystogram before catheter removal.materials and methods : we reviewed our prospectively collected rarp database of 230 consecutive patients . cystography was performed at low pressure by gravity instillation of diluted contrast through the catheter . patients were observed under fluoroscopy in lateral oblique position for any contrast leak at the site of anastomosis . all patients were followed for a minimum of 6 months , and the longest follow - up was 5 years.results:a total of 207 patients ( 90% ) underwent catheter removal on postoperative day 7 . nine patients ( 3.9% ) had extravasation on initial cystogram . two patients with leak had a history of transurethral resection of prostate ( turp ) and seven other had bladder neck reconstruction for wide bladder neck . three patients with minimal leak did not require catheter replacement . in rest of the 6 patient with leak , continued catheter drainage was done . no significant difference in the intraoperative variables , blood loss , duration of drain , length of hospital stay , and continence outcomes was noted between the patients with leak compared to rest of the patients . none of the patient needed any procedure / intervention related to the surgery and none developed bladder neck stenosis.conclusion:in usual circumstances , catheter removal can be done safely on a postoperative day 7 without routine cystography . selective use of check cystogram can be done in the case where bladder neck reconstruction is performed or those had a prior turp and a wide bladder neck .

the optimal time of catheter removal following radical prostatectomy ( rp ) is not defined . from a patient 's perspective , early removal of the urinary catheter is desirable due to catheter - related discomfort , penile pain , bladder irritation / spasm , pericatheter urine leaks , etc . , however , the surgeon 's concerns with early catheter removal are the possibility of anastomotic site leak and acute urinary retention . with experience and improvement in techniques , the duration of catheterization has decreased from an average of 21 days in open rp era to 710 days in cases where robot - assisted rp ( rarp ) is performed . in fact , there are numerous reports about the feasibility and safety of even earlier ( i.e. on the postoperative day 35 ) catheter removal after radical retropubic prostatectomy or rarp . with early catheter removal , some concerns have been raised about increased risk of acute urinary retention . therefore , in practice , the majority of the institutions perform catheter removal after 7 days of surgery . however , there is a variation in the practice of performing cystograms before catheter removal which is done primarily to rule out anastomotic site leak . with the improvement in anastomotic technique , it is rare to find anastomotic site leak after rarp , and it may not always be necessary to obtain a routine check cystogram . we , therefore , evaluated our 230 consecutive rarp patients and the outcomes of their cystogram findings to determine the indications for cystogram before catheter removal . we reviewed our prospectively collected rarp database of 230 consecutive patients who underwent robot - assisted rp by 2 surgeons from april 2010 to january 2015 . all the patients underwent rarp through transperitoneal approach using 4 arm da vinci surgical system . clinical characteristics included patient 's age , biopsy gleason score , clinical stage , and history of transurethral resection of prostate ( turp ) or other urethral / bladder neck surgery . intraoperative data recorded included : estimated blood loss , operative time , bladder neck reconstruction , visible leak upon bladder irrigation at completion of anastomosis , and placement of pelvic drains . bladder neck sparing was done whenever possible . in patients with a wide bladder neck , bladder neck reconstruction was performed by placing sutures at the 3 and 9 oclock positions . urethrovesical anastomosis was performed in 2 layers ( described earlier ) . at the end of anastomosis , a bladder fill test was performed ( using 120 ml of saline ) to assess intraoperative anastomotic leak . patients were called for office cystography and catheter removal , usually a week after surgery . cystography was performed at low pressure by gravity instillation of 150200 ml of diluted contrast through the catheter placed during rarp . patients were observed under fluoroscopy in lateral oblique position for any contrast leak at the site of anastomosis . in the absence of extravasation , the catheter was left in place until a subsequent cystogram revealed resolution , typically 1 week later . in patients with retention of urine after catheter removal , placement of a 16fr foley catheter all patients were followed for a minimum of 6 months , and the longest follow - up was 5 years . postoperative data included pathologic gleason score and stage , hospitalization time , and catheterization time . postoperative complications included urinary retention , urinary tract infection , bladder neck contracture , and urinary incontinence . continence status was defined as the use of either no pad or just a security liner . continence rate was assessed with the self - administered questionnaire during a visit in the outpatient department or by e - mail / postal mail at 6 week and at 3 , 6 , 9 , 12 , 18 , and 24 months and then once a year . continuous parametric variables were reported as the mean standard deviations or as the median values and interquartile range . a total of 207 patients ( 90% patients ) underwent catheter removal on the 7 postoperative day while 10% ( 23 patients ) had catheter removal on the 10 day or later . a decision about delay in catheter removal was usually made by the operating surgeon , based upon satisfaction with bladder neck reconstruction or observation of minimal leak on bladder fill test during surgery . two of these patients had prior bladder neck reconstruction for the wide neck , and other two underwent standard anastomosis . as a routine , patients with bladder neck reconstruction and/or intraoperative leak ( on bladder fill test ) had their catheter removed on the day 10 . only one patient out of four with an intraoperative leak on bladder fill test had leak on postoperative cystogram . demographic details of patients total of nine patients ( 3.9% ) had extravasation on initial cystogram . among the patient with anastomotic site leak , two patients had a history of turp and seven other had wide bladder neck ( resulting from wide margin due to basal tumor ) . out of the total nine patients with leak , 3 patients had a minimal amount of contrast leak ( which cleared on post void film ) ; therefore , catheter was still not replaced [ flow chart 1 ] . in rest of the 6 patient showing leak , continued catheter drainage was done for another week and repeat cystogram was done [ figure 1 ] . four out of nine patient showing leak needed catheter for a total of 14 days while the two patients needed the catheter for a total of 21 days . no significant difference in the intraoperative variables , blood loss , duration of drain , length of hospital stay , and continence outcomes was noted between the patients with anastomotic leak ( noted on cystogram ) when compared to rest of the patients . flow chart of outcome of cystogram and their management ( a ) cystogram showing minimal anastomotic leak on the postoperative day 7 . ( b ) resolution of leak after 7 days of indwelling catheter ( day 14 ) acute urinary retention after catheter removal was noted in 3 patients . only 1 out of 3 patients who had retention after catheter removal had bladder neck sparing surgery . none of the patient experienced immediate retention of urine . out of those who developed retention ( 3 cases ) , one occurred after a week of catheter removal , and the other two developed after 48 h. the catheter was easily replaced in an emergency without any problem . these patients were started on alpha - blocker and catheter removed after 3 days with successful voiding . no pad status at 3 months , 6 months , and 1 year was 76% , 86% , and 94% , respectively . all but one patient with urine leak was continent at 6 months ( one patient became continent by 9 months of follow - up ) . no significant difference in the intraoperative variables , blood loss , and duration of drain , length of hospital stay , prostate volume , grade or stage of the tumor , and continence outcomes was noted between the patients with leak compared to rest of the patients . cystogram at the time of catheter removal is an important tool that can help in documentation of healing at anastomotic site and identifying leak , if any . however , as noted in our study and also studied by other authors , the incidence of urine leak noted on cystogram done at day 7 is very low ( 25% ) . therefore , instead of using cystogram in all the patients , selective use of cystogram can prove to be cost - effective . compared to studies published from developed countries , our patients had a higher clinical stage and more cases of post - turp status , thus requiring wide bladder neck dissection and subsequent reconstruction . we found that there is specific utility of cystogram in the patients who have had a history of turp , wide bladder neck with intraoperative bladder neck reconstruction , or minimal leak noted on intraoperative bladder fill test . although not supported by any evidence , it is our practice to delay catheter removal to postoperative day 10 ( instead of day 7 ) , in patients who undergo bladder neck reconstruction . the optimal time for catheter removal ( or indwelling catheter drainage ) is still a point of scrutiny . understandably , catheter - related symptoms and discomfort are less with shorter duration of indwelling catheter . however , the risk of anastomotic leak , acute urinary retention , and the impact of urine leak on long - term continence and bladder neck contracture are some of the concerns with early catheter removal . most of the authors consider catheter removal after 7 days minimizes the risk of anastomotic leak as well as the chance of acute retention . interestingly , there are studies that have suggested even earlier catheter removal ( at 34 days ) without any significant increase in complication . have reported 1% cystogram leak rate after catheter removal at 24 days , but with a higher rate of acute retention . it is considered that anastomotic site urine leak can potentially delay the time to continence , and adversely impact the overall continence rates . several authors have studied the outcomes in their patients who had a varying degree of anastomotic site urine leak . all the studies based on robotic prostatectomy have concluded that urinary extravasation had no adverse impact on the long - term continence status . patil et al . reported that those patients who had a leak , 70% were dry at 3 months , compared to 90% in those with no leaks . at the 12-month follow - up , 95.2% of the patients with no leak were dry , compared with 94% of those with a leak , thus concluding that at 12 months there was no difference . in our study , the number of patient with anastomotic site leak was too small to make a comparative statement about the impact on recovery pattern of continence . however , all but one patient with urine leak had zero pad status at 6 months ( one patient became continent by 9 months ) . it is also postulated that urine leak ( especially prolonged duration ) will lead to a sustained inflammatory reaction , hampering wound healing , thus increasing the risk of delayed complication such as bladder neck contracture . in a large series reporting urine leak eight of 287 ( 2.8% ) patients it was further clarified that bladder neck contracture developed in patients with higher grade leaks . only three of our patient had retention of urine after few hour of catheter removal . it is likely that the cause of acute retention is mucosal edema or hematoma or increase bladder neck smooth muscle tone . the reported risk of acute retention is reportedly higher ( 6.721% ) with catheter removal at 4 days or less . therefore , results may not be generalizable . however , at the same time , constancy of anastomotic technique , operating surgeons and the follow - up methodology is the strength of this study . the results of our study suggest that in usual circumstances , urinary catheter can be safely removed on the 7 postoperative day after rarp without a routine cystography . selective use of check cystogram can be done in cases where bladder neck reconstruction is performed or those who have a history of turp with wide bladder neck and when an intraoperative leak noted on the bladder fill test .

double gallbladder is a rare finding in patients with symptomatic cholelithiasis or acute cholecystitis . the incidence has been described as 1 in every 4000 - 5000 patients during autopsy . to identify the gallbladder ( gb ) duplication prior to surgical removal of the gb is of upmost importance . it is not unusual to identify this diagnosis intraoperatively , but by using us , ercp or mrcp more than 50% of the cases are diagnosed preoperatively . the use of intraoperative cholangiogram helps to identify the anatomy and confirm the diagnosis during laparoscopic cholecystectomy in patients with gallbladder duplication .

successful laparoscopic treatment has been described previously in only a few cases . rarely the diagnosis is suspected preoperatively when using only ultrasound . other diagnostic tests like magnetic resonance cholangiopancreatography ( mrcp ) , endoscopic retrograde cholangiopancreatography ( ercp ) and intraoperative cholangiogram can be more accurate in the diagnosis of this condition . intraoperative cholangiogram represents a powerful tool for the surgeon when dealing with double gallbladder , it help to delineate the anatomy and confirms the diagnosis . ( 1,2 ) we present a case report of a patient with symptomatic cholelithiasis with double gallbladder treated laparoscopically using intraoperative cholangiogram . a 21 year old male otherwise healthy with no previous surgical history , presented to the emergency room with the chief complaint of several hours of right upper quadrant pain that started after a large meal . the patient described a history of several weeks of right upper quadrant pain after meals but normally it would subside without any intervention . on admission , the patient had normal and stable vital signs and on physical exam there was tenderness to palpation on the right upper quadrant but no peritoneal signs . his laboratory exams were unremarkable with a normal white blood cell count and normal liver function test . the patient was admitted to the hospital and he was taken to the operating room where he underwent a laparoscopic cholecystectomy using the standard 4 port technique with intraoperative cholangiogram ( fig 1 ) . also appreciated in the image : the common bile duct , the common hepatic duct and the left and right hepatic ducts . during the operation ( fig 23 ) the intraoperative cholangiogram helped to identify each cystic duct and the operation was otherwise uneventful . he was started on clear liquid diet postoperatively and he was discharged home on postoperative day one . gallbladder duplication is a very rare diagnosis . even though the true incidence reported during autopsy is about 1:40005000 the gallbladder primordium can split and form a gallbladder duplication but this only represents a congenital anomaly of the gallbladder . true gallbladder duplication has two cystic ducts and two cystic arteries . the true double gallbladder forms from an extra primordium during the embryonic development . the symptomatic patient with two gallbladder represents a diagnostic and treatment challenge to the general surgeon.(13 ) to aid in the diagnosis the surgeon can use ultrasound , mrcp , ercp and ultrasound . none of these tools are 100% sensitive and only in little more than 50% of the cases the diagnosis is made preoperatively.(2,3 ) in our case the ultrasound was questionable but certainly prompted the use of intraoperative cholangiogram to help define the anatomy . in figure 2 two cystic ducts can clearly be seen which makes our case a true gallbladder duplication treated successfully with standard laparoscopic technique . the differential diagnosis of a gb duplication seen in us is large , but the most common diagnosis are a phrygian cap , gallbladder diverticulum or a choledochal cyst.(3 ) once the diagnosis has been made by seen two cystic ducts in the symptomatic patient , the surgical removal of both gallbladders is indicated . there are only a few case reports of double gb successfully managed laparoscopically in the literature.(3 - 7 ) our case shows that the use of intraoperative cholangiogram confirms the diagnosis and helps to identify both cystic ducts . previous literature have shown conflicting and controversial results when trying to use intraoperative cholangiogram to prevent bile duct injuries , but there no large series or randomized studies evaluating the use of intraoperative cholangiogram in patients with double gallbladder . ( 8 - 10 ) we recommend routine use of ioc when dealing with abnormal anatomy or congenital anomalies of the gallbladder . the evidence is anecdotal but as shown on this case report clearly seen two cystic ducts accurately diagnosed a gallbladder duplication and prevented the surgeon from leaving an unidentified gallbladder which later can become a diagnostic enigma .

background : phantom limb pain ( plp ) is approximately a common condition after limb amputation , which potentially affects the quality of life . we aimed to evaluate anxiety and depression in patients with amputated limbs suffering from plp and to compare these psychological dysfunctions with that of patients with non - phantom chronic pain.methods:a total number of 16 male amputees with plp and 24 male age - matched patients with non - phantom chronic pain were recruited in this study , which was performed at khatam - al - anbia pain clinic , tehran , iran . a validated persian version of the hospital anxiety and depression scale ( hads ) was used to compare two psychological dysfunctions anxiety and depression between the two groups of study.results:the mean of total anxiety score was significantly lower in patients with plp ( 8.00 3.93 vs. 11.25 5.23 ; p = 0.041 ) and the prevalence of anxiety caseness ( hads - a score 11 ) was also lower in the plp group ( 25% vs. 58.3% ; p = 0.112 , power = 31.7% ) . the mean of total depression score was 7.69 5.51 and 9.38 6.11 in patients of plp and chronic pain groups , respectively ( p = 0.340 , power = 15% ) . consequently , the prevalence of depression caseness ( hads - d score 11 ) was lower in plp patients ( 37.5% vs. 50% ; p = 0.710 , power = 8%).conclusion : our results indicate that depression and anxiety are not more common in plp patients , whereas they are more prevalent in subjects with non - phantom chronic pain . these lower levels of anxiety and depression in plp compared with chronic pain is a new finding that needs to be evaluated further , which may lead to new insights into the pathogenesis of phantom pain in further studies .

phantom limb pain ( plp ) is classified as a kind of neuropathic pain that is induced after nerve injury . it is an after effect of amputation occurring in up to 50 - 85% of the patients,[13 ] and often leads to significant impairment in occupational and social functioning . daily pain is reported by 48% of patients with plp and it is supposed that these symptoms potentially affect their quality of life . more importantly , emotional factors are influential in patients experience of prolonged pain in a phantom limb after amputation . on the one hand , it is likely that phantom pain develops poor health - related quality of life such as emotional problems including anxiety and depression . on the other hand , it is also possible that poor quality of life due to emotional problems induces phantom pain and some conditions , such as anxiety and depression , may themselves worsen plp.[1810 ] although some psychological investigations have been performed in patients suffering from plp , controversial findings have been reported . although multiple psychological modalities have been attempted in managing plp[1119 ] and psychotherapy was reported to yield good results , katz and melzack found no significant difference in standardized tests of psychological dysfunction between patients who experienced phantom pain and those who did not . they concluded that the pain is more likely to vary with the experience of pre - amputation pain , even retaining many of its characteristics . a review of the literature on measures used to diagnose psychopathology found that many measures include items that confound emotional distress with the physical disorder and it is estimated that since the period 1996 - 2002 , the number of hads papers that have been published has increased almost fourfold . a recent review of the literature on the validity of hads clearly indicates that it is a well - performed questionnaire in assessing the symptom severity and caseness of anxiety disorders and depression in somatic , psychiatric , and primary care patients and even in the general population . however , the prevalence and severity of these psychological dysfunctions have not been compared in plp patients with other chronic pain sufferers . previous comparative studies have focused on the comparison of psychiatric disorders in plp patients with non - pain amputees in which the effect of pain itself was assessed . therefore , the aim of our study was to evaluate anxiety and depression in patients with amputated limbs suffering from phantom pain and to compare these psychological disorders with those of patients with non - phantom chronic pain using the hads questionnaire . thus , in contrast to previous investigations , it could be possible to evaluate the effect of amputation instead of the pain itself . this prospective analytical cross - sectional study was conducted in khatam - al - anbia pain clinic , tehran , iran , between 2008 and 2010 . a total number of 16 male amputees consecutively participated in the study as the plp group . for this purpose , patients with upper and lower limb amputations between the ages of 25 years and 65 years with traumatic amputations and with plp ( at least five points on a visual analogue scale [ vas ] for at least 3 months ) were included . plp was defined as pain perceived at parts of an extremity that were no longer present . therefore , the presence of plp and phantom sensation was based on the answers to the following questions : ( i ) do you feel the amputated part of your limb ? ( ii ) do you feel pain in the amputated part of your limb ? if the answer to the second question was yes the severity of the pain was investigated using the vas ( 0 - 10 cm ) . as a control group , 24 male age - matched patients who were experiencing non - phantom chronic pain were recruited and evaluated at the same period of time . those eligible were men aged 25 years or older with neuropathic pain of at least 3 months in duration caused by trauma or surgery , with allodynia or hyperalgesia , and with an average weekly pain intensity score greater than five on a 10 cm vas . it must be noted that all of the patients were administered the same analgesic protocol , had a stable analgesic regimen , and were followed in the khatam - al - anbia pain clinic . the study was approved by the medical ethics committee of khatam - al - anbia hospital . patients were told that their data and the results of their assessment were being using for research purposes , and each patient provided written informed consent . after each patient fulfilled the eligible criteria , a list of demographic , baseline , and pain characteristics were asked , which were recorded at the time of enrollment . these variables consisted of age , marital status , educational level , body mass index ( bmi ) , and pain characteristics including duration , average , highest and lowest intensity , and aggravating factors ( e.g. , somatic , psychotic , prolonged fix position , changed position , prolonged rest ) . pain intensity measurements were taken at baseline for all of the 40 recruited patients using a 10 cm vas . the patients were instructed to record the highest , the lowest , and the average intensity of their pain within the last 4 weeks prior to the study enrollment . they were asked to point out each intensity on a 10 cm vas , which theoretically ranged between 0 and 10 , where 0 indicates no pain and 10 corresponds to the worst pain imaginable . the hads was used to assess two psychological dysfunctions anxiety and depression in two groups of the patients . it has been shown that hads provides clinically meaningful results as a psychological screening tool , in clinical group comparisons and in studies with several aspects of disease and quality of life . as mentioned previously , the validity of this questionnaire was previously evaluated and it was indicated that hads is a well - performed questionnaire in assessing the symptom severity and caseness of anxiety disorders and depression in chronic pain sufferers . the persian version of the hads questionnaire was first validated by montazeri et al . in 2003 . they showed that the iranian version of the hads questionnaire was generally acceptable to almost all patients . checking the reliability of the hads , cronbach 's alpha coefficient was found to be 0.78 for the anxiety subscale and 0.86 for the depression subscale . using the known groups comparison and convergent analysis , the persian version of the hads questionnaire was shown to be valid . moreover , both anxiety and depression subscales discriminated well between subgroups of their sampled patients with different statuses . the hads questionnaire contains 14 items and consists of two subscales : anxiety and depression . each subscale consists of seven items , where the odd - numbered questions belong to the anxiety subscale ( hads - a ) and the even items belong to the depression subscale ( hads - d ) . each item is rated on a four - point scale ( scored between 0 and 3 ) , giving maximum scores of 21 for anxiety and depression . in addition , the total score of each subscale could be categorized using some cutoff points . scores of 11 or more on either subscale are considered to be a significant case of psychological morbidity , whereas scores of 8 - 10 represent borderline and 0 - 7 represent normal. all the 40 participants were asked to complete the hads questionnaire , following which the scores were calculated for each subscale for each patient . a sample size of 16 patients in each group was calculated to detect a difference of two units in the total score of the hads questionnaire in each subscale with an estimation of 5% for type i error and 20% for type ii error based on a two - sided independent samples t - test . descriptive statistics were used to determine frequency and percentage for categorical variables , and mean and standard deviation ( sd ) for continuous measurements . after checking the normal distribution with the kolmogorov - smirnov test , the differences in patient characteristics and hads scores ( including each item and total scores ) between the plp group and non - phantom chronic pain patients were analyzed with the mann whitney u test , independent samples t - test , fisher 's exact test , and the chi - square test , as appropriate . analyses were performed with the statistical package for the social sciences , version 16 ( spss inc . , this prospective analytical cross - sectional study was conducted in khatam - al - anbia pain clinic , tehran , iran , between 2008 and 2010 . a total number of 16 male amputees consecutively participated in the study as the plp group . for this purpose , patients with upper and lower limb amputations between the ages of 25 years and 65 years with traumatic amputations and with plp ( at least five points on a visual analogue scale [ vas ] for at least 3 months ) were included . plp was defined as pain perceived at parts of an extremity that were no longer present . therefore , the presence of plp and phantom sensation was based on the answers to the following questions : ( i ) do you feel the amputated part of your limb ? ( ii ) do you feel pain in the amputated part of your limb ? if the answer to the second question was yes the severity of the pain was investigated using the vas ( 0 - 10 cm ) . as a control group , 24 male age - matched patients who were experiencing non - phantom chronic pain were recruited and evaluated at the same period of time . those eligible were men aged 25 years or older with neuropathic pain of at least 3 months in duration caused by trauma or surgery , with allodynia or hyperalgesia , and with an average weekly pain intensity score greater than five on a 10 cm vas . it must be noted that all of the patients were administered the same analgesic protocol , had a stable analgesic regimen , and were followed in the khatam - al - anbia pain clinic . the study was approved by the medical ethics committee of khatam - al - anbia hospital . patients were told that their data and the results of their assessment were being using for research purposes , and each patient provided written informed consent . after each patient fulfilled the eligible criteria , a list of demographic , baseline , and pain characteristics were asked , which were recorded at the time of enrollment . these variables consisted of age , marital status , educational level , body mass index ( bmi ) , and pain characteristics including duration , average , highest and lowest intensity , and aggravating factors ( e.g. , somatic , psychotic , prolonged fix position , changed position , prolonged rest ) . pain intensity measurements were taken at baseline for all of the 40 recruited patients using a 10 cm vas . the patients were instructed to record the highest , the lowest , and the average intensity of their pain within the last 4 weeks prior to the study enrollment . they were asked to point out each intensity on a 10 cm vas , which theoretically ranged between 0 and 10 , where 0 indicates no pain and 10 corresponds to the worst pain imaginable . the hads was used to assess two psychological dysfunctions anxiety and depression in two groups of the patients . it has been shown that hads provides clinically meaningful results as a psychological screening tool , in clinical group comparisons and in studies with several aspects of disease and quality of life . as mentioned previously , the validity of this questionnaire was previously evaluated and it was indicated that hads is a well - performed questionnaire in assessing the symptom severity and caseness of anxiety disorders and depression in chronic pain sufferers . the persian version of the hads questionnaire was first validated by montazeri et al . in 2003 . they showed that the iranian version of the hads questionnaire was generally acceptable to almost all patients . checking the reliability of the hads , cronbach 's alpha coefficient was found to be 0.78 for the anxiety subscale and 0.86 for the depression subscale . using the known groups comparison and convergent analysis , the persian version of the hads questionnaire was shown to be valid . moreover , both anxiety and depression subscales discriminated well between subgroups of their sampled patients with different statuses . the hads questionnaire contains 14 items and consists of two subscales : anxiety and depression . each subscale consists of seven items , where the odd - numbered questions belong to the anxiety subscale ( hads - a ) and the even items belong to the depression subscale ( hads - d ) . each item is rated on a four - point scale ( scored between 0 and 3 ) , giving maximum scores of 21 for anxiety and depression . in addition , the total score of each subscale could be categorized using some cutoff points . scores of 11 or more on either subscale are considered to be a significant case of psychological morbidity , whereas scores of 8 - 10 represent borderline and 0 - 7 represent normal. all the 40 participants were asked to complete the hads questionnaire , following which the scores were calculated for each subscale for each patient . a sample size of 16 patients in each group was calculated to detect a difference of two units in the total score of the hads questionnaire in each subscale with an estimation of 5% for type i error and 20% for type ii error based on a two - sided independent samples t - test . descriptive statistics were used to determine frequency and percentage for categorical variables , and mean and standard deviation ( sd ) for continuous measurements . after checking the normal distribution with the kolmogorov - smirnov test , the differences in patient characteristics and hads scores ( including each item and total scores ) between the plp group and non - phantom chronic pain patients were analyzed with the mann whitney u test , independent samples t - test , fisher 's exact test , and the chi - square test , as appropriate . analyses were performed with the statistical package for the social sciences , version 16 ( spss inc . , sixteen male patients with plp and 24 age - matched males who suffered from non - phantom chronic pain were recruited in this study . a complete list of demographic , baseline , and pain characteristics of the patients are shown in table 1 . the mean age of the patients with plp was 48.88 7.47 year , which was similar to that of chronic pain sufferers ( 48.63 9.42 year , p = 0.930 ) . comparison of the baseline and pain characteristics between the two groups of the study regarding pain characteristics , the mean duration of pain was 12.00 10.36 year in plp patients , which was not significantly different from the pain duration in the other group ( 10.35 11.54 year , p = 0.315 ) . using vas , the mean of the average severity of pain was found to be 8.12 1.86 and 7.82 1.94 in phantom pain and non - plp patients , respectively ( p = 0.627 ) . although psychotic factors were the most involved pain - aggravating factors in the plp group ( 87.5% ) , prolonged fix position was more responsible for worsening of pain in patients of group b ( 95.2% ) . the results of hads scores in each item and overall anxiety and depression status are shown and compared between the two groups of the study in table 2 . as is seen , the mean of hads scores for each of the seven items of anxiety ( hads - a ) is higher in patients of group b who suffered from non - plp chronic pain , which is in favor of a more severe anxiety . consequently , the mean of total anxiety score was significantly lower in patients with plp ( 8.00 3.93 vs. 11.25 5.23 ; p = 0.041 ) . comparison of the hospital anxiety and depression scale scores in each item and overall anxiety and depression status between two groups of the study moreover , when the total anxiety score is categorized using the proposed cutoff points of previous studies , the prevalence of anxiety caseness ( hads - a score 11 ) was found to be higher in chronic pain sufferers ( 58.3% vs. 25% ; p = 0.112 , power = 31.7% ) . as listed in table 2 , except for item number 14 , the other hads scores of six items in the depression subcategory ( hads - d ) were also higher in patients with non - phantom chronic pain , which was in favor of a more severe depressed mood . the mean of total depression score was 7.69 5.51 and 9.38 6.11 in patients of the plp and chronic pain groups , respectively . however , this difference was not statistically significant ( p = 0.340 , calculated power = 15% ) . when the total depression score is categorized using the proposed cutoff points of previous studies , the prevalence of depression caseness ( hads - d score 11 ) was also found to be lower in patients with phantom pain ( 37.5% vs. 50% ; p = 0.710 , power = 8% ) . sixteen male patients with plp and 24 age - matched males who suffered from non - phantom chronic pain were recruited in this study . a complete list of demographic , baseline , and pain characteristics of the patients are shown in table 1 . the mean age of the patients with plp was 48.88 7.47 year , which was similar to that of chronic pain sufferers ( 48.63 9.42 year , p = 0.930 ) . comparison of the baseline and pain characteristics between the two groups of the study regarding pain characteristics , the mean duration of pain was 12.00 10.36 year in plp patients , which was not significantly different from the pain duration in the other group ( 10.35 11.54 year , p = 0.315 ) . using vas , the mean of the average severity of pain was found to be 8.12 1.86 and 7.82 1.94 in phantom pain and non - plp patients , respectively ( p = 0.627 ) . although psychotic factors were the most involved pain - aggravating factors in the plp group ( 87.5% ) , prolonged fix position was more responsible for worsening of pain in patients of group b ( 95.2% ) . the results of hads scores in each item and overall anxiety and depression status are shown and compared between the two groups of the study in table 2 . as is seen , the mean of hads scores for each of the seven items of anxiety ( hads - a ) is higher in patients of group b who suffered from non - plp chronic pain , which is in favor of a more severe anxiety . consequently , the mean of total anxiety score was significantly lower in patients with plp ( 8.00 3.93 vs. 11.25 5.23 ; p = 0.041 ) . comparison of the hospital anxiety and depression scale scores in each item and overall anxiety and depression status between two groups of the study moreover , when the total anxiety score is categorized using the proposed cutoff points of previous studies , the prevalence of anxiety caseness ( hads - a score 11 ) was found to be higher in chronic pain sufferers ( 58.3% vs. 25% ; p = 0.112 , power = 31.7% ) . as listed in table 2 , except for item number 14 , the other hads scores of six items in the depression subcategory ( hads - d ) were also higher in patients with non - phantom chronic pain , which was in favor of a more severe depressed mood . the mean of total depression score was 7.69 5.51 and 9.38 6.11 in patients of the plp and chronic pain groups , respectively . however , this difference was not statistically significant ( p = 0.340 , calculated power = 15% ) . when the total depression score is categorized using the proposed cutoff points of previous studies , the prevalence of depression caseness ( hads - d score 11 ) was also found to be lower in patients with phantom pain ( 37.5% vs. 50% ; p = 0.710 , power = 8% ) . as an approximately common condition after limb amputation , plp serves as a disabling chronic pain syndrome for which regular pain treatment is seldom effective . this painful condition may be the result of inappropriately stored or chronically activated pain memories that continue to disturb the subject even after the disease or injury has been successfully treated or the limb has been amputated.[2831 ] on the other hand , it seems that both the amputation of a limb and the consequent phantom pain may affect quality of life , especially with regard to psychological functions . the incidence of anxiety and depression symptoms after amputation has been reported to be 37% and 20% , respectively , and even as high as 41%[3336 ] and much higher than the rates in the community . alongside these considerable prevalence rates , there are some other evidences that show these psychological dysfunctions are not more prevalent among those amputees reporting phantom pain . moreover , research in this field is difficult because of the different measurement tools used for anxiety and depression as well as the lack of prospective studies . in the present study , it was hypothesized that the prevalence rates and scores of anxiety and depression could be more common in patients with plp compared with non - phantom chronic pain sufferers . regarding the proposed cutoff points for the caseness of anxiety and depression in the hads questionnaire , 25% and 37.5% of our patients with plp fulfilled the criteria , respectively . nevertheless , our results indicate that depression and anxiety are more common in plp patients , and these conditions are more prevalent in non - phantom chronic pain sufferers . in 1973 , parkes suggested that emotional and psychological factors are dominant in patients with prolonged pain due to phantom limb after amputation . in another study using the hads scale , badura - brzoza et al . declared that patients after limb amputation achieved a higher score in hads - a and hads - d compared with the control group and concluded that higher levels of anxiety and depression are noticed in patients suffering from plp . in contrast , katz and melzack found no significant difference in psychological dysfunction between patients who experienced phantom pain and those who did not . in another study by fisher and hanspal , the hads score was compared between 29 plp patients with 64 non - phantom pain amputees with the mean time of post - amputation of 9.7 years . the mean reported hads scores in their study were 4.66 and 3.45 for anxiety and depression subscales , respectively . they concluded that only a few patients experienced emotional distress after amputation , and anxiety is being reported more often than depression . in agreement with the studies of katz and melzack and fisher and hanspal , our results also confirmed the lower prevalence of depression and anxiety in patients with plp compared with the control group . however , it should be noted that in the study of katz and melzack and fisher and hanspal , the control group consisted of amputees who did not experience any plp , whereas in our study the patients with non - phantom chronic pain were considered as the control subjects . moreover , compared to their results , the mean score of anxiety and depression was higher in our plp patients ( 8.00 and 7.69 , respectively ) though it was considerably lower when compared with that of chronic pain sufferers ( 11.25 and 9.38 , respectively ) . a longitudinal study by singh et al . even showed that there is a dramatic drop in the incidence of psychological symptoms in individuals after amputation by the time of discharge from a rehabilitation ward . they have concluded that it may be due to adjustment after the emotional stress of limb loss as well as the learning of new skills to adjust to life after amputation . this explanation could be also indicated in our patients , as they received different special cares at our pain clinic , too . however , in another more recent study by singh et al . , it was shown that after an initial drop in incidence of depression and anxiety symptoms after amputation , there is then an increase again after 2 - 3 years post amputation . in contrast , our evaluated plp patients had a mean duration of 12 years post amputation and still showed a lower prevalence of anxiety and depression . to the best of our knowledge , our study is the first one to compare the prevalence of anxiety and depression in plp patients with a sample of non - phantom chronic pain sufferers . in addition to the lesser mean scores of these psychological dysfunctions by the hads questionnaire , it was shown that the caseness of anxiety ( 25% vs. 58.3% ) and depression ( 37.5% vs. 50% ) were also lower in plp patients compared with subjects with chronic pain . a review of the literature reveals that the considerable prevalence of depression in chronic pain sufferers has been previously found in some investigations such as that of magni et al . in subjects with chronic musculoskeletal pain where the authors suggest a two - way relationship similar to most of the previous studies , one of the important limitations of our study was the cross - sectional design and lack of longitudinal assessment . in addition , the low statistical power in the comparison of depression score emphasizes the need for a higher sample size in further investigations . the results of our study suggest that although the prevalence rates of anxiety and depression are considerable in patients with plp , they were not as frequent as what is observed in non - phantom chronic pain sufferers . these lower levels of anxiety and depression in plp compared to chronic pain is a new finding , which needs to be further evaluated and may lead to new insights into the pathogenesis of phantom pain in further studies .

double - orifice mitral valve ( domv ) is a very rare congenital anomaly that usually presents as mitral regurgitation . we present the case of a 39-year - old asymptomatic , healthy man with no previous medical history who was affected by isolated complete bridge type domv , incidentally detected by two - dimensional echocardiographic examination in the parasternal short - axis view . the mitral valve of the patient was normally functioning without any other coexistent cardiac abnormalities . isolated domv was also confirmed by cardiac magnetic resonance imaging . the patient is now followed up to detect possible complications .

isolated double - orifice mitral valve ( domv ) is an extremely rare congenital anomaly whose exact incidence has not been conclusively determined . interestingly , this cardiac abnormality rarely presents as an isolated finding , preferentially associated with valvular regurgitation rather than stenosis . the etiology of domv has been recognized in the abnormal fusion of the endocardial cushions and abnormal development of the mitral valve during the delamination process . transthoracic echocardiography is the modality of choice for the characterization of this anomaly ; in this respect , the parasternal short - axis view allows a full morphologic characterization of the anomaly and should always be performed in the case of a meaningful clinical suspect . we present the case of an asymptomatic patient with isolated domv who was referred for cardiac evaluation to achieve a certificate of fitness for competitive sports . a 39-year - old asymptomatic and healthy man was referred to our outpatients clinic for cardiac evaluation to achieve a certificate of fitness for competitive sports . he had no previous medical history , but presented a strong family history of early coronary artery disease . accordingly , a complete transthoracic two - dimensional echocardiography with color doppler examination was performed with a philips ie33 ultrasound machine ( philips healthcare , leiden , the netherlands ) . the echocardiographic examination in the parasternal short - axis view showed a distinctive morphology of an isolated domv [ figure 1 ] . the two mitral orifices were located in the posteromedial and posterolateral positions and their estimated areas were 2.20 cm and 1.98 cm , respectively [ figure 2 ] . there was a single mitral annulus with the two valvular orifices separated by a central fibrous bridge [ figure 1 ] . the echocardiographic examination seemed to show separated subvalvular structures for each of the two orifices [ figure 3 ] . a normal function of both the anterior and posterior leaflets of each orifice was present without evidence of either systolic mitral regurgitation jets or diastolic turbulence [ figure 4 ] . moreover , the two peak and the mean transmitral gradients were normal . similarly , a normally functioning mitral valve ( i.e. without stenosis or insufficiency ) was evident also in the four - chamber view . parasternal short - axis view at the level of the mitral valve in diastole showing the bridge of tissue dividing the mitral valve into two orifices parasternal short - axis view at the level of the mitral valve in diastole showing the areas of the orifices of the double - orifice mitral valve apical four - chamber view seems to show the two separate subvalvular structures of the double - orifice mitral valve apical four - chamber view with color doppler demonstrating the absence of mitral regurgitation the resting echocardiographic examination excluded the presence of any sign of either congenital or acquired morphological cardiac abnormalities . to definitively exclude the presence of any other cardiac or vascular anomaly , a cardiac magnetic resonance imaging evaluation was performed , which confirmed the findings of two - dimensional echocardiography as it showed domv and each of the two orifices with its own chordal apparatus [ figures 57 ] . the patient is now followed up on a yearly basis for the early recognition of any valvular alteration . cardiac magnetic resonance imaging demonstrating normal left ventricular volumes , apex hypertrabeculation , and absence of atrioventricular / ventricular septal defects cardiac magnetic resonance imaging demonstrating the two orifices of the double - orifice mitral valve in the absence of other cardiac abnormalities cardiac magnetic resonance imaging demonstrating the two orifices of the double - orifice mitral valve in the absence of other cardiac abnormalities this anomaly is characterized by the presence of a single mitral annulus with two orifices , each having independent subvalvular apparatus and papillary muscles . domv is an extremely rare condition , usually associated with other congenital heart anomalies ( above all , atrioventricular septal defect , coarctation of aorta , interrupted aortic arch , patent ductus arteriosus , and ventricular septal defect ) . it generally presents with symptoms related to mitral regurgitation or stenosis , but it can also present as an incidental finding . three specific types of domv can be described at two - dimensional echocardiography : ( 1 ) complete bridge type ( about 15% of domvs ) , in which both openings are visible from the leaflet edge and both orifices appear circular ( equal or unequal in size ) with normal subvalvular apparatus and papillary muscles ; ( 2 ) incomplete bridge type , in which the connection is seen only at the leaflet edge ; and ( 3 ) hole type ( the most frequent ) characterized by a small accessory orifice situated at either the posteromedial or anterolateral commissure and identifiable only at the mid - leaflet level . accordingly , our patient presented the rarest form of the anomaly , as represented by the complete bridge type domv . transesophageal echocardiography ( tee ) defines the anatomy of mitral valve better than transthoracic echocardiography and may help to differentiate domv from other conditions such as lesions of valvular leaflets provoked by endocarditis . however , despite these theoretical advantages , tee was not performed in the diagnostic work - up of the patient because of its invasiveness . on the other hand , once domv is nearly assured by two - dimensional echocardiography , cardiac magnetic resonance imaging evaluation becomes a mandatory step to confirm the clinical suspicion , and above all , to exclude any concurrent cardiac and vascular abnormalities . moreover , three - dimensional echocardiography is generally useful to add anatomical details to that obtained by two - dimensional echocardiography . however , it should be performed only in case of uncertain findings at the two - dimensional echocardiography , which is considered the screening test for domv . the management of domv is related to the type and severity of mitral valve dysfunction . asymptomatic domv usually requires no active intervention ; in case of severe mitral regurgitation , stenosis , or associated cardiac anomalies , surgical repair is needed . in all cases of domv , a long - term follow - up is required for the early detection of complications . thus , domv is a very rare anomaly which is usually detected by transthoracic two - dimensional echocardiographic examination in short - axis parasternal views . nevertheless , it can be rarely diagnosed in asymptomatic middle - aged and elderly patients . in any case , the presence of other coexistent cardiovascular abnormalities should be excluded .

mercury is added to the biosphere by anthropogenic activities raising the question of whether changes in the human chromatin , induced by mercury , in a parental generation could allow adaptation of their descendants to mercury . we review the history of andean mining since pre - hispanic times in huancavelica , peru . despite the persistent degradation of the biosphere today , no overt signs of mercury toxicity could be discerned in present day inhabitants . however , mercury is especially toxic to the autonomic nervous system ( ans ) . we , therefore , tested ans function and biologic rhythms , under the control of the ans , in 5 huancavelicans and examined the metal content in their hair . mercury levels varied from none to 1.014 ppm , significantly less than accepted standards . this was confirmed by microfocused synchrotron x - ray fluorescence analysis . biologic rhythms were abnormal and hair growth rate per year , also under ans control , was reduced ( p < 0.001 ) . thus , evidence of mercury 's toxicity in ans function was found without other signs of intoxication . our findings are consistent with the hypothesis of partial transgenerational inheritance of tolerance to mercury in huancavelica , peru . this would generally benefit survival in the anthropocene , the man - made world , we now live in .

the largest mercury processing in the western hemisphere was in huancavelica in the peruvian andes , now a town of 30,000 people situated at 3676 m altitude . the extensive mercury deposits have been mined since pre - hispanic times , they are just above the present town at ~4000 m. although the mine closed 30 years ago , mining continues on small scales . the legacy of 500 years of mercury mining is now found in the city trenches and waterways , which are lined with mercury but this legacy is less evident in today 's inhabitants of huancavelica . mining is a dangerous , unhealthy occupation but nowhere was it more health threatening than in colonial south america , especially in huancavelica . the widespread ecological impacts of mining in the andes such as landscape destruction and pollution through the introduction of mercury and other toxicants into the biosphere were overshadowed by the population shifts to the then sparsely inhabited highlands . there , the challenges of survival in the ambient hypoxia of altitude , at which mining generally occurs in the andes , were added to the deleterious health effects of mining . in 1928 , carlos monge of lima , peru , described chronic mountain sickness ( cms ) in a native miner from cerro de pasco , a mining town in peru situated at 4338 m above sea level . it occurs in high altitude natives ( hypoxia adapted individuals ) who for , as yet , unknown reasons lose their adaptation to their hypoxic homeland . recently , cms was found to be associated with deranged expression of hypoxia - related genes . in colonial times , less so now , mining required heavy physical labor which predisposes to cms . the toxicity of mercury , when added to the stress of inescapable hypoxia at high altitude and the dangers to health and predisposition to injury of mining in colonial huancavelica , earned the mine the infamous reputation as the mina de la muerte ( the mine of death ) . because of its importance to the spanish economy , mercury killed thousands of andeans . prior to the conquest , the locals used the huancavelica cinnabar ( mercury sulfide , hgs ) deposits for body paint and cosmetics for their women . but , after the spanish arrival , the lord of the local angaraes people betrayed the cinnabar deposits to amador de cabrera in 1563 . the riches of the deposits enticed newly arriving colonists to stake claims begin mining cinnabar , and distill mercury which they then sold to mexico 's silver refineries . demand for mercury increased when the silver mines at potosi began using mercury to amalgamate the ores in 1570 . the subsoil now belonged to the crown which granted its use to miners upon payment of taxes . under the new rules the huancavelica treasury contracted with former claim holders to keep the mine operating , setting the amount of mercury to be produced and the price to be paid . a change in the production of silver which required mercury for amalgamation of the ore increased the demand for labor in the mines . toledo committed the colonial government to provide adequate manpower for the mercury producers . to do this , he copied inca rules which required adult males to take turns on public projects called a mit'a ( turn ) and ordered neighboring provinces to send 900 mitayos . the population in the surrounding areas decreased because of disease and the demands of the mita . this forced the government to reduce the size of the mita to 620 by 1645 and expand the area from which mitayos were committed into the labor force . mitayos received a low wage but served only for 2 months at a time , taken here as evidence for governmental awareness of the devastating health effects of mercury mining . huancavelica became notorious as a dangerous workplace by the late 16th century and the spanish crown received reports that the mita was abusive , unchristian , and unhealthy . the high altitude of the mine added the stresses of ambient hypoxia , extreme temperatures and harsh weather to the hazards of mining , such as landslides and tunnel collapse . by 1600 the reputation of the huancavelica mine as the mine of death was well deserved . the franciscan miguel agia , in favor of the continuation of the mita , admitted that work in the mine was equivalent to sending [ indians ] to die . and the protector of indians damin de jeria likened the tunnel of the mine to a luis de velasco , another viceroy , wrote to the king about the health aspects of the huancavelica mine in 1600:these quicksilver ores , when they extract them in the mines , they give out a dust that enters itself into the indians as they breath and settles in the chest , of such evil quality , that it causes them a dry cough and light fever and at the end death without repair , because the doctors have it for an incurable evil . these quicksilver ores , when they extract them in the mines , they give out a dust that enters itself into the indians as they breath and settles in the chest , of such evil quality , that it causes them a dry cough and light fever and at the end death without repair , because the doctors have it for an incurable evil . he wanted to close the mine but because of its economic importance , he pleaded for guidance from the crown [ 8 , 9 ] . de jeria wrote to the viceroy that although the mitayos had committed no crimes they were forced to endure conditions worse than those imposed on criminals in prisons and galleys . he argued that huancavelica mining will eventually cause economic collapse because its continuance , costing so many lives of so many indians that which may be lost from not producing mercury is gained in the conservation of the indians , for without them there will be neither quicksilver , nor silver , nor the common good , nor peru . by 1630 , the count of chinchn , then viceroy , wrote to the king about the mita : it is a most terrible matter because a free , innocent , defenseless , poor and afflicted people are condemned to a notorious risk of death their fear of the mita was such that trustworthy people who have seen it have reported to me that they take these miserable indians by force against all their will from their houses and take them in iron collars and chains more than 100 leagues to put them in this risk and from this has resulted their own mothers maiming and crippling their sons to preserve them . it is a most terrible matter because a free , innocent , defenseless , poor and afflicted people are condemned to a notorious risk of death their fear of the mita was such that trustworthy people who have seen it have reported to me that they take these miserable indians by force against all their will from their houses and take them in iron collars and chains more than 100 leagues to put them in this risk and from this has resulted their own mothers maiming and crippling their sons to preserve them . pick - men and ore carriers developed silicosis ( deposition of dust and silica particles in the lungs ) rapidly because of enormous dust levels in the mine , but this also affected work in other mines . in huancavelica , additionally , mercury killed by its inhalation . after returning to their huts , they transferred the dust to their living quarters and contaminated their families by prolonging exposure and mercury absorption through the skin and food . the symptoms of mercury poisoning were known to the workers who called it the huancavelica sickness or the evil of huancavelica . although the usual treatment for poisoned miners was bloodletting to put their bodily humors back into balance , this increased their susceptibility to intercurrent illnesses . eventually the graves of mitayos were said to contain puddles of mercury after their bodies decayed [ 4 , 7 ] . the threat of mercury poisoning was even greater to those who worked the ovens to heat the ore and extract the mercury as the vapors cooled . and indians were made to open the ovens before they had completely cooled to increase mercury extraction by reloading them with fresh ore . those rendered unable to work because of mercury poisoning were known as azogados ( azogue = quicksilver ) . weight loss , tremors , excessive salivation , ulcers of the mouth , restlessness , anemia , and eventually severe depression which often lead to alcoholism . many miners had to be fed by their relatives because of the violent tremors which could not be controlled to bring food to their mouths . the prince of santo buono ( viceroy 17161720 ) urged the abolition of the mita . although initially rejected by the crown and the council of the indies it was finally approved on humanitarian grounds . by 1740 skilled miners were now able to stand and swing sledge hammers in the mining shafts which , by now , had acquired improved ventilation . ulloa , governor of huancavelica , wrote in 1760 that miners were now free of mercury intoxication because the ore contained so little quicksilver but poisoning still occurred at the ovens when workers failed to let them cool completely before reloading them . ulloa reported that azogados descended to warmer lowlands , drank huge amounts of chicha , a beer made from maize , worked very hard in the fields , and sweated out the mercury . the documented improvement was , likely , due to the diuretic effect of the beer which expelled the mercury concentrated in the kidneys . after some weeks of heavy drinking and hard physical labor the shakes , excessive salivation and mouth ulcers disappeared and some miners returned to work in the huancavelica mine . although the mine was especially deadly because of its mercury , most spaniards at that time thought that the indians were destined to die anyway if this was necessary to obtain the metal . camargo wrote to the king in 1595 : they are barbarous people and without knowledge of god ; in their lands they are only occupied in idolatries and drunkenness and other vices of great filthiness these people going to work the mines first seems to me a service to god and to your holy catholic royal majesty and for the good of the natural indians themselves , because the mines they teach them doctrine and make them hear mass and they deal with spaniards by which they become ladinos [ hispanicized] . losing their barbarity , and they have no place for the idolatries of their lands by being outside of those sites where they do them and being occupied , which is the principal thing . they are barbarous people and without knowledge of god ; in their lands they are only occupied in idolatries and drunkenness and other vices of great filthiness these people going to work the mines first seems to me a service to god and to your holy catholic royal majesty and for the good of the natural indians themselves , because the mines they teach them doctrine and make them hear mass and they deal with spaniards by which they become ladinos [ hispanicized] . losing their barbarity , and they have no place for the idolatries of their lands by being outside of those sites where they do them and being occupied , which is the principal thing . camargo affirmed his great desire to [ also ] die in the service of your holy catholic royal majesty . some improvements in health occurred with new mining methods but these failed to take into account the huge pollution of the biosphere of huancavelica which persists to the present . the refining ovens were along the river where the escaping vapors settled on the ground , in the water , and blew across town depositing mercury on everything . these vapors were converted into methyl mercury by bacterial activity which then easily entered the food chain and which is especially injurious to the nervous system . the best - described mass poisoning occurred in minamata bay , japan , in the 1950s . but intoxications restricted to single families due to ingestion of mercury with longterm consequences have also been reported . mercury poisoning in infants ( pink disease , also known as acrodynia ) associated with the use of teething powders containing mercury , especially in england and australia , has been eliminated by the removal of the metal from these remedies . pain , paresthesias , and hypotonia allowed the assumption of characteristic postures by the affected infants . these symptoms reflect the especially injurious effects of mercury on the autonomic nervous system ( ans ) . the syndrome has not been found in recent epidemics of mercury poisoning in japan , but it reemerged in an epidemic of methyl mercury ( used as a fungicide ) poisoning employed for the sterilization of diapers in argentinean babies ( for review see ) . mercury compounds occur in the environment and their levels are rising due to anthropogenic activities , especially the burning of fossil fuels . coal contains large amounts of mercury , which is released into the atmosphere upon burning and it is then transported , possibly , around the globe . even low concentrations of mercury can cause subclinical effects , and slightly increased levels of mercury in hair are associated with decreased academic performance . the world health organization ( who ) places the level at which toxicity occurs at 50 ppm of mercury in hair and advises that healthy people should have no more than 5 ppm of mercury in their hair . however , normal mercury levels in hair are difficult to come by because they are dependent on the amount and type of fish eaten , on location ( especially where the environmental mercury burden may be high ) on the sampling methods , and on analytical methods . thus , in populations that have been exposed to high burdens of mercury for centuries , like those in huancavelica , the effects of such exposure are difficult to assess . hair is renewed throughout life ; it has a well - defined variability in growth influenced by diet , age , blood flow to the skin , and hormonal and metabolic signals which are , ultimately , regulated by the ans . but hair also reflects biologic rhythms because of clock - like signals that affect its growth through the intermediary of the ans . recurrent circa - annual periods of slow and fast rhythms in the hydrogen isotope ratios of human and animal hair can be recognized . using power spectral analysis of the ratios along the length of the hairs can reveal the effects of life at altitude and toxin accumulation on biologic rhythms . here we report a study of hydrogen isotope ratios in the hair and of heart rate variability in the same residents of huancavelica , peru , to assess their biologic rhythms and ans function . we carried out this study to determine whether centuries of ancestral and present exposure to increased mercury in the biosphere could affect ans function and give evidence for continuing deleterious effects on the health in modern huancavelica residents . the demographics of the 5 subjects and mercury and copper levels in their hair are given in table 1 . because the length of the hair reflects the duration of the stored information on biologic rhythms , our study was confined to women who generally have longer hair . we confirmed these very low levels in the hair from two huancavelica subjects using the x26a hard x - ray microprobe at the brookhaven synchrotron . no detectable mercury in single hairs from each subject was reported by the x - ray microprobe . to strengthen our conclusions about the surprisingly low mercury levels in the hair in huancavelica residents we used a control from the usa . in table 1 cold vapor atomic absorption spectrometry was used to measure total mercury ( organic + inorganic ) and inductively coupled plasma - atomic emission spectrometry for the other element ( one sample was misplaced by the analytical company performing the assays no values for hg are available ; here labeled lost ) . to assess the impact of individual metals on the hydrogen isotope ratios of low frequency / high frequency ( lf / hf ) power bands , a measure of ans control of biologic rhythms these power bands were affected by both copper and mercury levels in the hair clearly separating the lf and hf power according to mercury and copper levels . the annual growth rate reported for normal human hair is ~16 cm / year . in our controls ( n = 4 ) as determined from the sinusoidal variation in hydrogen isotope ratios in our samples from the usa and europe , it was 14.2 3.7 sd cm / year . the growth rate of hair from huancavelica ( n = 5 ) was 6.4 1.7 sd cm / year ( p < 0.001 ) ( figure 2 ) . the standard deviation of the variance of the power spectra of the hydrogen isotope ratios between controls from other parts of the world and that from huancavelica is shown in figure 3 . remarkably , this variance is significantly less in huancavelica residents ( p = 0.01 ) . pooling of hydrogen isotope power spectra from huancavelica and control hair is shown in figure 4 . the only statistically significant difference between these spectra is in the low - power frequencies ( p < 0.05 ) , though the power of high frequencies appears also to be less ( ns ) . the annual growth rates of hair can be shown to occur in fast and slow periods of weekly cycles ( figure 5 ) . the shorter fast period , and very prolonged slow period which correspond to the rest phase of hair growth , are consistent with the reduced annual hair growth in huancavelica residents . we compared the power spectra derived from heart rate variability with those obtained from the hydrogen isotope ratios in the hair , in the same subjects ( figure 6 ) . the low- and high - power frequency bands are of comparable length but , not surprisingly , the spectral power is different because of the different time scales heart rate variability in milliseconds , hydrogen isotope ratios in days . because of the complexity of the interactions between mercury and biologic rhythms as derived from hydrogen isotope ratios along the length of the hairs we used fractal analysis to illustrate the differences between those living in a mercury - polluted environment and a control ( figure 7 ) . these results are consistent with severe dysfunction of the ans evidenced also by the reduced annual hair growth in huancavelica residents . the most famous mercury poisoned character was the mad hatter , the one from the mad tea party in alice in wonderland . he was partner to the march hare , mad as only a march hare can be ( though the hare was not poisoned by mercury ) . however , the phrases mad as a hatter and mad as a march hare were in common use in 1837 , well before the publication of alice in wonderland in 1865 . hatters did go mad because felt used for making hats was cured by mercurious nitrate and the mercury vapors inhaled by hatters gave them the characteristic symptoms reported also by azogados ( quicksilver poisoned miners from huancavelica ) centuries earlier who noted the shakes , hallucinations , and psychosis as found in hatters centuries later . mercury was used in europe throughout medieval times for medicinal purposes or as mercury sulphide , an ingredient of the bright red ink used by scribes in monasteries . increased levels were found in medieval bones of those who had either employed it for the treatment of leprosy or syphilis , or with increased occupational exposure such as pharmacists . mercury is a neurotoxin but its damaging effects on humans are unpredictable and dependent , in part , on its chemical speciation and interactions with other metals in the environment and in the diet . organic mercury which is the primary source of mercury exposure is principally derived from the diet . huancavelica is listed amongst the ten most polluted places in the world and despite its listing , our measurement of mercury levels in the hair of current huancavelica residents was noticeably low ( table 1 ) and , clinically , huancavelica residents showed no signs of mercury poisoning as recorded in hatters and azogados centuries earlier . however , measurements of mercury levels in the hair are not good indicators of mercury 's toxicity . while levels of mercury can be accurately measured in the blood , the hair has no blood supply and the levels derived from hair represent metabolism over time and reflect the chronobiology of the individuals rather than the blood levels of this metal . in modern times , hair is also frequently washed with shampoos and dried with hair driers ; these manipulations can add or remove mercury and thus compound the difficulties of assaying the mercury levels in the hair . here we show that people living in a heavily mercury - polluted environment , in huancavelica , may have acceptable mercury levels , by international standards , in their hair and no overt clinical symptoms of intoxication . nevertheless , the effects of mercury exposure are evident from the reduced rate of hair growth and disturbed biologic rhythms in these people . taken together these findings give unequivocal evidence of the toxicity of mercury to the human autonomic nervous system the most vulnerable part of the nervous system to mercury 's damage , in modern huancavelica , peru . the high - frequency / low - frequency ( hf / lf ) ratios are measures of the control by the autonomic nervous system ( ans ) of biologic rhythms . we find that hg and cu both affected the power bands of the hf and lf . mercury does interact with other metals to produce its effect on biologic rhythms in huancavelica and such interactions might account for the unpredictable effects of hg on individual physiology in other parts of the world too ( figure 1 ) . the growth rate of human scalp hair is variable but on average ~16 cm / year . we suggest that the altered biologic rhythms in modern huancavelica residents may have contributed to this failure to renew the hair at an appropriate rate ( figure 2 ) . the standard deviation of the variance of the power spectra of the hydrogen isotope ratios along the length of the hairs between our controls from other parts of the world and that from huancavelica is shown in figure 3 . normal rhythmic oscillations in biology are marked by high variability ( large variances ) ; less variability degrades performance of the system . the reduction in variance we observed is yet another expression of the deranged biologic rhythms in our huancavelica subjects . the low - frequency peak of the power spectra reflects primarily the sympathetic nervous system 's control of autonomic variability . a decrease or absence of this peak in heart rate variability and in the power spectra derived from hydrogen isotope ratios in the hair in the same individuals is seen in ans diseases such as pure autonomic failure ( paf ) , manifested by widespread failure of sympathetic nervous system function . our identification of significant reduction in low - frequency power in the hydrogen isotope ratios in huancavelica subjects ( figure 4 ) further supports ans impairment in these individuals . long - term rhythms recurring at weekly , monthly , or yearly intervals are well known in animal and human physiology . the recurring annual fast periods reflect growth while the slow periods reflect the rest stages in hair growth . the shortened fast period rhythms in huancavelica residents ' hair ( figure 5 ) are in keeping with the reduced hair growth rate and further support the altered ans function ( and biologic rhythms ) in these people . to confirm our findings from hydrogen isotope derived biologic rhythms , we measured heart rate variability using 2 minutes of recording of cardiac action ( ~150 heart beats ) in the same subjects ( figure 6 ) . averaged power spectra showed comparable durations of low- and high - frequency bands and spectral power did not differ significantly . we have previously shown that spectra obtained from human heart rate variability were comparable to those derived from power spectra obtained from hydrogen isotope ratios in the hair and from growth lines from teeth . our results confirmed that biologic rhythms as gleaned from power spectral analysis reflected the pulsing of life , the varying rhtymicity of physiologic functions even when the ans has been affected by disease or mercury as in huancavelica residents . . they can show complex natural phenomena in graphic form such as ice crystals on a window pane and the intricate contours of mountains and ridges , and river branching . in biology they illustrate the 3/4 scaling of metabolism with body size , the complexity of heart rate control , of blood circulation , growth , development , and lifespan . here we used mandelbrot fractal sets to show the complex relationships between hg , its effects on the ans 's control of human biologic rhythms , and the variability of hydrogen isotope ratios along the length of the hair in our subjects ( figure 7 ) . the fractals derived from all huancavelica subjects clearly reflect derangement in biologic rhtymicity and the lack of variance which characterizes their exposure to hg , supporting our contention of continuing influence of this metal on human life in modern huancavelica . taken together our results show that survival in a heavily , mercury , polluted environment is possible without overt signs of mercury intoxications such as those found during mercury epidemics of the past or in miners during the spanish exploitation of peru 's mineral riches . nevertheless , careful analyses of the function of the most mercury vulnerable part of the nervous system gives evidence of continued effects in contemporaneous huancavelica residents . how can we reconcile the clear evidence of hg 's effects on biologic rhythms and ans function with the unexpected low levels of the metal in the hair of these same individuals and their apparent lack of overt signs of intoxication ? first , there is the fact that hg in hair is a poor indicator of its possible toxic effects . secondly , we propose a parallel between how human height and life expectancy increase in a short span of ~300 years after the industrial revolution . this well - documented phenomenon has been explained by several contributing factors such as technological advances and physiological adaptation . the history of mining in the high andes is replete with anecdotes of how technical advances in the extraction of ores improved miners ' survival and at the same time , in huancavelica , increased the yield of hg extraction . while the cause of the shortened lifespan in huancavelica miners remained a mystery for long , nevertheless , the miners themselves devised useful adaptations which allowed them to return to work after total incapacitation , extending their lifespan . just like in the early years of the industrial revolution in europe , improved health , better nutrition through obtaining the means of continuing to work , increased productivity of the mines and of the miners themselves , implied greater resilience , increase in longevity , and improved fertility in the high andes . human genetic potentials that could produce drastic changes in the relatively short span of 1215 generations in europeans during the industrial revolution may also have been operative in the mining towns of the high andes for more than 500 years . the andean miners had the opportunity to adapt to enormous environmental pollution and now their descendants tolerate , relatively , but not entirely , unscathed , the persistent hg in the huancavelica biosphere . nevertheless , our multilevel analyses clearly showed that even 21 generations is insufficient for complete adaptation to the horrendous mercury pollution . the microbiome , the symbiotic bacteria in the gut of the flies , profoundly affected their physiology . the molecular mechanisms that underlie the interactions of gut bacteria with their host are beginning to be unraveled in the flies . in humans since one route of entry of mercury in humans is through the gut , the microbiome might be the first to encounter the toxin and the bacteria adapt to increasing levels of ingested mercury . the survival of a functioning microbiome in the human gut like in the flies is , in turn , closely linked to the survival of the host . adaptation over a period of some 500 years to enormous environmental mercury loads in huancavelica may have ultimately been the results of bacterial ( microbiome ) adaptation to mercury . for this proposal , the nutritional and smoking habits of paternal grandparents could influence the health of their grandchildren and in australia the mercury sensitivity of paternal grandparents who had pink disease as infants , affected their grandchildren . a third possible explanation is found in the theory of tolerance to disease or to environmental stressors such as toxins . this theory has the most support from infectious diseases , where tolerance to bacterial invaders has been shown to offer evolutionary advantages by avoiding the costly means to eliminate the infectious agents . in this setting , tolerance to invaders offers a defense strategy that favors survival in heavily contaminated surroundings . similarly , cellular responses to stress can offer survival advantages in stressful environments such as the hypoxia of altitude or excessive heat by inducing reactive oxygen species ( ros ) or activating numerous heat - shock proteins , respectively . resistance and tolerance are thought to be complimentary strategies for living also in heavily polluted environments such as in huancavelica . analyzing the molecular pathways which allow relatively normal survival in a heavily mercury , polluted environment after five hundred years of mercury exposure may offer insights into strategies of coping with the climate and environmental changes wrought by the anthropocene , the man - made world , we now live in . the ethics committee of the new mexico health enhancement and marathon clinics ( nmhemc ) research foundation reviewed and approved the study . informed written consent was obtained from all participants . this was a field study , and as such suffers from limitations common to such studies such as small number of subjects , poor adherence to study protocols , and low number of controls . nevertheless , wherever statistical analyses were possible these were performed and gave clear and meaningful results . microfocused synchrotron x - ray fluorescence analyses were performed at beamline x26a at the national synchrotron light source , brookhaven national laboratory , upton , ny , usa . beamline x26a utilizes a bending magnet source on the nsls 2.8 gev electron storage ring , which operates at a current of 300 ma . for these experiments a monochromatic x - ray beam was used , monochromatized using a si(111 ) channel - cut monochromator and tuned to an incident beam energy of 12.5 kev . the beam was focused to a spot size of 5 m in the vertical x 8 m in the horizontal using a pair of dynamically bent , grazing - incidence mirrors , each 100 mm long , arranged in a kirkpatrick - baez ( kb ) geometry . x - ray fluorescence from the human hairs was measured using a combined set of three energy dispersive detectors ; a canberra 9-element hpge array detector placed 90 to the incident beam within the plane of the storage ring to minimize backgrounds from compton scattering . the other two detectors are single element radiant vortex - ex silicon drift diode detectors also at 90 to the incident beam , but each sitting 45 above and below the plane of the storage ring , respectively . the 9-element hpge array provides an active area of 900 mm and each silicon drift diode detector has an active area of 50 mm . all eleven detector elements are integrated simultaneously using the xmap series of compact pci - based digital spectrometers produced by x - ray instrumentation associates ( xia ) interfaced through epics and controlled through in - house client software written in idl . incident beam intensity was monitored using an ion - chamber upstream of the focusing optics and all images were corrected for changes in incident beam flux through normalization to the change in ion - chamber counts over time . hydrogen isotope ratios were determined using the continuous - flow high - temperature - reduction technique . hair was wrapped in silver foil and placed into the combustion chamber of a mass spectrometer using a carlo erba as 200-ls autosampler . the stable isotopic compositions of low - mass elements such as hydrogen are reported as delta ( ) values in parts per thousand ( ) . values are calculated as follows : in ( ) = ( r sample / r standard 1 ) 1000 , where r is the ratio of the heavy to the light isotope in the sample . the stable isotope standard for hydrogen is reported relative to the standard mean ocean water ( smow ) . isotope composition is reported in relation to this standard which has been defined as 0 . approximately 150 rr intervals were measured for each subject . the statistical analysis was similar to that used for the hydrogen isotope power spectra analysis ( see below ) and the frequency bands were identical to those chosen for the hydrogen isotope bands of the power spectra derived from the hair of the same subject . data derived from each hair were entered into the mandelbrot set - online generator by dawid makiela ( http://www.mandelbrot.ovh.org/ ) . x 1 : mercury content ; y1 : low - frequency / high - frequency ratio of power spectrum ; x2 : elemental content ( cu ) ; y2 : total power of low , mid- and high frequencies ; x : total low power ; y : total high power ; r : sum of low , mid , and high frequencies of the power spectrum obtained from the hydrogen isotope ratios along the length of each hair . maximum number of iterations was 100 . to strengthen our conclusions about biologic rhythms derived from the hair in huancavelica residents we used an aged control from the usa . the variance of biologic rhythms decreases with advancing age thus an old control subject , us resident , should serve better as a standard for comparison to the biologic rhythms derived from the hair of young mercury exposed subjects from huancavelica . in order to determine the relationship between metals in hair and the hydrogen isotope spectra in hair , we computed pearson and spearman correlations of each metal with measures of the spectra . the log - transformed quantities of hg and cu were found to be related to the spectral power in the high - frequency band we identify two clusters of high - frequency power ( low and high power ) in the hg - cu scatter plot of the 5 huancavelica subjects ( see figure 1 ) . the fact that periodicity of this sinusoid is 52 weeks allows us to compute annual growth rates in centimeters . fitting the annual sinusoid for huancavelica subject # 3 by nonlinear regression yielded the function of length along the hair in cm : ( 1)predicted dd=91.1315 + 1.5832sin ( .8850cm+1.3243 ) . one can use the frequency of the fitted sinusoid to estimate the growth rate that matches the 0.0193 cycles / week = 1 cycle/52 weeks , whose periodicity is 52 weeks . so ( freq/2)*(growth / year)/(52 weeks / year ) set equal to 1 cycle/52 weeks implies the following : ( 2)growthyear=2(frequency of fitted annual sinusoid ) . here the average growth rate for the 5 huancavelica subjects was 6.4 1.7 sd cm / year . this is compared to the average growth rate of ~16 cm / year in human hair by a one - sample t - test ( see figure 2 ) . all power spectra were computed using the finite fourier transform , decomposes time series into a sum of sine and cosine waves of different amplitudes and wavelengths ( proc spectra from sas ) . the spectral variance for a given time series of hydrogen isotope ratios in hair is the total power ( area under the periodogram ) . the comparison of these standard deviations ( square root of the variance ) for the 5 huancavelica subjects to our sample of controls are made by a two - sample t - test ( see figure 3 ) . the spectral power in the low - frequency band and their standard errors are computed as described in priestly . these are then compared by t - test ( see figure 4 ) . fast and slow periods of hair growth in weekly cycles is computed from the frequencies of higher power observed in the periodogram . the form of the calculation is as follows : ( 3)period = dt(freq)(growth ) , where dt is the increment of the series ( see figure 5 ) . for figure 6 , the power spectra for both heart rate interval ( rr ) and hydrogen isotope ratio time series measured in the same 5 huancavelica subjects ( the 5 series

heparin - binding protein ( hbp ) , also known as azurocidin , has multiple functions in the inflammatory process , especially during severe infections . beside its antimicrobial properties , hbp may induce vascular leakage leading to extravascular efflux , which is an important pathophysiologic event in the development of septic shock . not surprisingly , high hbp plasma levels are found in severe sepsis patients and in septic shock patients as well as in serious infections associated with endothelial damage . in the present issue of critical care , linder and colleagues demonstrate new aspects of hbp daily monitoring in icu patients . the authors observed that high hbp plasma levels are associated with an increased mortality rate in both septic and nonseptic critically ill patients , indicating that hbp may be a reliable prognostic biomarker . however , there are some limitations hindering rapid translation of these interesting findings into the daily routine . first , the group of nonseptic critically ill patients ( n = 28 ) enrolled in the study was rather small as compared with the septic group ( n = 151 ) . moreover , 50% of nonseptic patients developed infection while hospitalized in the icu , and to classify them as truly nonseptic patients is problematic . second , there is a lack of a routine diagnostic method for hbp analysis . nevertheless , if the results of the present study are validated in large clinical trials in different icu populations and cost - effectiveness data become available , the serial hbp measurements will have a promising future .

in the present issue of critical care , linder and colleagues present a new study in which they assess the clinical importance of serial measurements of heparin - binding protein ( hbp ) plasma levels in critically ill septic and nonseptic patients . they found that hbp plasma levels are significantly higher in patients with severe sepsis and septic shock in comparison with patients with a nonseptic critical condition . the authors also demonstrated that hbp plasma levels obtained at admission to the icu and during the last individual sampling are higher in nonsurvivors as compared with survivors in both the septic group and the whole study group . moreover , the high baseline hbp plasma levels in septic patients were associated with an increased 28-day mortality rate . altogether , these results indicate that serial hbp measurements might be very helpful in stratification of icu patients . however , there are some issues that should be raised before the study results can be translated into the daily routine . the study was designed to compare hbp plasma levels in patients with severe sepsis and septic shock with levels in patients with noninfectious critical illness . however , these two groups of patients were not equal in size . also , a significant proportion of patients from the nonseptic group developed infection and was treated with antibiotics . this raises the question of whether the comparison between septic and nonseptic icu patients is hindered by this treatment . nevertheless , the study design is logical because finding a biomarker that would predict development of any shock state is highly desirable . previous studies have demonstrated significant predictive values of elevated levels of lactate , cortisol and il-6 in the blood of patients with different etiologies of shock [ 2 - 4 ] . however , these biomarkers have some limitations : lactate levels are less influenced by arterial sampling , endogenous cortisol levels are downregulated by corticosteroids used in the treatment of septic shock or by relative adrenal insufficiency , and il-6 analysis is not generally available in regular hospital laboratories . other routinely measured biomarkers - such as procalcitonin , c - reactive protein , neutrophil and lymphocyte counts - have only a limited value in prognostic scoring of the critically ill patients and are mostly used in the early diagnostics of bacterial etiology of critical illness [ 6 - 8 ] . notably , hbp has antibacterial activity , which includes a direct microbicidal effect , and also helps neutrophils to migrate into the focus of infection . similarly to hbp , c - reactive protein and il-6 play an active role during the immune responses against infections : c - reactive protein is an inflammation opsonin , and the major function of il-6 is amplification as well as downregulation of inflammatory reactions , depending on the concentrations . regarding procalcitonin , there are only limited data from animal studies - which demonstrate that immunoneutralization of procalcitonin improved survival in experimental porcine sepsis . additionally , elevated cortisol levels in peripheral blood during sepsis are considered an integral part of compensatory anti - inflammatory response syndrome , leading to downregulation of exaggerated systemic immune responses . from the functional point of view , in comparison with the abovementioned biomarkers , hbp therefore plays the most complex role in severe sepsis and septic shock - highlighting its potential for clinical use . in conclusion , the serial measurements of hbp plasma levels can be a useful tool for close monitoring of critically ill septic patients . however , availability of a routine diagnostic method for hbp analysis is essential to confirm these interesting data . the authors thank dr e david mcintosh ( imperial college , london , uk ) for help with the manuscript .

introduction : spontaneous reporting of adverse drug reactions ( adrs ) is the basis of pharmacovigilance . in fact , adrs are associated with a high degree of morbidity and mortality . however , underreporting by all healthcare professionals remains the major problem in italy and in the rest of the world . the dissemination of pharmacovigilance knowledge among italian healthcare professionals , and the new pharmacovigilance regulations may promote the early detection and reporting of adrs . this review examines the legislative framework concerning the pharmacovigilance in italy.materials and methods : the information was collected from scientific articles and the websites of the italian ministry of health and the italian medicines agency ( agenzia italiana del farmaco , aifa).results : the pharmacovigilance system , both in italy and europe , has undergone profound changes . european legislation on pharmacovigilance has been changed in 2010 according to the eu regulation 1235/2010 and directive 2010/84/eu . basically , the changes tend to increase the efficiency , speed and transparency of pharmacovigilance activities . the new regulation ( 1235/2010 ) and the directive ( 2010/84/eu ) aim to strengthen the system of pharmacovigilance , establish more precisely who is obliged to do what , and allow faster and easier circulation and retrieval of information about adrs.conclusion:a greater knowledge on what is the italian pharmacovigilance legislation will be useful to improve the status of adrs reporting and spread the culture of spontaneous reporting .

the information gathered during pre - marketing drug development is inevitably incomplete with regard to possible adverse reactions . this is mainly due to : animal testing is insufficient to predict human safety;patients enrolled in clinical trials are selected and limited in number;the conditions of use are different from those in clinical practice;the period of testing is limited . animal testing is insufficient to predict human safety ; patients enrolled in clinical trials are selected and limited in number ; the conditions of use are different from those in clinical practice ; the period of testing is limited . clinical trials often lack important information about rare but serious adverse reactions , chronic toxicity , use in special groups ( such as children , women , elderly or pregnant ) or interactions with other drugs . therefore , post - marketing surveillance activities are important to allow the early detection of unexpected and/or serious adverse reactions . it is estimated that only 6 - 10% of all adrs are reported : this underestimation is a major problem . although the extent of under - reporting is widely variable depending on the estimates , it is certain that the number of reported adrs represents only a small percentage of the total number of occurring adrs . adrs have a great impact on public health and represent a significant economic burden on both healthcare systems and society . in fact , it is estimated that in the united states , the total cost of adrs may be comparable to that of diabetes , as well as it represents the fourth leading cause of death by disease . in the european union ( eu ) , it is estimated that 5% of all hospital admissions are due to adrs , which are the fifth leading cause of hospital death : approximately 197,000 deaths per year in the eu are caused by adrs , and the total cost to the society of adrs in the eu is about 79 billion . based on these alarming data , on july 21 , 2012 , a new eu pharmacovigilance legislation came into force in order to strengthen and rationalize the eu pharmacovigilance system , with the overall objectives of better protection of public health , ensuring proper functioning of the internal market and simplifying the existing procedures . pharmacovigilance was officially born in december 1961 with the publication of a letter ( case report ) in the lancet by w. mcbride , the australian doctor who first suspected a causal link between serious fetal deformities ( phocomelia ) and thalidomide drug used during pregnancy : thalidomide was used as an antiemetic and sedative agent in pregnant women . in 1968 , programme for international drug monitoring , a pilot project aimed to the centralization of world data on adrs . in particular , the main aim of the who programme is to identify the earliest possible pharmacovigilance signals . as of april 2013 , 112 countries have joined the who programme for international drug monitoring , and in addition 32 countries participating in the who programme for international drug monitoring , with year of joining * all member countries contribute sending at least quarterly individual case safety reports ( icsrs ) to the who global icsr database system , named vigibase . it is updated with incoming icsrs on a continuous basis . the who worldwide database of adrs is located in uppsala in sweden , and it is developed and maintained by the uppsala monitoring center for pharmacovigilance ( umc ) on behalf of the who.by april 2013 over 8 million reports were contained in the database . the term pharmacovigilance was proposed in the mid-70s by a french group of pharmacologists and toxicologists to define the activities promoting the assessment of the risk of side effects potentially associated with drug treatment . pharmacovigilance is defined by the who as the science and activities relating to the detection , assessment , understanding and prevention of adverse effects , or any other problem in the field of medicine . the monitoring of spontaneous suspected adrs reports represents the key component of the integrated systems of pharmacovigilance . pharmacovigilance has four main objectives : recognize , as quickly as possible , new adrs;improve and increase the information about already known or suspected adrs;assess the benefits of a drug compared to other ones or other therapies;communicate the information in order to improve the therapeutic practice . recognize , as quickly as possible , new adrs ; improve and increase the information about already known or suspected adrs ; assess the benefits of a drug compared to other ones or other therapies ; communicate the information in order to improve the therapeutic practice . the main aim of pharmacovigilance is to provide an alarm signal through the early detection of new adrs . in pharmacovigilance a signal of suspected causality is defined as follows : information that arises from one or multiple sources , including observations and experiments , which suggests a new potentially causal association , or a new aspect of a known association , between an intervention and an event or set of related events , either adverse or beneficial , which would command regulatory , societal or clinical attention , and is judged to be of sufficient likelihood to justify verifiable and , when necessary , remedial actions . ad hoc studies will be performed to test this hypothesis and possibly provide additional information , not achievable through spontaneous reporting systems , such as the incidence and relative risk for evaluated adrs . . the main limitations of spontaneous reporting are related to under - reporting , variable quality of the reported data and lack of information on drug exposure . there are differences between countries ( and also between regions within countries ) in the occurrence of adrs and other drug - related problems . this may be due to many factors such as : disease and prescribing practices;genetics , diet , traditions of the communities;processes of drugs production influencing the quality of the pharmaceutical composition;drugs distribution and use including indications , doses and availability;the use of traditional medicines and supplements ( for example , herbal remedies ) , which may constitute specific toxicological problems . the data resulting from the whole country or single region may have greater relevance and educational value and may affect the national regulations . disease and prescribing practices ; genetics , diet , traditions of the communities ; processes of drugs production influencing the quality of the pharmaceutical composition ; drugs distribution and use including indications , doses and availability ; the use of traditional medicines and supplements ( for example , herbal remedies ) , which may constitute specific toxicological problems . the data resulting from the whole country or single region may have greater relevance and educational value and may affect the national regulations . according to the circumstances , the information obtained in a country ( for example , the country of origin of the drug ) may not be relevant in other ones . at european level , government agencies responsible for pharmacovigilance in member states are in contact with each other and with the european medicines agency ( ema ) . ema has established a web - based european network ( eudravigilance ) for reporting andexchangingsuspected adrs reports , during the development ( pre - authorization phase ) and following the marketing a - authorization ( post - authorization phase ) of medicinal products in the european economic area . eudravigilance is an european database , which supports : the early detection of possible safety signals associated with medicinal products for human use.the continual monitoring and evaluation of potential safety issues in relation to reported adverse reactions.the decision making process , based on a broader knowledge of the adverse reaction profile of medicinal products . the early detection of possible safety signals associated with medicinal products for human use . the continual monitoring and evaluation of potential safety issues in relation to reported adverse reactions . the decision making process , based on a broader knowledge of the adverse reaction profile of medicinal products . in the early 80 's , the italian rules on the safety of marketed drugs identified the manufacturers as responsible for the communication to the ministry of health about possible drug - related adverse effects . in fact , in 1980 , in italy , the marketing - authorization holders were required to transmit to the ministry of health a periodical report in which they must specify the nature and frequency of any toxic effects and secondary consequences or correlated with drugs use . according to the ministerial decree ( dm ) of june 23 , 1981 , ( article 8) and the dm of july 28 , 1984 , the data collection forms on drugs use doctors could also decide to send the form directly to the ministry of health . in 1987 , two major changes occurred : the term pharmacovigilance appeared and the local health units were involved . since then , the doctors were required to notify the local health units , which , in turn , communicated to the ministry information about the most serious cases and deaths . in 1997 , the national system of pharmacovigilance was established . by the legislative decree of april 8 , 2003 , n. 95 was clarified that the detectors can be doctors and healthcare professionals , who may report suspected serious or unexpected adverse reactions to vaccines and drugs undergoing intensive monitoring . in december 2003 , a new pharmacovigilance form was approved and is still currently in use [ form 1 ] . with the legislative decree no . 129 - 134 regions began to provide , within its competences , the dissemination of information to healthcare personnel and the training of operators in the field of pharmacovigilance . the healthcaresettings must nominate a qualified person responsible for pharmacovigilance ( qppv ) for adrs reports management . physicians and other healthcare professionals are required to report all suspected serious and/or unexpected adrs , which they learn as part of their activity . they are in duty bound to report all suspected adverse reactions , serious , not serious , expected and unexpected from all vaccines and medicines under intensive monitoring . all healthcare professionals are required to send reports of suspected adverse reactions by means of the appropriate form , promptly , to qppv . upon verifying completeness and consistency of the reporting form data , the rafv must store the report , no later than seven days from the date of receipt , in the database of the national pharmacovigilance network . the national pharmacovigilance system is headed by the italian medicines agency ( agenzia italiana del farmaco , aifa ) , acting in accordance with the rules laid down at eu level by the ema . it is a public body that operates autonomously , transparently and according to cost - effectiveness criteria , under the direction of the ministry of health and under the supervision of the ministry of health and the ministry of economy . aifa collaborates with the regions , the national institute of health , research institutes , patients associations , healthcare professionals , scientific societies , the pharmaceutical industry and the distributors . specifically , the agency : guarantees access to the drugs and their safe and appropriate use in order to protect public health;provides an unified national pharmaceutical system in agreement with the regions;provides pharmaceutical expenditure governance in the context of economic and financial compatibility and competitiveness of the pharmaceutical industry;ensures innovation , efficiency and simplification of the marketing authorization procedures , in order to allow rapid access to innovative drugs and drugs used for rare diseases;strengthens the relationships with the agencies of other member countries , with the ema and other international organizations;encourages and rewards investments in research and development ( r and d ) in italy , promoting and rewarding innovation;interacts with the community of patients associations , the medical / scientific world , pharmaceutical companies and distributors;promotes pharmaceutical knowledge and culture . guarantees access to the drugs and their safe and appropriate use in order to protect public health ; provides an unified national pharmaceutical system in agreement with the regions ; provides pharmaceutical expenditure governance in the context of economic and financial compatibility and competitiveness of the pharmaceutical industry ; ensures innovation , efficiency and simplification of the marketing authorization procedures , in order to allow rapid access to innovative drugs and drugs used for rare diseases ; strengthens the relationships with the agencies of other member countries , with the ema and other international organizations ; encourages and rewards investments in research and development ( r and d ) in italy , promoting and rewarding innovation ; interacts with the community of patients associations , the medical / scientific world , pharmaceutical companies and distributors ; promotes pharmaceutical knowledge and culture . the mission of the aifa in pharmacovigilance is to ensure a positive risk / benefit ratio for all authorized drugs through the continuous monitoring of all safety information and adrs . these data arise from different sources : spontaneous reports of suspected adrs , clinical trials , scientific literature , reports submitted by the pharmaceutical companies , and other sources . in particular , all data of spontaneous adrs reporting are managed and coordinated through the national network of pharmacovigilance ( rete nazionale di farmacovigilanza , rnf ) , a database that allows the collection , management and analysis of spontaneous reports of suspected adrs . it is active since november 2001 . the rnfisan aifa - managed , extensive network throughout the national territory that includes the regional authorities and the autonomous provinces of trento and bolzano , the regional centers of pharmacovigilance , 204 local health authorities , 112 hospitals , 43 research institutes , more than eight hundreds pharmaceutical companies and obviously aifa . through this network , periodicallyt he rnf data are transmitted to eudra vigilance database ( european database of suspected adrs reports ) , managed by the ema . eudravigilance collects all data provided at national level by the eu countries in a single database , which , in turn , communicates with vigibase , the who global icsr database . in december 2010 , european legislation on pharmacovigilance was changed with the adoption of the eu regulation 1235/2010 and directive 2010/84/eu amending , as regards pharmacovigilance , regulation ( ec ) 726/2004 and directive 2001/83/ec , respectively . this new legislation was the biggest change to the regulation of human medicines in the eu since 1995 . ema is responsible for implementing much of the new legislation , which has been effective since july 2012 . in october 2012 , the eu 's new pharmacovigilance legislation was further amended by : regulation ( eu ) no 1027/2012 ( applies from 5 june 2013);directive 2012/26/eu ( applies from 28 october 2013 ) . regulation ( eu ) no 1027/2012 ( applies from 5 june 2013 ) ; directive 2012/26/eu ( applies from 28 october 2013 ) . new legislation will greatly improve public - health protection and , ultimately , save lives by enhancing the overall efficiency and effectiveness of the pharmacovigilance system . basically , changes tend to increase the efficiency , speed and transparency of pharmacovigilance tasks through rules that aim to : strengthening pharmacovigilance systems ( more clearly defined roles and responsibilitiesof the many actors involved);streamline the activities among the member states ( distribution of tasks and work sharing , with less duplication of effort;increasing engagement of the patients and healthcare professionals;improving the communication systems of decision - making processes , giving proper justification to the public;increasing transparency . strengthening pharmacovigilance systems ( more clearly defined roles and responsibilities of the many actors involved ) ; streamline the activities among the member states ( distribution of tasks and work sharing , with less duplication of effort ; increasing engagement of the patients and healthcare professionals ; improving the communication systems of decision - making processes , giving proper justification to the public ; increasing transparency . first of all , the definition of adverse reaction has been changed and broadened to ensure that it now covers noxious and unintended effects resulting not only from the authorized use of a medicine at normal doses , but also from medication errors and uses outside the terms of the marketing authorization ( off - label use ) , including overdose , misuse and abuse of the product , and from occupational exposure . as a consequence , therefore , there will be an increase of reports and resulting a greater monitoring activity . in all eu countries , the patients will be able to report directly suspected adverse reactions . in italy , this possibility has already been considered since some years back by a paper form , however , from now on , also in accordance with the new directive , reports may also be sent by the web . all reports of adverse reactions will merge into the european database eudravigilance , but with different timing depending on the severity of the reaction ( within 15 days for serious reports and within 90 days for non - serious ones ) . reports from pharmaceutical companies will also be transmitted to the eudravigilance database . regarding some medicinal products authorized through the centralized procedure , it is already possible to consult the european database of adverse reactions at the following website http://www.adrreports.eu/. in summary , directive 2010/84/eu and regulation ( eu ) 1235/2010 aim to maintain , reinforce and further develop the eudravigilance database as the single or at least the main- point of receipt , storage and exchange of information concerning the safety of medicinal products for human use authorized in the eu . the data collected in the eudravigilance database will be monitored by the ema in cooperation with the member states , while monitoring of the data originated at the national level will be carried out by the member state involved ; these activities are aimed at the identification of changes in risk or new risks through the signals analysis . the methodology for the identification and management process of the signal were defined in the implementing regulation ( eu ) n. 520/2012 of 19 june 2012 , concerning the performance of pharmacovigilance activities provided for in regulation ( ec ) n.726/2004 of the european parliament and of the council and directive 2001/83/ec of the european parliament and of the council . as afore mentioned , the new legislation aims to guarantee greater transparency and improve communication . in fact , on its web portal , aifa makes available to the public at least the following information : public assessment reports , together with a summary there of;summaries of product characteristics and package leaflets;summaries of risk management plans;list of medicinal products that are subject to additional monitoring;information about the various procedures for the reporting of suspected adrs to the competent authorities by the healthcare professionals and patients , including forms with web data entry mask . public assessment reports , together with a summary there of ; summaries of product characteristics and package leaflets ; summaries of risk management plans ; list of medicinal products that are subject to additional monitoring ; information about the various procedures for the reporting of suspected adrs to the competent authorities by the healthcare professionals and patients , including forms with web data entry mask . medicines subject to additional monitoring will be the products containing new active substances not included in the authorized medicines in the eu before the january 1 2011 ; in particular , biologics and biosimilars , but the list may also include products whose license is subject to particular conditions or products authorized in exceptional circumstances ; e.g. , products subject to safety studies after the granting of the marketing authorization ( post - authorisation safety studies ) . these medicinal products subject to additional monitoring will be identifiable from the package leaflet that will bear the statement this medicinal product is subject to additional monitoring . this statement will be preceded by a black symbol and followed by an explanatory sentence explaining the concept of additional monitoring . the additional monitoring list will be regularly kept up to date by the ema in collaboration with member states . the new legislation has empowered the relevant authorities to impose on the marketing authorization holders the obligation to perform post - authorization safety and/or efficacy studies . these studies would be required at the time of the granting of the marketing authorization or later . according to another key point of the 2010 reform , each member state and each marketing authorization holder is obliged to adopt a pharmacovigilance system with the aim of analyzing suspected adrs and implementing provisions in order to prevent them . at last , the new eu pharmacovigilance legislation has established within the ema a new scientific committee , the pharmacovigilance risk assessment committee ( prac ) in which all member states are represented . the role of the prac in protecting public health requires close interaction with the agency 's committee for medicinal products for human use ( chmp ) and the coordination group for mutual recognition and decentralized procedures - human ( cmdh ) . its members include experts from regulatory authorities in eu member states and independent scientific experts who provide additional expertise in particular scientific areas . members from the patient organizations and healthcare professionals will be included in the near future further to appointment from the european commission . in its role in monitoring the safety of human medicines , the prac assesses all aspects of risk management , including the detection , assessment , minimization and communication of the risk of adrs , giving due consideration to the benefits of the medicine . the prac is also involved in designing and evaluating post - authorization safety studies and in conducting pharmacovigilance audits . when the prac has concluded an evaluation related to the safety of medicines , it prepares a recommendation that is sent either to the chmp ( if it concerns at least one centrally authorized medicine ) or to the cmdh ( if it only concerns medicines that are not centrally authorized in the eu ) . the regional authorities are responsible for the development and promotion of active pharmacovigilance programs , which agreed with aifa and subsequently , allow the regions to access to funds . in this context , aifa has promoted several projects and studies in italy , with the aim of increasing knowledge on drugs and the culture of pharmacovigilance the funding of pharmacovigilance projects by aifa has contributed to the improvement of spontaneous reporting . in italy , the situation of spontaneous adrs reports in 2010 showed an increase of 39% in comparison to 2009 . this result is in line with the average annual increase of 30% observed in the last five years , and it also demonstrates the crucial role of active pharmacovigilance programs , funded by aifa , in increasing knowledge on drugs , to better define their safety profile and to improve their safe use in clinical practice . pharmacovigilance was officially born in december 1961 with the publication of a letter ( case report ) in the lancet by w. mcbride , the australian doctor who first suspected a causal link between serious fetal deformities ( phocomelia ) and thalidomide drug used during pregnancy : thalidomide was used as an antiemetic and sedative agent in pregnant women . in 1968 , the world health organization ( who ) promoted the programme for international drug monitoring , a pilot project aimed to the centralization of world data on adrs . in particular , the main aim of the who programme is to identify the earliest possible pharmacovigilance signals . as of april 2013 , 112 countries have joined the who programme for international drug monitoring , and in addition 32 countries participating in the who programme for international drug monitoring , with year of joining * all member countries contribute sending at least quarterly individual case safety reports ( icsrs ) to the who global icsr database system , named vigibase . it is updated with incoming icsrs on a continuous basis . the who worldwide database of adrs is located in uppsala in sweden , and it is developed and maintained by the uppsala monitoring center for pharmacovigilance ( umc ) on behalf of the who.by april 2013 over 8 million reports were contained in the database . the term pharmacovigilance was proposed in the mid-70s by a french group of pharmacologists and toxicologists to define the activities promoting the assessment of the risk of side effects potentially associated with drug treatment . pharmacovigilance is defined by the who as the science and activities relating to the detection , assessment , understanding and prevention of adverse effects , or any other problem in the field of medicine . the monitoring of spontaneous suspected adrs reports represents the key component of the integrated systems of pharmacovigilance . pharmacovigilance has four main objectives : recognize , as quickly as possible , new adrs;improve and increase the information about already known or suspected adrs;assess the benefits of a drug compared to other ones or other therapies;communicate the information in order to improve the therapeutic practice . recognize , as quickly as possible , new adrs ; improve and increase the information about already known or suspected adrs ; assess the benefits of a drug compared to other ones or other therapies ; communicate the information in order to improve the therapeutic practice . the main aim of pharmacovigilance is to provide an alarm signal through the early detection of new adrs . in pharmacovigilance a signal of suspected causality is defined as follows : information that arises from one or multiple sources , including observations and experiments , which suggests a new potentially causal association , or a new aspect of a known association , between an intervention and an event or set of related events , either adverse or beneficial , which would command regulatory , societal or clinical attention , and is judged to be of sufficient likelihood to justify verifiable and , when necessary , remedial actions . following signal , subsequent ad hoc studies will be performed to test this hypothesis and possibly provide additional information , not achievable through spontaneous reporting systems , such as the incidence and relative risk for evaluated adrs . . the main limitations of spontaneous reporting are related to under - reporting , variable quality of the reported data and lack of information on drug exposure . there are differences between countries ( and also between regions within countries ) in the occurrence of adrs and other drug - related problems . this may be due to many factors such as : disease and prescribing practices;genetics , diet , traditions of the communities;processes of drugs production influencing the quality of the pharmaceutical composition;drugs distribution and use including indications , doses and availability;the use of traditional medicines and supplements ( for example , herbal remedies ) , which may constitute specific toxicological problems . the data resulting from the whole country or single region may have greater relevance and educational value and may affect the national regulations . disease and prescribing practices ; genetics , diet , traditions of the communities ; processes of drugs production influencing the quality of the pharmaceutical composition ; drugs distribution and use including indications , doses and availability ; the use of traditional medicines and supplements ( for example , herbal remedies ) , which may constitute specific toxicological problems . the data resulting from the whole country or single region may have greater relevance and educational value and may affect the national regulations . according to the circumstances , the information obtained in a country ( for example , the country of origin of the drug ) may not be relevant in other ones . at european level , government agencies responsible for pharmacovigilance in member states are in contact with each other and with the european medicines agency ( ema ) . ema has established a web - based european network ( eudravigilance ) for reporting andexchangingsuspected adrs reports , during the development ( pre - authorization phase ) and following the marketing a - authorization ( post - authorization phase ) of medicinal products in the european economic area . eudravigilance is an european database , which supports : the early detection of possible safety signals associated with medicinal products for human use.the continual monitoring and evaluation of potential safety issues in relation to reported adverse reactions.the decision making process , based on a broader knowledge of the adverse reaction profile of medicinal products . the early detection of possible safety signals associated with medicinal products for human use . the continual monitoring and evaluation of potential safety issues in relation to reported adverse reactions . the decision making process , based on a broader knowledge of the adverse reaction profile of medicinal products . in the early 80 's , the italian rules on the safety of marketed drugs identified the manufacturers as responsible for the communication to the ministry of health about possible drug - related adverse effects . in fact , in 1980 , in italy , the marketing - authorization holders were required to transmit to the ministry of health a periodical report in which they must specify the nature and frequency of any toxic effects and secondary consequences or correlated with drugs use . according to the ministerial decree ( dm ) of june 23 , 1981 , ( article 8) and the dm of july 28 , 1984 , the data collection forms on drugs use doctors could also decide to send the form directly to the ministry of health . in 1987 , two major changes occurred : the term pharmacovigilance appeared and the local health units were involved . since then , the doctors were required to notify the local health units , which , in turn , communicated to the ministry information about the most serious cases and deaths . in 1997 , the national system of pharmacovigilance was established . by the legislative decree of april 8 , 2003 , n. 95 was clarified that the detectors can be doctors and healthcare professionals , who may report suspected serious or unexpected adverse reactions to vaccines and drugs undergoing intensive monitoring . in december 2003 , a new pharmacovigilance form was approved and is still currently in use [ form 1 ] . with the legislative decree no . 129 - 134 regions began to provide , within its competences , the dissemination of information to healthcare personnel and the training of operators in the field of pharmacovigilance . the healthcaresettings must nominate a qualified person responsible for pharmacovigilance ( qppv ) for adrs reports management . physicians and other healthcare professionals are required to report all suspected serious and/or unexpected adrs , which they learn as part of their activity . they are in duty bound to report all suspected adverse reactions , serious , not serious , expected and unexpected from all vaccines and medicines under intensive monitoring . all healthcare professionals are required to send reports of suspected adverse reactions by means of the appropriate form , promptly , to qppv . upon verifying completeness and consistency of the reporting form data , the rafv must store the report , no later than seven days from the date of receipt , in the database of the national pharmacovigilance network . the national pharmacovigilance system is headed by the italian medicines agency ( agenzia italiana del farmaco , aifa ) , acting in accordance with the rules laid down at eu level by the ema . it is a public body that operates autonomously , transparently and according to cost - effectiveness criteria , under the direction of the ministry of health and under the supervision of the ministry of health and the ministry of economy . aifa collaborates with the regions , the national institute of health , research institutes , patients associations , healthcare professionals , scientific societies , the pharmaceutical industry and the distributors . specifically , the agency : guarantees access to the drugs and their safe and appropriate use in order to protect public health;provides an unified national pharmaceutical system in agreement with the regions;provides pharmaceutical expenditure governance in the context of economic and financial compatibility and competitiveness of the pharmaceutical industry;ensures innovation , efficiency and simplification of the marketing authorization procedures , in order to allow rapid access to innovative drugs and drugs used for rare diseases;strengthens the relationships with the agencies of other member countries , with the ema and other international organizations;encourages and rewards investments in research and development ( r and d ) in italy , promoting and rewarding innovation;interacts with the community of patients associations , the medical / scientific world , pharmaceutical companies and distributors;promotes pharmaceutical knowledge and culture . guarantees access to the drugs and their safe and appropriate use in order to protect public health ; provides an unified national pharmaceutical system in agreement with the regions ; provides pharmaceutical expenditure governance in the context of economic and financial compatibility and competitiveness of the pharmaceutical industry ; ensures innovation , efficiency and simplification of the marketing authorization procedures , in order to allow rapid access to innovative drugs and drugs used for rare diseases ; strengthens the relationships with the agencies of other member countries , with the ema and other international organizations ; encourages and rewards investments in research and development ( r and d ) in italy , promoting and rewarding innovation ; interacts with the community of patients associations , the medical / scientific world , pharmaceutical companies and distributors ; promotes pharmaceutical knowledge and culture . the mission of the aifa in pharmacovigilance is to ensure a positive risk / benefit ratio for all authorized drugs through the continuous monitoring of all safety information and adrs . these data arise from different sources : spontaneous reports of suspected adrs , clinical trials , scientific literature , reports submitted by the pharmaceutical companies , and other sources . in particular , all data of spontaneous adrs reporting are managed and coordinated through the national network of pharmacovigilance ( rete nazionale di farmacovigilanza , rnf ) , a database that allows the collection , management and analysis of spontaneous reports of suspected adrs . it is active since november 2001 . the rnfisan aifa - managed , extensive network throughout the national territory that includes the regional authorities and the autonomous provinces of trento and bolzano , the regional centers of pharmacovigilance , 204 local health authorities , 112 hospitals , 43 research institutes , more than eight hundreds pharmaceutical companies and obviously aifa . through this network , periodicallyt he rnf data are transmitted to eudra vigilance database ( european database of suspected adrs reports ) , managed by the ema . eudravigilance collects all data provided at national level by the eu countries in a single database , which , in turn , communicates with vigibase , the who global icsr database . in december 2010 , european legislation on pharmacovigilance was changed with the adoption of the eu regulation 1235/2010 and directive 2010/84/eu amending , as regards pharmacovigilance , regulation ( ec ) 726/2004 and directive 2001/83/ec , respectively . this new legislation was the biggest change to the regulation of human medicines in the eu since 1995 . ema is responsible for implementing much of the new legislation , which has been effective since july 2012 . in october 2012 , the eu 's new pharmacovigilance legislation was further amended by : regulation ( eu ) no 1027/2012 ( applies from 5 june 2013);directive 2012/26/eu ( applies from 28 october 2013 ) . regulation ( eu ) no 1027/2012 ( applies from 5 june 2013 ) ; directive 2012/26/eu ( applies from 28 october 2013 ) . new legislation will greatly improve public - health protection and , ultimately , save lives by enhancing the overall efficiency and effectiveness of the pharmacovigilance system . basically , changes tend to increase the efficiency , speed and transparency of pharmacovigilance tasks through rules that aim to : strengthening pharmacovigilance systems ( more clearly defined roles and responsibilitiesof the many actors involved);streamline the activities among the member states ( distribution of tasks and work sharing , with less duplication of effort;increasing engagement of the patients and healthcare professionals;improving the communication systems of decision - making processes , giving proper justification to the public;increasing transparency . strengthening pharmacovigilance systems ( more clearly defined roles and responsibilities of the many actors involved ) ; streamline the activities among the member states ( distribution of tasks and work sharing , with less duplication of effort ; increasing engagement of the patients and healthcare professionals ; improving the communication systems of decision - making processes , giving proper justification to the public ; increasing transparency . first of all , the definition of adverse reaction has been changed and broadened to ensure that it now covers noxious and unintended effects resulting not only from the authorized use of a medicine at normal doses , but also from medication errors and uses outside the terms of the marketing authorization ( off - label use ) , including overdose , misuse and abuse of the product , and from occupational exposure . as a consequence , therefore , there will be an increase of reports and resulting a greater monitoring activity . in all eu countries , the patients will be able to report directly suspected adverse reactions . in italy , this possibility has already been considered since some years back by a paper form , however , from now on , also in accordance with the new directive , reports may also be sent by the web . all reports of adverse reactions will merge into the european database eudravigilance , but with different timing depending on the severity of the reaction ( within 15 days for serious reports and within 90 days for non - serious ones ) . reports from pharmaceutical companies will also be transmitted to the eudravigilance database . regarding some medicinal products authorized through the centralized procedure , it is already possible to consult the european database of adverse reactions at the following website http://www.adrreports.eu/. in summary , directive 2010/84/eu and regulation ( eu ) 1235/2010 aim to maintain , reinforce and further develop the eudravigilance database as the single or at least the main- point of receipt , storage and exchange of information concerning the safety of medicinal products for human use authorized in the eu . the data collected in the eudravigilance database will be monitored by the ema in cooperation with the member states , while monitoring of the data originated at the national level will be carried out by the member state involved ; these activities are aimed at the identification of changes in risk or new risks through the signals analysis . the methodology for the identification and management process of the signal were defined in the implementing regulation ( eu ) n. 520/2012 of 19 june 2012 , concerning the performance of pharmacovigilance activities provided for in regulation ( ec ) n.726/2004 of the european parliament and of the council and directive 2001/83/ec of the european parliament and of the council . as afore mentioned , the new legislation aims to guarantee greater transparency and improve communication . in fact , on its web portal , aifa makes available to the public at least the following information : public assessment reports , together with a summary there of;summaries of product characteristics and package leaflets;summaries of risk management plans;list of medicinal products that are subject to additional monitoring;information about the various procedures for the reporting of suspected adrs to the competent authorities by the healthcare professionals and patients , including forms with web data entry mask . public assessment reports , together with a summary there of ; summaries of product characteristics and package leaflets ; summaries of risk management plans ; list of medicinal products that are subject to additional monitoring ; information about the various procedures for the reporting of suspected adrs to the competent authorities by the healthcare professionals and patients , including forms with web data entry mask . medicines subject to additional monitoring will be the products containing new active substances not included in the authorized medicines in the eu before the january 1 2011 ; in particular , biologics and biosimilars , but the list may also include products whose license is subject to particular conditions or products authorized in exceptional circumstances ; e.g. , products subject to safety studies after the granting of the marketing authorization ( post - authorisation safety studies ) . these medicinal products subject to additional monitoring will be identifiable from the package leaflet that will bear the statement this medicinal product is subject to additional monitoring . this statement will be preceded by a black symbol and followed by an explanatory sentence explaining the concept of additional monitoring . the additional monitoring list will be regularly kept up to date by the ema in collaboration with member states . the new legislation has empowered the relevant authorities to impose on the marketing authorization holders the obligation to perform post - authorization safety and/or efficacy studies . these studies would be required at the time of the granting of the marketing authorization or later . according to another key point of the 2010 reform , each member state and each marketing authorization holder is obliged to adopt a pharmacovigilance system with the aim of analyzing suspected adrs and implementing provisions in order to prevent them . at last , the new eu pharmacovigilance legislation has established within the ema a new scientific committee , the pharmacovigilance risk assessment committee ( prac ) in which all member states are represented . the role of the prac in protecting public health requires close interaction with the agency 's committee for medicinal products for human use ( chmp ) and the coordination group for mutual recognition and decentralized procedures - human ( cmdh ) . its members include experts from regulatory authorities in eu member states and independent scientific experts who provide additional expertise in particular scientific areas . members from the patient organizations and healthcare professionals will be included in the near future further to appointment from the european commission . in its role in monitoring the safety of human medicines , the prac assesses all aspects of risk management , including the detection , assessment , minimization and communication of the risk of adrs , giving due consideration to the benefits of the medicine . the prac is also involved in designing and evaluating post - authorization safety studies and in conducting pharmacovigilance audits . when the prac has concluded an evaluation related to the safety of medicines , it prepares a recommendation that is sent either to the chmp ( if it concerns at least one centrally authorized medicine ) or to the cmdh ( if it only concerns medicines that are not centrally authorized in the eu ) . recently , the financial law no . 296/2006 has provided to the aifa an ad hoc fund to be allocated for activities related to pharmacovigilance . the regional authorities are responsible for the development and promotion of active pharmacovigilance programs , which agreed with aifa and subsequently , allow the regions to access to funds . in this context , aifa has promoted several projects and studies in italy , with the aim of increasing knowledge on drugs and the culture of pharmacovigilance the funding of pharmacovigilance projects by aifa has contributed to the improvement of spontaneous reporting . in italy , the situation of spontaneous adrs reports in 2010 showed an increase of 39% in comparison to 2009 . this result is in line with the average annual increase of 30% observed in the last five years , and it also demonstrates the crucial role of active pharmacovigilance programs , funded by aifa , in increasing knowledge on drugs , to better define their safety profile and to improve their safe use in clinical practice . in conclusion , the italian pharmacovigilance system is based on local activities coordinated at national level by aifa under the control of the ministry of health . this national system represents a link among local experiences and its counterparts at the european level . therefore , aifa has the double opportunity to transfer and receive knowledge from other eu countries ; furthermore , it is responsible in italy of the entire campaign to improve pharmacovigilance knowledge and increase the number of adrs reports up to the gold standard level established by the who . this system represents a good structure , however , several points need to be improved . this is particularly relevant for italy , where too many differences exist between regional and local activities . the financial support received by aifa and the future projects should help to achieve a more comprehensive pharmacovigilance activity .

aim . the aim of this study was to determine tumor necrosis factor - alpha ( tnf- ) levels in periapical exudates and to evaluate their relationship with radiological findings . methodology . periapical exudates were collected from root canals of 60 single - rooted teeth using absorbent paper points . tnf- levels were determined by enzyme - linked immunosorbent assays . the samples were divided into three groups according to the periapical radiolucent area . results . nonparametric kruskal - wallis test revealed significant differences between tnf- concentrations in control group ( 40 , 5728 , 15 pg / ml ) and group with larger radiolucent areas ( 2365 , 79582 , 95 pg / ml ) , as well as between control and canals with small radiolucent areas ( 507 , 66278 , 97 ) ( p<.05 ) . conclusions . the levels of tnf- increase significantly in teeth with periapical pathosis , from smaller to bigger lesions . this research and its results have shown that objective analysis of the tnf- levels enables establishment of a relationship between different concentrations of tnf- and different radiological changes .

periapical pathoses of pulpal origin develop in response to microbial irritants in the root canal systems . bacterial cell wall components react with monocytes , macrophages , other cells of the immune system , as well as with fibroblasts , leading to the production of proinflammatory cytokines , such as il-1 , il-1 , tnf- , il-6 , and il-8 . irritants may be mechanical or chemical , but are most often bacterial . the interaction between these irritants and host defensive cells results in release of numerous mediators that , through the root canal system , are capable of initiating immunologic reactions in periapical tissues resulting in the formation on inflammatory periapical lesions . these reactions include immune response mediated by cells , through the actions of t lymphocytes and cytokines , and hummoral immune response mediated by antibodies , activated by b - lymphocyte products . tnf- is a monocyte - derived protein that has a wide range of proinflammatory and immunomodulatory effects on a number of different cell populations . tnf- is a cytokine which stimulates bone resorption , prostaglandin synthesis , and protease production by many cell types , including fibroblasts and osteoblasts . the local production of pge2 , il1- , and tnf- has been demonstrated in periapical lesions [ 4 , 5 ] . tnf- is a cytokine initially identified as a causative of hemorrhagic necrosis in certain tumors and was later shown to be the same molecule as cachectin , a serum product earlier known as a mediator of wasting in chronic disease . in addition to its diverse bioactivities , tnf- is the only molecule other than il-1 that is presently known to have osteoclast - activating function . although extensive research has been performed in the area of periapical inflammatory mediators , only few studies focused on the role and levels of tnf- in periapical exudate [ 6 , 7 ] . the aim of this study was to determine the levels of tnf- in periapical exudates and to evaluate possible relationships between this cytokine and radiological findings in the involved teeth . the patients with noncontributory medical histories and diagnosed with apical periodontitis were selected for the study as follows . group 1 consisted of 20 subjects whose single - rooted teeth had the diagnosis of chronic apical periodontitis , with dull or absent pain , and size of the lesion < 1 cm . group 2 consisted of 20 subjects whose single - rooted teeth were diagnosed with chronic apical periodontitis , with dull or absent pain , but with the lesion size of > 1 cm.group 3 was control and comprised of 20 subjects whose investigated single - rooted teeth showed symptoms of acute periapical condition accompanied by excruciating pain , necrotic pulp , and radiological thickening of the apical periodontal ligament . primary access to the pulp chamber was gained using slow - speed round dental burs , with placement of rubber dam isolation . after determination of the working length , the necrotic pulp was removed and the root canal was enlarged to iso size 40 . a sterile paper point was inserted into the root canal up to the instrumentation length and held in place for 1 minute to absorb the exudate for the sample [ 6 , 7 ] . paper points were immersed into sterile ependorf vials containing phosphate buffered saline ( pbs ) and stored at 70c until further analysis [ 8 , 9 ] . the volume of the fluid was calculated from a standard curve and expressed as l . size of the radiolucent area was determined by two independent examiners at the time exudate samples were collected . periapical lesions were classified as small , when the longer diameter was shorter than 1 cm , while the remaining lesions were classified as large . in the control group , the radiological thickening of the periodontal ligament was present . group 1 consisted of 20 subjects whose single - rooted teeth had the diagnosis of chronic apical periodontitis , with dull or absent pain , and size of the lesion < 1 cm . group 2 consisted of 20 subjects whose single - rooted teeth were diagnosed with chronic apical periodontitis , with dull or absent pain , but with the lesion size of > 1 cm . group 3 was control and comprised of 20 subjects whose investigated single - rooted teeth showed symptoms of acute periapical condition accompanied by excruciating pain , necrotic pulp , and radiological thickening of the apical periodontal ligament . determination of the tnf- levels in supernatant of periapical exudate is based on utilization of monoclonal antibodies specific for tnf- which bind competitively to tnf-. investigation is based on sandwich all subjects were informed of the aims and procedures of research . within the research , they are guaranteed in respect of their basic ethical and bioethical principles : personal integrity ( independence , righteousness , well - being , and safety ) as regulated by nrnberg codex and the most recent version of helsinki declaration . only those subjects who gave a written permission in the form of informed consent the present study demonstrated the presence of tnf- in all periapical exudate samples . differences in concentrations of tnf- between control group ( 40 , 57 28 , 15 pg / ml ) , the group with large radiolucent areas ( 2365 , 79 582 , 95 pg / ml ) , as well as between control and the canals associated with small radiolucent areas ( 507 , 66 278 , 97 ) were statistically significant ( p < .05 ) . there was also statistically significant difference between the samples with large radiolucent areas and small radiolucent areas . eia data for tnf- were categorized according to radiological diagnosis , using the nonparametric kruskal - wallis test ( see figure 1 ) . soluble mediators produced and secreted by various inflammatory , immunologically - active , and structural cells , commonly referred to as cytokines , play the leading role in pathogenesis of infectious disease , acting side - by - side with other inflammatory mediators : kinines , vasoactive amines , components of the complement system , and metabolites of arachidonic acid . secretion of the cytokines is initiated with the purpose of activating immunological response to irritants and increasing local concentrations of inflammatory cells in order to prevent further colonization of bacteria within the tissues . enhanced reaction of the host to various antigens results in bone resorption and formation of granulomatous tissue , which are the typical features of periapical lesion . in this investigation , levels of the proinflammatory cytokine tnf- in periapical exudate were analyzed due to its leading role in pathogenesis of periapical lesions . these exudates present the inflammatory exudate from the periapical tissues and theoretically contain locally produced and secreted factors . they resulted from the inflammatory response and contain host mediators related in response to the infection . highest concentrations of tnf- were detected in periapical lesions with large radiolucent areas , while these levels were significantly lower in the periapical lesions with smaller radiolucent areas . in the control group diagnosed as acute periodontitis , with radiological changes consistent with periodontal ligament thickening , which differs in clinical symptoms from the other two experimental groups , and is characterized by radioloical changes consistent with periodontal ligament thickening , the levels of the investigated cytokine demonstrated the lowest levels . these results show different values from the results obtained by ataoglu et al . ; however , these authors compared the levels of two kinds of cytokines : il-1 and tnf-. in the work of ataoglu et al . , il-1 was detected in all samples of periapical exudates and its levels were significantly higher than tnf- levels ; however , the authors focused their attention only on the tnf- levels in periapical exudates demonstrated in all tissue specimens with significant differences regarding certain experimental groups . a study by kjeldsen et al . using elisa technique also showed significantly higher concentrations of tnf- in crevicular fluid in patients with chronic adult periodontitis . it is likely that chronic periodontal infection may evoke an immune response that may result in the production of slightly higher levels of tnf-. it was proved that tnf- leads to bone resorption through osteoclast activation and stimulation of the secretion of proteolytic enzymes , plasminogen activator ( pa ) , and matrix metalloproteinases ( mmp ) , which are in charge of destroying extracellular matrix of the bone tissue . . showed that there is a small fraction of tnf--positive cells within the granuloma with a macrophage - like morphology . ultrastructural analyses showed that there are some macrophages which have adjusted the extracellular secretion ; therefore , these macrophages might be the main source of cytokines in the tissue . wang et al . showed that both tnf- and il-1 are secreted in the infected rat pulps and periapical lesions . the cells appeared as soon as 2 days following the pulp chamber opening and their numbers increased steadily until the day 30 . these findings demonstrate the presence of il-1- and tnf--secreting cells in the pulp and periapical tissues immediately following the pulp exposure , which supports the assumption that the abovementioned cytokines play a role in pathogenesis of pulp and periapical pathoses . in the analysis of tnf- levels in healthy , symptomatic , and asymptomatic human pulps , the highest concentrations of tnf- , with regard to clinical classification , were found in symptomatic reversible pulpitis , and the difference was statistically significant in comparison with irreversible asymptomatic pulpitis and healthy pulp . the highest concentrations of tnf- , with regard to histological classification , were found in histologically confirmed moderate inflammation , followed by severe inflammation , mild inflammation , and finally healthy pulp . conclusion can be drawn that cytokine tnf- could be an objective marker in radiologically confirmed periapical lesions . the lowest levels were demonstrated in control group , which was characterized by radiological thickening of the periodontal ligament , while somewhat greater concentrations were found in teeth with periapical radiolucencies of < 1 cm diameter ; finally , the greatest concentrations were measured in teeth with radiolucent lesions > 1 cm , which confirms the relationship between tnf- and the emergence and development of periapical lesions . objective analysis of tnf- levels enables establishment of a relationship between different concentrations of this cytokine and different radiological changes .

traditional management of gallstone pancreatitis ( gp ) has been to perform cholecystectomy during the same hospital admission after resolution . however , when gp develops in the immediate postoperative period from a major colorectal operation , cholecystectomy may be fraught with difficulty due to the inflammatory response that occurs . thus , delaying cholecystectomy until the inflammatory response subsides may be worthwhile , and it maximizes the chances of completing the cholecystectomy laparoscopically . we have described our management of 2 patients with gp occurring after colorectal operations , which required proximal diverting ileostomy . in both cases , we deferred management of gp with either endoscopic retrograde cholangiopancreatography ( ercp ) or medical conservative measures during the acute attack and performed laparoscopic cholecystectomy during ostomy reversal surgery utilizing the existing ostomy takedown site for port placement . both patients tolerated this management well .

the traditional management of gallstone pancreatitis ( gp ) has been to perform cholecystectomy during the same hospital admission after resolution . however , when gp develops in the immediate postoperative period from a major colorectal operation , cholecystectomy may be fraught with difficulty due to the inflammatory response that occurs . thus , delaying cholecystectomy until the inflammatory response subsides may be worthwhile , and it maximizes the chances of completing the cholecystectomy laparoscopically . the best management strategy for gp under these circumstances , we have described our management of 2 patients with gp occurring after colorectal operations requiring proximal diverting ileostomy . a 31-year - old male with adenomatous polyposis coli gene mutation and familial adenomatous polyposis presented prophylactic surgical treatment . he underwent an uncomplicated laparoscopic total proctocolectomy with ileal j - pouch anal anastomosis and diverting loop ileostomy . on postoperative day ( pod ) 8 , he developed acute epigastric and right upper quadrant ( ruq ) pain . laboratory studies revealed a white blood cell ( wbc ) count of 14,000 , serum lipase and amylase of 1300 , and normal bilirubin . he was placed on bowel rest with iv hydration until pancreatitis improved clinically and biochemically . cholecystectomy was deferred until planned ileostomy reversal . to decrease the risk of recurrent episodes of gp , the patient then underwent ileostomy reversal and cholecystectomy as described below 6 weeks later . a 44-year - old male with a history of sigmoid colectomy for endoscopically unresectable adenoma presented to us with rectal bleeding . laboratory studies revealed a wbc count of 13,000 , lipase and amylase above 400 , and normal bilirubin . gp was diagnosed and then managed non - operatively , similar to our first case . he refused ercp / s . after discharging , the patient returned in 6 weeks for ileostomy reversal and cholecystectomy as described below . in both cases , anastomosis was found to be healed on pouchogram and pouchoscopy at 6 weeks postoperatively . after establishing general anesthesia and administration of pre - incisional antibiotics , the proximal and distal portions were marked with different colored sutures and placed within the abdomen . a 12-mm balloon - tipped port was placed through the residual defect for the laparoscope . three 5 mm ports were placed in the ruq away from any intra - abdominal adhesions [ figure 1 ] . the two limbs of ileum were eviscerated in proper anatomic position using the colored sutures . a 31-year - old male with adenomatous polyposis coli gene mutation and familial adenomatous polyposis presented prophylactic surgical treatment . he underwent an uncomplicated laparoscopic total proctocolectomy with ileal j - pouch anal anastomosis and diverting loop ileostomy . on postoperative day ( pod ) 8 , he developed acute epigastric and right upper quadrant ( ruq ) pain . laboratory studies revealed a white blood cell ( wbc ) count of 14,000 , serum lipase and amylase of 1300 , and normal bilirubin . he was placed on bowel rest with iv hydration until pancreatitis improved clinically and biochemically . cholecystectomy was deferred until planned ileostomy reversal . to decrease the risk of recurrent episodes of gp , endoscopic retrograde cholangiopancreatography with sphincterotomy ( ercp / s ) was performed . a 44-year - old male with a history of sigmoid colectomy for endoscopically unresectable adenoma presented to us with rectal bleeding . laboratory studies revealed a wbc count of 13,000 , lipase and amylase above 400 , and normal bilirubin . gp was diagnosed and then managed non - operatively , similar to our first case . he refused ercp / s . after discharging , the patient returned in 6 weeks for ileostomy reversal and cholecystectomy as described below . in both cases , anastomosis was found to be healed on pouchogram and pouchoscopy at 6 weeks postoperatively . after establishing general anesthesia and administration of pre - incisional antibiotics , the proximal and distal portions were marked with different colored sutures and placed within the abdomen . a 12-mm balloon - tipped port was placed through the residual defect for the laparoscope . three 5 mm ports were placed in the ruq away from any intra - abdominal adhesions [ figure 1 ] . the two limbs of ileum were eviscerated in proper anatomic position using the colored sutures . about 20% of patients harboring asymptomatic gs develop symptoms requiring cholecystectomy 3 - 5 years following colorectal surgery . the mechanism is unclear , but studies have implicated changes in the enterohepatic circulation and cholesterol metabolism . the potential for gs related symptoms in these patients caused some to recommend cholecystectomy as an incidental procedure during the time of the index colorectal operation . proponents feel that this practice spares patients future complications and the morbidity of another operation . previous studies do not focus on the immediate postoperative period or comment on gp specifically . therefore , the true incidence of this particular postoperative complication is unknown . in the absence of good data , most patients presenting with gp undergo cholecystectomy during the same hospital admission to prevent the potential morbidity of future bouts . in our cases , ercp / s can be used as a temporizing procedure to prevent recurrent episodes of gp while awaiting cholecystectomy . in addition , both of our patients had stomas that were planned to be reversed 6 - 8 weeks after the initial operation and , thus , provided the opportunity for a combined operation . ports for laparoscopic procedures are routinely placed at sites of planned stomas in order to minimize the number of incisions . using the stoma site for the camera avoids an extra incision and is away from the midline , where adhesions are expected from previous operations . we feel that delaying the completion of the anastomosis until after cholecystectomy and marking the proximal and distal limbs are important to avoid trauma to the anastomosis and to prevent un - intended twisting of the bowel . complications attributable to gs in the immediate postoperative period following colorectal surgery are difficult to manage . when the complication is gp , the episode is mild and resolves with non - operative measures , it may be preferable to delay cholecystectomy until the postoperative inflammatory response subsides . when the index operation is the first stage of a planned 2-stage operation , delaying the cholecystectomy seems even more attractive . as seen in these two cases , the fascial aperture of the ileostomy site can be easily used for the laparoscope , and combining the two operations is straightforward .

the maintenance of quality of life ( qol ) in patients with high - grade glioma is an important endpoint during treatment , particularly in those with glioblastoma multiforme ( gbm ) given its dismal prognosis despite limited advances in standard therapy . it has proven difficult to identify new therapies that extend survival in patients with recurrent gbm , so one of the primary aims of new therapies is to reduce morbidity , restore or preserve neurologic functions , and the capacity to perform daily activities . apart from temozolomide , cytotoxic chemotherapeutic agents do not appear to significantly impact response or survival , but produce toxicity that is likely to negatively impact qol . new biological agents , such as bevacizumab , can induce a clinically meaningful proportion of durable responses among patients with recurrent gbm with an acceptable safety profile . emerging evidence suggests that bevacizumab produces an improvement or preservation of neurocognitive function in gbm patients , suggestive of qol improvement , in most poor - prognosis patients who would otherwise be expected to show a sudden and rapid deterioration in qol .

high - grade gliomas , who grade iii or iv [ 1 , 2 ] , are the most common primary brain tumors in adults [ 1 , 3 ] . who grade iv tumors are almost exclusively ( 8090% ) glioblastoma multiforme ( gbm ) , which are the most common high - grade glioma ( 4045% ) [ 1 , 3 ] , the most common form of brain tumor overall ( 1215% ) [ 1 , 3 ] , and the most aggressive malignant primary brain tumor . despite limited significant advances in standard therapy , notably temozolomide , median overall survival ( os ) remains low : 15 months for newly - diagnosed gbm [ 5 , 6 ] and 57 months for recurrent / relapsed gbm [ 710 ] . in a clinical trial setting , the current standard of care ( radiotherapy plus temozolomide followed by 6 cycles of adjuvant temozolomide ) provided 2- and 5-year survival rates of 27 and 10% for patients with newly diagnosed gbm . given the poor prognosis of gbm , the primary objectives of therapy are to reduce morbidity , restore or preserve neurologic functions and the capacity to perform daily activities as long as possible . the aim of this review is to examine the impact of gbm therapy on qol , neurocognitive function , and their correlates . it is well recognized that impairment of neurocognitive functioning , resulting in behavioral , emotional , and intellectual difficulties , occurs in nearly all patients with brain tumors and eventually compromises their independence . this impairment is related to a combination of various factors , including the tumor itself , tumor - related epilepsy , treatment , and patient - related factors ( e.g. , age , psychological distress ) [ 1316 ] . however , there have been relatively few well - powered longitudinal studies of neurocognitive function in patients with high - grade glioma . not surprisingly , neurocognitive function assessments have been incorporated as major components of patient assessments , along with common and widely used questionnaires to assess health - related qol ( hr - qol ) , e.g. , european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 ) , and functional assessment of cancer therapy ( fact ) cancer - specific scales . indeed , neurocognitive function has been shown to be a valid predictor of long - term qol [ 2224 ] . as life expectancy in high - grade glioma , and particularly in recurrent gbm , is so short , issues relating to qol are immensely important to patients and their caregivers . this is especially important in relation to new treatments in recurrent gbm that do not yet have evidence supporting their contribution to extended survival but may significantly delay the expected steep qol deterioration occurring after progression following standard therapies . unfortunately , qol data are difficult to collect in cancer patients because they may be unwilling to complete the questionnaire when they are feeling unwell . furthermore , repeated application of lengthy , formal hr - qol questionnaires can represent a major and impractical burden for patients . also , the analysis of qol data is challenging due to the high rates of non - random missing qol values that may be linked to patients qol status , and if ignored may introduce bias in the interpretation of results . interpretation of the impact of standard and new therapies on qol in gbm patients is consequently problematic , even when attempting to classify their effect into the three broad categories of negative , positive , or neutral . to overcome these challenges , changes in neurocognitive functioning assessment of neurocognitive function can therefore represent a practical surrogate for formal qol assessment of patients with recurrent gbm . in addition , besides grade and age , performance status in patients with newly diagnosed glioma is an independent prognostic marker . thus , it is plausible to assume that neurocognitive function , irrespective of clinical stage , may also have prognostic implications even after initiation of therapy and during the course of treatment . hr - qol in patients with high - grade glioma has recently been reviewed in detail , which noted problems associated with interpretation of different studies and the paucity of robust hr - qol information derived from well - powered randomized controlled trials . among the seven randomized controlled trials of new treatments published from 2002 to 2007 they identified for high - grade glioma , there was no or little difference between treatment groups at baseline or follow - up evaluation . these authors suggested that standard multidimensional hr - qol questionnaires might therefore contain too many items and consequently lack sensitivity to detect qol changes in patients with high - grade glioma . simpler , practical , and more sensitive instruments ( such as cognitive function ) are therefore needed to study qol changes in relation to therapy in high - grade glioma , and , thus , the confounding factor of missing substantial follow - up data ( primarily related to dropouts ) needs to be addressed . neurosurgery and/or radiotherapy are still fundamental elements of standard therapy for patients with high - grade glioma . it is well recognized that surgery may initially , at least temporarily , improve qol dramatically in a significant proportion of patients with severe symptoms related to increased intracranial pressure . conversely , radiotherapy may decrease qol in some patients from adverse effects such as hair loss , fatigue , somnolence , or cognitive problems . it is clear that tumor recurrence and short - term survival are confounding variables , but it is generally agreed that short - term memory and progression of dementia are observed in many patients treated with brain irradiation . the impact of adjunctive therapy with corticosteroids and antiepileptic medication has also been extensively studied . while the presence and severity of epileptic seizures and/or the use of antiepileptic medication have been significantly associated with cognitive deficits in patients with low - grade gliomas , more so than the effects of radiotherapy [ 2830 ] , the effects of antiepileptic medication on neurocognitive functioning and qol have been less extensively studied in patients with high - grade glioma , although some studies have reported a negative impact . the negative effects of corticosteroid use on neurocognitive function and/or qol are well documented in healthy subjects [ 32 , 33 ] and in various disease states , such as leukemia . while it appears accepted that the use of high - dose corticosteroids has a clinically significant negative impact on neurocognitive function in glioma , there would appear to be no published information in this indication . it is documented that corticosteroid use in primary brain tumors and/or metastases caused adverse effects [ 35 , 36 ] which would be expected to decrease qol . assessment of qol is important with cytotoxic chemotherapy , particularly when survival benefit may be marginal and has to be balanced against any negative contribution of significant toxicity . among newly diagnosed gbm patients randomized to radiotherapy alone or radiotherapy plus temozolomide , the addition of temozolomide had no significant negative effect on qol measures , except on social functioning ( p = 0.0052 ) . similarly , among first - relapse gbm patients , temozolomide had no significant negative effect on qol , although responders to temozolomide had improvement in most qol domain scores , e.g. , global , motor dysfunction , emotional function , drowsiness , future uncertainty , and communication deficit , until eventual disease progression [ 3840 ] . these overall findings with temozolomide in gbm are concordant with the recent cochrane review that evaluated randomized controlled trial data assessing temozolomide in patients with high - grade glioma . when used as a comparator for temozolomide among the aforementioned trials , procarbazine was reported to have a negative impact on hr - qol domain scores , e.g. , drowsiness , communications deficit , motor dysfunction , role functioning , social functioning , and physical functioning , regardless of disease progression status [ 38 , 39 ] . the impact of combined procarbazine , ccnu ( lomustine ) , and vincristine ( pcv ) chemotherapy after radiotherapy compared with radiotherapy alone on hr - qol measures was determined in the eortc 26951 trial of patients with anaplastic oligodendroglioma ( n = 368 ) : a major negative impact on hr - qol ( nausea / vomiting , appetite loss , and drowsiness ) was found during and shortly after pcv treatment . however , when hr - qol measures were used to assess the impact of pcv chemotherapy after radiotherapy compared with radiotherapy alone in the radiation therapy oncology group ( rtog ) trial 94 - 02 of patients with mixed anaplastic oligodendroglioma , scores were similar longitudinally and between treatments for survivors . hr - qol ( spitzer quality of life index ) score showed continual deterioration when measured longitudinally in the rtob 98 - 03 study of escalating doses of conformal three - dimensional radiation and carmustine in gbm patients . supplementation of surgery and radiotherapy with ccnu chemotherapy provided no benefit in terms of quality of life or change in clinical performance . finally , there are very limited qol data from randomized controlled trials with implanted carmustine - impregnated wafers in primary or recurrent high - grade glioma according to a recent cochrane review . bevacizumab ( avastin ) , a humanized monoclonal antibody that binds to and inhibits the activity of vegf , is the first approved antiangiogenic cancer treatment . bevacizumab acts synergistically with cytotoxic chemotherapy or biological agents in the treatment of various tumors , e.g. , colorectal , lung , renal , and breast cancer [ 4750 ] . recently , studies in patients with recurrent high - grade glioma or gbm have indicated promising results with durable response using the combination of bevacizumab and irinotecan [ 5167 ] , and additionally in combination with etoposide , nitrosourea , fotemustine , or erlotinib . positive results have also been reported with single - agent bevacizumab in recurrent high - grade glioma [ 57 , 61 , 7274 ] , and in combination with adjuvant temozolomide in newly diagnosed gbm [ 7579 ] . during these studies of bevacizumab - based therapy in high - grade glioma , it was reported that one of the consequences of bevacizumab therapy is a steroid - sparing effect in a proportion of patients , which would be expected to positively impact qol . various studies have indicated a reduction or elimination of corticosteroid use with bevacizumab - based therapy in patients with recurrent high - grade glioma after prior treatment [ 53 , 5661 , 63 , 64 , 73 ] . for example , in the randomized phase ii clinical trial comparing bevacizumab alone or in combination with irinotecan in 167 patients with recurrent , treatment - refractory gbm , patients who were taking corticosteroids at baseline showed a trend to take stable or decreasing doses over time , e.g. , median corticosteroid dose was reduced by about 75% after 6 months ( fig . 1 ) . the steroid - sparing effect associated with bevacizumab - based therapy appeared associated with clinical response in high - grade glioma [ 58 , 64 ] and clinical benefit such as improved neurological symptoms in high - grade glioma or recurrent gbm .fig . 1change in median corticosteroid use following treatment with bevacizumab with or without irinotecan in patients with recurrent glioblastoma multiforme ( adapted with permission from friedman et al . ) change in median corticosteroid use following treatment with bevacizumab with or without irinotecan in patients with recurrent glioblastoma multiforme ( adapted with permission from friedman et al . ) a potentially positive impact of bevacizumab - based therapy on neurocognitive function , performance status and/or qol has also started to emerge from reports of clinical studies among gbm patients [ 52 , 59 , 63 , 64 ] . for example , in a retrospective analysis of recurrent gbm patients treated with bevacizumab ( n = 44 ) or without bevacizumab ( n = 79 ) at a single us institution , it was reported that bevacizumab - treated patients took longer to increase dexamethasone dose ( median 149 vs. 130 days , p = 0.04 ) and also maintained their karnofsky performance status ( kps ) for longer ( median 252 vs. 120 days , p = 0.006 ) ; subgroup analysis indicated that the difference in these effects were more pronounced in patients aged > 55 years . in another study of 22 consecutive patients with recurrent gbm treated with bevacizumab plus irinotecan , cognitive function was assessed by the blessed orientation - memory - concentration test ( bomc ) and functional status was assessed by kps , barthel index ( bi ) , and instrumental activities of daily living ( iadl ) prior to each cycle of treatment . improvement in bomc score was seen in 15 patients ( 62% ) , with median improvement of 7 points . improvement in functional status was seen in 18 patients ( 85.7% ) , with median improvement in kps by 10 points , bi by 8 points , and iadl by 2 points . the overall clinical response rate with bevacizumab plus irinotecan was 95% and was associated with significant improvements in cognitive functional and functional status . larger scale controlled clinical trials of bevacizumab - based therapy in gbm patients were consequently undertaken or are planned / on - going . among these is the recently reported brain study ( avf3708 g ) , an open - label , multicenter , randomized , phase ii trial of two concurrent arms treated with single - agent bevacizumab ( n = 85 ) or bevacizumab plus irinotecan ( n = 82 ) in patients with first- or second - relapse gbm who had been previously treated with temozolomide initially or at relapse . primary endpoint results have been reported : estimated 6-month pfs rates were 43 and 50% in single - agent bevacizumab and bevacizumab plus irinotecan arms , respectively , and objective response rates were 28 and 38% , respectively . these results supported the activity of bevacizumab in recurrent gbm patients given that the 6-month pfs rate was considerably higher than the 15% rate expected for salvage chemotherapy and/or chemotherapy alone . the authors noted a trend for patients who were taking corticosteroids at baseline to take stable or decreasing doses over time , but they made no formal comparison . more compellingly data on corticosteroid use during this study have recently been reported . at baseline , 51 and 53% of patients received systemic corticosteroids in the single - agent bevacizumab and bevacizumab plus irinotecan arms , respectively . more than 75 and 65% of patients in the single - agent and combination arms , respectively , who did not receive corticosteroids at baseline continued to receive no corticosteroids after baseline . sustained reduction in corticosteroid use was defined in this study as a 50% dose reduction for 50% of time on study treatment up to 52 weeks . among patients with complete or partial response in the single - agent bevacizumab and bevacizumab plus irinotecan arms , 57 and 65% , respectively , had a sustained reduction in corticosteroid use compared with 17 and 38% , respectively , among those with stable or progressive disease . neurocognitive function of patients treated with single - agent bevacizumab in the brain study ( n = 85 ) has been analyzed . patients were assessed with the hopkins verbal learning test - revised ( hvlt - r ) , trail making test parts a ( tmta ) and b ( tmtb ) , and the controlled oral word association ( cowa ) test . assessments were conducted at baseline and then every 6 weeks while patients remained on study treatment up to 52 weeks . change in neurocognitive function from baseline to week 6 was categorized as improved , stable , or declined , using the reliable change index . between 93 and 98% of patients completed each test at baseline and 7378% completed each test at both baseline and week 6 . the majority of patients demonstrated stable performance on each test at week 6 , relative to baseline . with the exception of the cowa test , 1628% of patients demonstrated improved performance on one or more tests at week 6 ( table 1 ) . preliminary results suggest that the majority of patients with recurrent gbm who were treated with bevacizumab alone in the brain study demonstrated stable or improved neurocognitive function during the first 6 weeks of treatment ( 81100% across all tests ) . changes across tasks and associations with measures of clinical efficacy were also explored ( table 2).table 1neurocognitive changes in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain studyassessmentchange in performance at week 6 relative to baseline ( per reliable change index)improved ( % ) declined ( % ) stable ( % ) hvlt - r total recall ( n = 74)161866hvlt - r delayed recall ( n = 70)141670hvlt - r delayed recognition ( n = 69)221959tmta ( n = 73)231166tmtb ( n = 65)281162cowa ( n = 70)3097hvlt - r hopkins verbal learning test - revised , tmta trail making tests part a , tmtb trail making test part b , cowa controlled oral word association testtable 2stabilization or improvement in neurocognitive function in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain studystable / improved neurocognitive function on all tests n ( % ) deterioration in neurocognitive function in at least one test n ( % ) responders at time of irf response ( n = 24)18 ( 75%)6 ( 25%)pfs > 6 months at week 24 ( n = 27)19 ( 70%)8 ( 30%)patients at time of investigator pd ( n = 49)15 ( 31%)34 ( 69%)irf independent radiology facility , pfs progression - free survival , pd progressive diseasetwo patients had missing neurocognitive data and were dropped from the analysiseight patients had missing neurocognitive data and were dropped from the analysis neurocognitive changes in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain study hvlt - r hopkins verbal learning test - revised , tmta trail making tests part a , tmtb trail making test part b , cowa controlled oral word association test stabilization or improvement in neurocognitive function in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain study irf independent radiology facility , pfs progression - free survival , pd progressive disease two patients had missing neurocognitive data and were dropped from the analysis eight patients had missing neurocognitive data and were dropped from the analysis in a recent retrospective analysis , 12 patients with gbm and poor neurologic function ( kps < 60% ) due to bulky disease were treated in an up - front setting with bevacizumab following induction temozolomide in an attempt to improve their ability to tolerate chemoradiation . median kps improved from 50 to 70% and their median dexamethasone dose was reduced from 12 to 4 mg / day . five of 11 evaluable patients ( 45% ) had a partial response , 4 ( 35% ) a minor response , and 1 ( 10% ) stable disease . the tumor response , rapidly improved neurologic function , and reduced steroid requirement allowed the majority of patients ( 75% ) to complete definitive radiotherapy . little significant information appears to have been published on the effects of other investigational biological agents ( e.g. , cilengitide , cediranib , sorafenib , sunitinib ) on corticosteroid use , neurocognitive function , or qol in patients with high - grade glioma or more specifically those with gbm . in a recent phase ii study of cediranib , an oral pan - vegfr tyrosine kinase inhibitor , was administered as monotherapy ( 45 mg / day ) in 31 patients with recurrent gbm and resulted in encouraging proportions of radiographic partial responses of 57 and 27% on 3- and 2-dimensional mri , respectively ) and 6-month pfs ( 26% ) . furthermore , among 15 patients receiving corticosteroids on study entry , the dose was reduced ( n = 10 ) or discontinued ( n = 5 ) . neurosurgery and/or radiotherapy are still fundamental elements of standard therapy for patients with high - grade glioma . it is well recognized that surgery may initially , at least temporarily , improve qol dramatically in a significant proportion of patients with severe symptoms related to increased intracranial pressure . conversely , radiotherapy may decrease qol in some patients from adverse effects such as hair loss , fatigue , somnolence , or cognitive problems . it is clear that tumor recurrence and short - term survival are confounding variables , but it is generally agreed that short - term memory and progression of dementia are observed in many patients treated with brain irradiation . the impact of adjunctive therapy with corticosteroids and antiepileptic medication has also been extensively studied . while the presence and severity of epileptic seizures and/or the use of antiepileptic medication have been significantly associated with cognitive deficits in patients with low - grade gliomas , more so than the effects of radiotherapy [ 2830 ] , the effects of antiepileptic medication on neurocognitive functioning and qol have been less extensively studied in patients with high - grade glioma , although some studies have reported a negative impact . the negative effects of corticosteroid use on neurocognitive function and/or qol are well documented in healthy subjects [ 32 , 33 ] and in various disease states , such as leukemia . while it appears accepted that the use of high - dose corticosteroids has a clinically significant negative impact on neurocognitive function in glioma , there would appear to be no published information in this indication . however , it is documented that corticosteroid use in primary brain tumors and/or metastases caused adverse effects [ 35 , 36 ] which would be expected to decrease qol . assessment of qol is important with cytotoxic chemotherapy , particularly when survival benefit may be marginal and has to be balanced against any negative contribution of significant toxicity . among newly diagnosed gbm patients randomized to radiotherapy alone or radiotherapy plus temozolomide , the addition of temozolomide had no significant negative effect on qol measures , except on social functioning ( p = 0.0052 ) . similarly , among first - relapse gbm patients , temozolomide had no significant negative effect on qol , although responders to temozolomide had improvement in most qol domain scores , e.g. , global , motor dysfunction , emotional function , drowsiness , future uncertainty , and communication deficit , until eventual disease progression [ 3840 ] . these overall findings with temozolomide in gbm are concordant with the recent cochrane review that evaluated randomized controlled trial data assessing temozolomide in patients with high - grade glioma . when used as a comparator for temozolomide among the aforementioned trials , procarbazine was reported to have a negative impact on hr - qol domain scores , e.g. , drowsiness , communications deficit , motor dysfunction , role functioning , social functioning , and physical functioning , regardless of disease progression status [ 38 , 39 ] . the impact of combined procarbazine , ccnu ( lomustine ) , and vincristine ( pcv ) chemotherapy after radiotherapy compared with radiotherapy alone on hr - qol measures was determined in the eortc 26951 trial of patients with anaplastic oligodendroglioma ( n = 368 ) : a major negative impact on hr - qol ( nausea / vomiting , appetite loss , and drowsiness ) was found during and shortly after pcv treatment . however , when hr - qol measures were used to assess the impact of pcv chemotherapy after radiotherapy compared with radiotherapy alone in the radiation therapy oncology group ( rtog ) trial 94 - 02 of patients with mixed anaplastic oligodendroglioma , scores were similar longitudinally and between treatments for survivors . hr - qol ( spitzer quality of life index ) score showed continual deterioration when measured longitudinally in the rtob 98 - 03 study of escalating doses of conformal three - dimensional radiation and carmustine in gbm patients . supplementation of surgery and radiotherapy with ccnu chemotherapy provided no benefit in terms of quality of life or change in clinical performance . finally , there are very limited qol data from randomized controlled trials with implanted carmustine - impregnated wafers in primary or recurrent high - grade glioma according to a recent cochrane review . bevacizumab ( avastin ) , a humanized monoclonal antibody that binds to and inhibits the activity of vegf , is the first approved antiangiogenic cancer treatment . bevacizumab acts synergistically with cytotoxic chemotherapy or biological agents in the treatment of various tumors , e.g. , colorectal , lung , renal , and breast cancer [ 4750 ] . recently , studies in patients with recurrent high - grade glioma or gbm have indicated promising results with durable response using the combination of bevacizumab and irinotecan [ 5167 ] , and additionally in combination with etoposide , nitrosourea , fotemustine , or erlotinib . positive results have also been reported with single - agent bevacizumab in recurrent high - grade glioma [ 57 , 61 , 7274 ] , and in combination with adjuvant temozolomide in newly diagnosed gbm [ 7579 ] . during these studies of bevacizumab - based therapy in high - grade glioma , it was reported that one of the consequences of bevacizumab therapy is a steroid - sparing effect in a proportion of patients , which would be expected to positively impact qol . various studies have indicated a reduction or elimination of corticosteroid use with bevacizumab - based therapy in patients with recurrent high - grade glioma after prior treatment [ 53 , 5661 , 63 , 64 , 73 ] . for example , in the randomized phase ii clinical trial comparing bevacizumab alone or in combination with irinotecan in 167 patients with recurrent , treatment - refractory gbm , patients who were taking corticosteroids at baseline showed a trend to take stable or decreasing doses over time , e.g. , median corticosteroid dose was reduced by about 75% after 6 months ( fig the steroid - sparing effect associated with bevacizumab - based therapy appeared associated with clinical response in high - grade glioma [ 58 , 64 ] and clinical benefit such as improved neurological symptoms in high - grade glioma or recurrent gbm .fig . 1change in median corticosteroid use following treatment with bevacizumab with or without irinotecan in patients with recurrent glioblastoma multiforme ( adapted with permission from friedman et al . ) change in median corticosteroid use following treatment with bevacizumab with or without irinotecan in patients with recurrent glioblastoma multiforme ( adapted with permission from friedman et al . ) a potentially positive impact of bevacizumab - based therapy on neurocognitive function , performance status and/or qol has also started to emerge from reports of clinical studies among gbm patients [ 52 , 59 , 63 , 64 ] . for example , in a retrospective analysis of recurrent gbm patients treated with bevacizumab ( n = 44 ) or without bevacizumab ( n = 79 ) at a single us institution , it was reported that bevacizumab - treated patients took longer to increase dexamethasone dose ( median 149 vs. 130 days , p = 0.04 ) and also maintained their karnofsky performance status ( kps ) for longer ( median 252 vs. 120 days , p = 0.006 ) ; subgroup analysis indicated that the difference in these effects were more pronounced in patients aged > 55 years . in another study of 22 consecutive patients with recurrent gbm treated with bevacizumab plus irinotecan , cognitive function was assessed by the blessed orientation - memory - concentration test ( bomc ) and functional status was assessed by kps , barthel index ( bi ) , and instrumental activities of daily living ( iadl ) prior to each cycle of treatment . improvement in bomc score was seen in 15 patients ( 62% ) , with median improvement of 7 points . improvement in functional status was seen in 18 patients ( 85.7% ) , with median improvement in kps by 10 points , bi by 8 points , and iadl by 2 points . the overall clinical response rate with bevacizumab plus irinotecan was 95% and was associated with significant improvements in cognitive functional and functional status . larger scale controlled clinical trials of bevacizumab - based therapy in gbm patients were consequently undertaken or are planned / on - going . among these is the recently reported brain study ( avf3708 g ) , an open - label , multicenter , randomized , phase ii trial of two concurrent arms treated with single - agent bevacizumab ( n = 85 ) or bevacizumab plus irinotecan ( n = 82 ) in patients with first- or second - relapse gbm who had been previously treated with temozolomide initially or at relapse . primary endpoint results have been reported : estimated 6-month pfs rates were 43 and 50% in single - agent bevacizumab and bevacizumab plus irinotecan arms , respectively , and objective response rates were 28 and 38% , respectively . these results supported the activity of bevacizumab in recurrent gbm patients given that the 6-month pfs rate was considerably higher than the 15% rate expected for salvage chemotherapy and/or chemotherapy alone . the authors noted a trend for patients who were taking corticosteroids at baseline to take stable or decreasing doses over time , but they made no formal comparison . more compellingly data on corticosteroid use during this study have recently been reported . at baseline , 51 and 53% of patients received systemic corticosteroids in the single - agent bevacizumab and bevacizumab plus irinotecan arms , respectively . more than 75 and 65% of patients in the single - agent and combination arms , respectively , who did not receive corticosteroids at baseline continued to receive no corticosteroids after baseline . sustained reduction in corticosteroid use was defined in this study as a 50% dose reduction for 50% of time on study treatment up to 52 weeks . among patients with complete or partial response in the single - agent bevacizumab and bevacizumab plus irinotecan arms , 57 and 65% , respectively , had a sustained reduction in corticosteroid use compared with 17 and 38% , respectively , among those with stable or progressive disease . neurocognitive function of patients treated with single - agent bevacizumab in the brain study ( n = 85 ) has been analyzed . patients were assessed with the hopkins verbal learning test - revised ( hvlt - r ) , trail making test parts a ( tmta ) and b ( tmtb ) , and the controlled oral word association ( cowa ) test . assessments were conducted at baseline and then every 6 weeks while patients remained on study treatment up to 52 weeks . change in neurocognitive function from baseline to week 6 was categorized as improved , stable , or declined , using the reliable change index . between 93 and 98% of patients completed each test at baseline and 7378% completed each test at both baseline and week 6 . the majority of patients demonstrated stable performance on each test at week 6 , relative to baseline . with the exception of the cowa test , 1628% of patients demonstrated improved performance on one or more tests at week 6 ( table 1 ) . preliminary results suggest that the majority of patients with recurrent gbm who were treated with bevacizumab alone in the brain study demonstrated stable or improved neurocognitive function during the first 6 weeks of treatment ( 81100% across all tests ) . changes across tasks and associations with measures of clinical efficacy were also explored ( table 2).table 1neurocognitive changes in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain studyassessmentchange in performance at week 6 relative to baseline ( per reliable change index)improved ( % ) declined ( % ) stable ( % ) hvlt - r total recall ( n = 74)161866hvlt - r delayed recall ( n = 70)141670hvlt - r delayed recognition ( n = 69)221959tmta ( n = 73)231166tmtb ( n = 65)281162cowa ( n = 70)3097hvlt - r hopkins verbal learning test - revised , tmta trail making tests part a , tmtb trail making test part b , cowa controlled oral word association testtable 2stabilization or improvement in neurocognitive function in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain studystable / improved neurocognitive function on all tests n ( % ) deterioration in neurocognitive function in at least one test n ( % ) responders at time of irf response ( n = 24)18 ( 75%)6 ( 25%)pfs > 6 months at week 24 ( n = 27)19 ( 70%)8 ( 30%)patients at time of investigator pd ( n = 49)15 ( 31%)34 ( 69%)irf independent radiology facility , pfs progression - free survival , pd progressive diseasetwo patients had missing neurocognitive data and were dropped from the analysiseight patients had missing neurocognitive data and were dropped from the analysis neurocognitive changes in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain study hvlt - r hopkins verbal learning test - revised , tmta trail making tests part a , tmtb trail making test part b , cowa controlled oral word association test stabilization or improvement in neurocognitive function in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain study irf independent radiology facility , pfs progression - free survival , pd progressive disease two patients had missing neurocognitive data and were dropped from the analysis eight patients had missing neurocognitive data and were dropped from the analysis in a recent retrospective analysis , 12 patients with gbm and poor neurologic function ( kps < 60% ) due to bulky disease were treated in an up - front setting with bevacizumab following induction temozolomide in an attempt to improve their ability to tolerate chemoradiation . median kps improved from 50 to 70% and their median dexamethasone dose was reduced from 12 to 4 mg / day . five of 11 evaluable patients ( 45% ) had a partial response , 4 ( 35% ) a minor response , and 1 ( 10% ) stable disease . the tumor response , rapidly improved neurologic function , and reduced steroid requirement allowed the majority of patients ( 75% ) to complete definitive radiotherapy . little significant information appears to have been published on the effects of other investigational biological agents ( e.g. , cilengitide , cediranib , sorafenib , sunitinib ) on corticosteroid use , neurocognitive function , or qol in patients with high - grade glioma or more specifically those with gbm . in a recent phase ii study of cediranib , an oral pan - vegfr tyrosine kinase inhibitor , was administered as monotherapy ( 45 mg / day ) in 31 patients with recurrent gbm and resulted in encouraging proportions of radiographic partial responses of 57 and 27% on 3- and 2-dimensional mri , respectively ) and 6-month pfs ( 26% ) . furthermore , among 15 patients receiving corticosteroids on study entry , the dose was reduced ( n = 10 ) or discontinued ( n = 5 ) . maintenance of qol in patients with high - grade glioma is an important endpoint during treatment , and more so for gbm because of the particularly poor prognosis with short life expectancy at this stage of the disease . however , reliable serial measurement of qol in patients with high - grade glioma is notoriously difficult , relating to many factors but particularly dropout bias or inability to repeatedly complete complex forms . it would appear that there is a progressive decrease in qol during the course of high - grade glioma that substantially accelerates once the disease relapses . this is also expressed as deterioration peaks driven by the administered therapies ( e.g. , radiotherapy ) or by the exacerbation of accompanying syndromes ( e.g. , brain edema , neurological symptoms , psychiatric disturbances ) . it has proven difficult to identify new therapies that extend os and pfs in patients with recurrent gbm after failure of previous therapy . most alternative cytotoxic chemotherapeutic agents do not seem to significantly impact response or survival , yet may produce adverse effects that have a likely negative impact on qol . however , among the new biological agents , bevacizumab has been shown to induce a clinically meaningful proportion of durable responses among patients with an acceptable safety profile . furthermore , data are emerging that bevacizumab induces improvement or preservation of neurocognitive function , suggestive of qol improvement , in the majority of poor - prognosis patients who would otherwise be expected to show a sudden , rapid deterioration in qol . further studies are underway to confirm these findings and better understand the natural history of the qol of these patients .

myelolipoma is a rare benign tumor of adrenal gland and rarer in children . myelolipoma contains adipose tissue and myeloid precursor producing white blood cells ( wbc ) , red blood cells ( rbc ) and megakaryocytes . asymptomatic tumor does not require treatment whereas symptomatic tumor needs operation . we are reporting a rare adrenal myelolipoma in a child with review of literature .

adrenal myelolipoma is a tumor - like lesion composed of variable amounts of mature adipose tissue and bone marrow elements . in spite of wbc and rbc precursors such as megakaryocytes giercke in 1905 first described and 24 years later , oberling coined the term myelolipoma. only two cases under the age of 16 are reported and to the best of our knowledge , this patient is the youngest pediatric case of adrenal myelolipoma reported in the english literature . an 11-year - old girl presented with recurrent pain abdomen for 1 year , which was gradually increasing in frequency and severity for last 5 months . ultrasonography ( usg ) of abdomen showed a well - encapsulated and hypo echoic lesion in right suprarenal region measuring 27.1 24.3 mm . contrast enhanced computed tomography ( cect ) scan of abdomen confirmed the lesion to be arising from right adrenal gland with fat density measuring 1.8 2.3 cm [ figure 1 ] . gross examination of the specimen showed reddish yellow colored friable mass , soft in consistency ; and on microscopy , islands of hematopoietic tissue were found scattered among the fat cells [ figure 2 ] . cect reveal small fat containing lesion in right adrenal suggestive of myelolipoma histopathology showing fat lobules ( f ) and megakaryocyte ( m ) adrenal myelolipoma is a tumor - like lesion composed of variable amounts of mature adipose tissue and bone marrow elements . in spite of wbc and rbc precursors such as megakaryocytes myelolipoma may occur in retroperitoneum around kidney and pre - sacral space or in liver ; however , adrenal gland is the most common site . most of the cases of myelolipomas are asymptomatic . usually diagnosed incidentally at imaging for other purposes and the reported incidence are reaching up to 7% of the adrenal masses . majority of incidentally discovered myelolipomas are small and asymptomatic though large symptomatic lesions are also reported but in adult population . symptoms may be due to large tumor causing the pressure effect , tumor necrosis and intralesional hemorrhage . malignant potential of adrenal myelolipoma has not been reported . in our study , the child presented with pain abdomen and , on investigation diagnosis was confirmed . the lesion is itself hormonally inactive but may be associated with metabolically active adrenal lesion like cushing syndrome , conn 's syndrome , congenital adrenal hyperplasia etc . myelolipoma can be regarded as an exception to the mandatory metabolic work - up of a newly discovered adrenal mass . large myelolipoma usually associated with hematological disorders like hereditary spherocytosis , thalassemia intermedia , thalassemia major , sickle cell anemia and usually bilateral if we get a case of large or bilateral adrenal myelolipoma we have to investigate for chronic hematological disorders . before removal the of tumor , it may be necessary for bone marrow transplantation to avoid aggravation of hemolytic anemia . most accepted theory regarding etiologies are that it arises in response to infection , stress or necrosis causing metaplasia of reticuloendothelial cells of the capillaries of adrenal gland . focal fat density , peripheral calcification and hemorrhage within the tumor can be better identified with cect scan . myelolipoma contains adipocytes with hematopoietic elements , consisting of myeloid and erthyroid precursors , as well as , megakaryocytes . these hematopoietic elements are scattered in the adrenal tissue and separated by sheets and large clusters of mature adipocytes admixed with hemorrhagic foci . though open surgery is the classical method , laparoscopic adrenalectomy has now become the standard of care for the treatment of functioning and non - functioning adrenal tumors , introduced by gagner in 1992 . small asymptomatic myelolipomas can be treated conservatively but the symptomatic one should be treated with surgical excision and/or adrenalectomy .

various anatomic anomalies have been considered the causes of nutcracker syndrome ( ncs ) . posterior ncs refers to the condition , in which vascular narrowing was secondary to the compression of the retroaortic left renal vein while it is crossing between the aorta and the vertebral column . here , we report an unusual case of posterior ncs associated with a complicated malformation of the interrupted left inferior vena cava with azygos continuation and retroaortic right renal vein , diagnosed by both color doppler ultrasonography and ct angiography .

nutcracker syndrome is generally referred to as the compression of the left renal vein between the superior mesenteric artery and aorta ( 1 ) . this compression leads to lrv hypertension and results in direct communication of the renal vein with the calyceal cavity and the varicosities of the collateral pathway of the lrv ( 2 ) . renal varicosities or abnormal branching of the superior mesenteric artery from the aorta are the most common factors considered responsible for this condition ( 1 , 2 ) . the other causes , such as compression of the retroaortic lrv between the aorta and spine ( which is referred to as ' posterior ncs ' in contrast to the more common ' anterior ncs ' ) or aberrant right renal artery constricting the lrv , are uncommon ( 3 , 4 ) . however , three recently reported cases of nutcracker phenomenon associated with the left inferior vena cava ( livc ) suggest that some anomalies of the inferior vena cava ( ivc ) can also impair lrv blood outflow and result in ncs ( 4 - 6 ) . here we reported a case of complicated malformation of livc with interrupted ivc and azygos continuation and retroaortic right renal vein causing posterior ncs , which has been diagnosed by both color doppler ultrasound and computed tomographic angiography ( cta ) . to the best of our knowledge , this is the first report of an association between a complicated anomaly of the livc and ncs . a 42-year - old female patient was referred to our hospital for renal ultrasonographic scanning . she had a past medical history of intermittent microscopic hematuria or clots for nearly 10 years ' duration . the morphology of urinary red cells by phase - contrast microscopy revealed 53% dysmorphic cells . however , oblique transverse scanning of the upper abdomen showed two tubular structures on both sides of the abdominal aorta above the level of the renal vein . we traced the vessels in an effort to scrutinize every segment of these two tubular structures and found that the right common iliac vein crossed the vertebra and formed the left - sided infrarenal ivc ( l - ivc ) along with the left common iliac vein . the right renal vein ( rrv ) traveled retroaortically and joined the l - ivc ( fig . the post - anterior diameter of the hilar portion and that of the retroaortic stenotic portion of the rrv were 8.8 mm and 2.0 mm , respectively , revealed by grey - scale ultrasound , which , with peak velocities , was demonstrated by a spectral doppler of 26 cm / s and 87 cm / s , respectively . the diameter ratio and the peak velocity ratio between the dilated portion and the stenotic portion were 4.4 and 3.4 , respectively . after collecting blood from the right and left common iliac veins and the renal veins , the l - ivc crossed the midline retroaortically and flowed into the right - sided suprarenal ivc ( r - ivc ) with turbulent , high - velocity flow ( fig . the diameters of the l - ivc at the level of the lrv and the dilated portion of the lrv near the hilus and the diameter of the ivc at the stenotic portion were 18.6 mm , 14.3 mm and 2.3 mm , respectively , with the peak velocities of 30 cm / s , 27 cm / s and 149 cm / s , respectively . correspondingly , the diameter ratio between the dilated portion of lrv and the stenotic portion of l - ivc was 6.2 , accompanied by the peak velocity ratio of 5.5 . 1d ) . below the level of the rrv , no tubular structure was found at the right lateral portion of the aorta . the results demonstrated that the rrv traveled retroaortically to the l - ivc . after collecting blood from the right and left common iliac veins and the renal veins , the l - ivc traversed retroaortically to join the r - ivc above the level of the celiac artery . the r - ivc ascended with the azygos instead of joining to the hepatic vein . in addition , paravertebral collateral veins in the right lumbar region traveled upward tortuously and drained into the r - ivc ( fig . 1e - g ) . the findings of both color doppler ultrasound and cta revealed that the anomaly of the ivc in our patient was livc with continuation of the azygos , existence of a retroaortic rrv , and absence of the posthepatic segment of the ivc ( fig . cystoscopy was performed to evaluate otherwise asymptomatic episodes of microscopic hematuria , which demonstrated no positive findings . analysis the data obtained by image findings , together with the patient 's typical history and other laboratory findings , she was diagnosed as the posterior ncs associated with a complicated malformation of the livc . the normal ivc is composed of four segments : hepatic , suprarenal , renal , and infrarenal . its evolutionary process involves the development , regression , and anastomosis of three pairs of fetal veins : the posterior cardinal veins , subcardinal veins , and supracardinal veins , in the embryonic life during the sixth to eighth week of gestation ( 7 ) . the livc is the result of the persistence of the left supracardinal vein with regression of the right supracardinal vein . moreover , this case was more complicated because of the continuation with the azygos , the existence of the retroaortic rrv , and the absence of the posthepatic segment of the ivc . consequently , the pathology may be accompanied by the atrophy of the right subcardinal vein , the persistence of the dorsal limb as well as the regression of the ventral limb of the right renal collar , and the failure to form the right subcardinal - hepatic anastomosis ( 7 ) . furthermore , interruption of the ivc with azygos continuation is often associated with congenital heart disease , polysplenia , and less commonly , asplenia . in our patient no congenital heart diseases or other anomalies were found , so we have reason to believe that this condition is extremely rare . diagnosis of the anomalies of ivc may have significant clinical implications , especially during retroperitoneal surgery or in the treatment of thromboembolic diseases ( 5 - 7 ) . the majority of abnormal ivcs are clinically asymptomatic and only recognized incidentally during radiologic investigations performed for other reasons or during surgeries on the aorta or retroperitoneal structures or during postmortem examinations . in our literature review , we found only three cases of the anomalies of the ivc presenting as ncs . ( 5 ) first described data on an 80-year - old woman , who had a liver metastatic adenocarcinoma , and was incidentally found to have the anomaly of the livc . the authors believed that the compression of the livc between the aorta and superior mesenteric artery was responsible for her hematuria , a symptom of ncs . regardless of the various causes of ncs , an outlet obstruction of the lrv is the outcome of ncs . its typical symptoms , such as hematuria , left flank and abdominal pain , and varicoceles , are closely related to a backward venous renal hypertension due to the outflow impairment of the lrv and the development of compensating collateral circulation . in our present case , the aberrant vessels of the ivc resulted in two potential entrapments of the renal vein : 1 ) the pathway where the l - ivc drains into the r - ivc between the aorta and vertebra indirectly causing the hypertension of the lrv and 2 ) the location where the rrv runs to the l - ivc retroaortically . the data obtained by sonography revealed that the ratios of the diameter and of the peak velocity in rrv between the dilated portion and the stenotic portion were 4.4 and 3.4 , respectively , whereas those between the dilated segment of lrv and the narrowest segment of l - ivc were 6.2 and 5.5 , respectively . ct angiography also demonstrated the existence of obvious dilatation of lrv , which we believed to be the main factor of the occurrence of the hematuria . in addition , the distension of the lrv and tortuously paravertebral collateral veins in the right lumbar region could explain the patient 's feeling of a continuous dull lumbago for a long time . clinically , the diagnosis of ncs is based on the assessment of the pathologic anatomy and physiology ( 8) . the newer imaging modalities such as cta and magnetic resonance angiography have powerful abilities in providing accurate , three - dimensional reconstructive images in any desired plane and allowing better identification of these anomalies . spectral doppler ultrasound can give more details of the hemodynamics of the lrv at the site of compression . the case we presented here was first diagnosed by color doppler ultrasound and then identified by cta scanning with a high - quality delineated picture of the anomaly of the ivc . the dilated lrv as well as the l - ivc draining into the r - ivc in our patient were clearly visualized by ct and color doppler ultrasound . kim 's study showed that the cutoff values for ncs as the ratios of both antero - posterior diameter and peak velocity between the distended and narrowed segments of the lrv are greater than 5 , with sensitivity of 69% , 80% and specificity of 89% , 94% , respectively . but when combined with the cutoff value of more than 5 of the ratio of the antero - posterior diameter and the ratio of peak velocities divided by 2 , the sensitivity and the specificity can be elevated to 90% and 100% , respectively ( 9 ) . in our case , both the diameter ratio and the peak velocity ratio between the dilated segment of lrv and the narrowest segment of l - ivc were greater than 5 with a combined value of 5.9 . moreover , the morphology of urinary red cells is less than 75% , which is thought to be non - specific in nature ( 10 ) . in review of our patient 's typical history and clinical and radiologic findings , the diagnosis was most likely posterior ncs associated with complicated malformation of the livc . in conclusion , a congenital anomaly of the ivc , although rare , may be one of the contributing factors for the development of ncs . with the aid of ultrasonography and the newer cross - sectional imaging modalities , variant venous anatomy should be identified and its clinical significance , which had not been mentioned in previous reports , should be evaluated thoroughly .

objectiveto evaluate the feasibility and clinical and angiographic outcomes of stent - assisted embolization for complex middle cerebral artery ( mca ) aneurysms.methodsthe records of 23 consecutive patients with 24 mca aneurysms , who underwent stent - assisted embolization of the aneurysm , were retrospectively evaluated.resultsfifteen aneurysms were treated with one stent and 8 were treated using more than two stents ( 5 a stent - within - a - stent , 1 triple stents , and two y - stent ) . angiographically , complete or near complete occlusion was achieved in 15 aneurysms ( 65.2% ) , residual neck in five ( 21.7% ) , and residual aneurysm in three ( 13.1% ) . five aneurysms demonstrated thrombosis within the stent during the procedure and hospitalization , and were resolved by intraarterial and intravenous tirofiban injection . symptomatic thromboembolic complications were developed in five patients and permanent deficits demonstrated in two patients with modified rankin scale 1 and 2 , respectively . treatment - related permanent morbidity and mortality rates were 8.3% and 0% with relatively high complication rate . angiographic follow - up was available in 17 aneurysms at 6 - 31 months ( mean , 13.2 months ) and showed stable or improved in 15 ( 88.2% ) and major and minor recurrence in one , respectively.conclusioncomplex mca aneurysms could be treated by stent - assisted coiling and showed lower recanalization rate during mid - term follow - up by effective flow diversion due to various stent - assisted techniques . our results warrant further study with a longer follow - up period in a larger sample .

since the international subarachnoid trial , coil embolization of aneurysms is now accepted as a primary optional treatment19 ) . however , endovascular treatment ( evt ) of middle cerebral artery ( mca ) aneurysms has been less often applied in many institutions because of the feasibility of surgical clipping afforded by the superficial anatomy of mca aneurysms and favorable outcomes8,24 ) . a large series of evt of mca aneurysms was recently reported with favorable results and a low recurrence rate4,11,27 ) , but evt of wide - necked and fusiform mca aneurysms remains still challenging . after self - expandable stents became available , many of complex aneurysms have been treated by coil embolization1,3,17,18,21,23 ) . to our knowledge , there are a few reports regarding stent - assisted evt of mca aneurysms , but they treated complex mca aneurysms with one self expandable intracranial stent6,26,29 ) . the aim of this study was to evaluate the feasibility and clinical and angiographic outcomes of stent - assisted coiling for complex mca aneurysms using more than one stent . the institutional review board of our hospital approved this retrospective study , and the requirement for written informed consent was waived . we treated 101 mca aneurysms with coil embolization in 98 patients between april 2006 and august 2011 . twenty - four aneurysms in 23 patients were treated with stent - assisted coil embolization . our study involved 13 males and 10 females , and the mean age of the patients was 56.9 years ( range , 42 - 70 years ) . nineteen of 24 were incidental aneurysms that had not been treated , and two were recanalized aneurysms previously coiled , and two were recurred after surgical clipping . the number of unruptured aneurysms were 23 and ruptured aneurysm was only one which was ruptured after surgical clipping . the mean size of the aneurysms was 7 mm ( range , 3 - 13.4 mm ) , and mean size of the aneurysm neck was 6.2 mm ( range , 2.4 - 12.7 mm ) . the mean sac - to - neck ratio ( snr ) was 1.02 ( range , 0.53 - 1.45 ) and the mean height - to - neck ratio was 0.75 ( range , 0.48 - 1.3 ) . all aneurysms had wide neck and/or shallow depths , and six aneurysms were " ultra - wide necked " circumferential aneurysm which is involving more than 180 degree of parent artery lumen with no visible neck . in circumferential five aneurysms had incorporated parent vessel branch . there were 17 aneurysms in mca bifurcation , 3 proximal inferior divisional branch , 2 distal m1 , and one proximal superior divisional branch . patients were premedicated with 75 mg of clopidogrel ( plavix ; bristol - myers squibb , new york , ny , usa ) and 100 mg of aspirin for 5 - 7 days . if antiplatelet premedication was not done , on a day before treatment , patients were premedicated with a loading dose of clopidogrel ( 300 mg ) and aspirin ( 100 mg ) . after coiling , patients were then kept under dual antiplatelet therapy daily for 6 months ( clopidogrel , 75 mg , and aspirin , 100 mg ) followed by aspirin daily alone for 12 months . in ruptured aneurysm , all procedures were performed using a monoplane and biplane angiographic system ( integris v-5000 or allura xper , philips healthcare , best , the netherlands ) with the patient under local anesthesia . anticoagulation was initiated by injection of a bolus of 3000 to 5000 iu heparin intravenously at the beginning of the procedure , followed by continuous infusion of heparin at the rate of 1000 iu / h . activated coagulation time was maintained between two and three times the baseline value during and for the next 24 to 48 hours after the procedure . in one ruptured aneurysm , a bolus of 3000 iu heparin was injected intravenously after two coils were inserted in the aneurysm , and followed by same heparinization . endovascular coiling was performed via a single femoral route in all cases with an envoy 6 f ( cordis endovascular systems , miami lakes , fl , usa ) or a shuttle 6 f guiding catheter ( cook , inc . , after completion of coiling , a closing device ( angioseal ; st jude medical , st paul , mn , usa , or perclose ; abott , diegem , belgium ) was used to seal the access site in all cases . we initially tried to treat all complex aneurysms using single or double microcatheter or balloon - assisted techniques without stent - assistance . if parent artery or branching vessel is compromised by frame coils or coil frame is unstable , we performed stent - assisted coiling during same procedure . fifteen aneurysms were treated using one self - expandable intracranial stent ( neuroform stent , boston scientific , natick , ma , usa ; or enterprise self - expanding stent , cordis , miami lakes , fl , usa ) . a stent placed into the parent vessel , bridging the aneurysm neck . stent placement was performed either before coiling ( taking advantage of the open cell design of neuroform stents ) or with the jailing technique ( using enterprise stents ) when the size of the artery allowed safe navigation of two simultaneous microcatheters . in selected cases , the stent was deployed after coiling at the end of the procedure with the aim of securing the parent artery . six aneurysms were treated with a stent - within - a - stent ( overlapping two stents ) when aneurysm treatment was incomplete with one stent due to very wide neck or fusiform aneurysm for more insertion of coils and effective flow diversion . the stent - within - a - stent technique involves stent - assisted coiling followed by overlapping stent(s ) . a 0.021-inch microcatheter is navigated across the aneurysm neck portion to a distal branch of the parent artery . a second microcatheter is inserted into the aneurysm sac , and the first enterprise stent is deployed , bridging the aneurysm neck . after the first stent is deployed , the microcatheter is re - advanced to the initial position over the stent - loading wire , which is left in situ within the deployed stent . when the first deployed stent was neuroform stent , a micorwire was re - advanced through the deployed stent into the m2 branch and the microcatheter was repositioned in the distal portion of the first deployed stent . then coil embolization is performed as compactly as possible , including the aneurysm neck portion , which encompasses the parent artery more than 180 degrees . finally , a second enterprise stent is introduced and is deployed in an overlapping manner . two mca bifurcation aneurysms were treated with y - stent if one stent was insufficient for covering the aneurysm neck which was involving both branches . a microcatheter was navigated into the mca bifurcation aneurysm with or without frame coiling and another microcatheter was placed in the distal superior or inferior divisional branch at the most acute angle from the mca bifurcation . a 021 prowler select plus microcatheter was placed at the distal mca through a long exchangeable wire , and the first stent was deployed from the m2 and the m1 of mca . the second stent was navigated through the interstices of the initial stent with its distal end in the another bifurcation branch of mca and the proximal portion overlapping the first stent in the m1 of mca . coiling was performed using axium coils ( ev3 , plymouth , ma , usa ) , guglielmi detachable coils and target coils ( boston scientific , natick , ma , usa ) , hydrocoils ( microvention , aliso viejo , ca , usa ) , or a combination of coils . in the event of a periprocedural thromboembolic complication , tirofiban ( aggrastat , medicure pharma , somerset , nj , usa ) was slowly injected intra - arterially as a loading dose ( 500 mcg/10 ml ) and then administered intravenously , at an initial rate of 0.4 mcg / kg / min for 30 minutes and then continued at 0.1 mcg / kg / min . the clinical outcomes of patients were evaluated during hospitalization after procedure and then re - evaluated at the first time of angiographic control , which was generally performed at 6 - 12 months by dsa . additional controls by mra were then scheduled at 2 years , 3 years , and 5 years . in our institution , the mra sequences ( time - of - flight ) were analyzed by using source images ; maximum - intensity - projection and contrast enhanced mra images were irrelevant due to major artifacts caused by the stents . in cases of unreliable mr imaging findings , dsa was performed . morbidity - mortality rates and angiographic results were retrospectively analyzed at initial and midterm follow - ups . we used the raymond classification scale22 ) to compare the initial and midterm angiographic results . we defined the midterm results as follows : 1 ) a stable result showed no change from the initial results ; 2 ) the improved state demonstrated further occlusion of the remnant neck or residual aneurysm ; 3 ) a minor recurrence revealed no need for retreatment ( aneurysms changed from the class 1 to class 2 ) ; and 4 ) a major recurrence required requiring retreatment ( aneurysms changed from class 1 to class 3 or from class 2 to class 3 ) . the mean size of the aneurysms was 7 mm ( range , 3 - 13.4 mm ) , and mean size of the aneurysm neck was 6.2 mm ( range , 2.4 - 12.7 mm ) . the mean sac - to - neck ratio ( snr ) was 1.02 ( range , 0.53 - 1.45 ) and the mean height - to - neck ratio was 0.75 ( range , 0.48 - 1.3 ) . all aneurysms had wide neck and/or shallow depths , and six aneurysms were " ultra - wide necked " circumferential aneurysm which is involving more than 180 degree of parent artery lumen with no visible neck . in circumferential aneurysm , five aneurysms had incorporated parent vessel branch . there were 17 aneurysms in mca bifurcation , 3 proximal inferior divisional branch , 2 distal m1 , and one proximal superior divisional branch . patients were premedicated with 75 mg of clopidogrel ( plavix ; bristol - myers squibb , new york , ny , usa ) and 100 mg of aspirin for 5 - 7 days . if antiplatelet premedication was not done , on a day before treatment , patients were premedicated with a loading dose of clopidogrel ( 300 mg ) and aspirin ( 100 mg ) . after coiling , patients were then kept under dual antiplatelet therapy daily for 6 months ( clopidogrel , 75 mg , and aspirin , 100 mg ) followed by aspirin daily alone for 12 months . in ruptured aneurysm , all procedures were performed using a monoplane and biplane angiographic system ( integris v-5000 or allura xper , philips healthcare , best , the netherlands ) with the patient under local anesthesia . anticoagulation was initiated by injection of a bolus of 3000 to 5000 iu heparin intravenously at the beginning of the procedure , followed by continuous infusion of heparin at the rate of 1000 iu / h . activated coagulation time was maintained between two and three times the baseline value during and for the next 24 to 48 hours after the procedure . in one ruptured aneurysm , a bolus of 3000 iu heparin was injected intravenously after two coils were inserted in the aneurysm , and followed by same heparinization . endovascular coiling was performed via a single femoral route in all cases with an envoy 6 f ( cordis endovascular systems , miami lakes , fl , usa ) or a shuttle 6 f guiding catheter ( cook , inc . , after completion of coiling , a closing device ( angioseal ; st jude medical , st paul , mn , usa , or perclose ; abott , diegem , belgium ) was used to seal the access site in all cases . we initially tried to treat all complex aneurysms using single or double microcatheter or balloon - assisted techniques without stent - assistance . if parent artery or branching vessel is compromised by frame coils or coil frame is unstable , we performed stent - assisted coiling during same procedure . fifteen aneurysms were treated using one self - expandable intracranial stent ( neuroform stent , boston scientific , natick , ma , usa ; or enterprise self - expanding stent , cordis , miami lakes , fl , usa ) . stent placement was performed either before coiling ( taking advantage of the open cell design of neuroform stents ) or with the jailing technique ( using enterprise stents ) when the size of the artery allowed safe navigation of two simultaneous microcatheters . in selected cases , the stent was deployed after coiling at the end of the procedure with the aim of securing the parent artery . six aneurysms were treated with a stent - within - a - stent ( overlapping two stents ) when aneurysm treatment was incomplete with one stent due to very wide neck or fusiform aneurysm for more insertion of coils and effective flow diversion . the stent - within - a - stent technique involves stent - assisted coiling followed by overlapping stent(s ) . a 0.021-inch microcatheter is navigated across the aneurysm neck portion to a distal branch of the parent artery . a second microcatheter is inserted into the aneurysm sac , and the first enterprise stent is deployed , bridging the aneurysm neck . after the first stent is deployed , the microcatheter is re - advanced to the initial position over the stent - loading wire , which is left in situ within the deployed stent . when the first deployed stent was neuroform stent , a micorwire was re - advanced through the deployed stent into the m2 branch and the microcatheter was repositioned in the distal portion of the first deployed stent . then coil embolization is performed as compactly as possible , including the aneurysm neck portion , which encompasses the parent artery more than 180 degrees . finally , a second enterprise stent is introduced and is deployed in an overlapping manner . two mca bifurcation aneurysms were treated with y - stent if one stent was insufficient for covering the aneurysm neck which was involving both branches . a microcatheter was navigated into the mca bifurcation aneurysm with or without frame coiling and another microcatheter was placed in the distal superior or inferior divisional branch at the most acute angle from the mca bifurcation . a 021 prowler select plus microcatheter was placed at the distal mca through a long exchangeable wire , and the first stent was deployed from the m2 and the m1 of mca . the second stent was navigated through the interstices of the initial stent with its distal end in the another bifurcation branch of mca and the proximal portion overlapping the first stent in the m1 of mca . coiling was performed using axium coils ( ev3 , plymouth , ma , usa ) , guglielmi detachable coils and target coils ( boston scientific , natick , ma , usa ) , hydrocoils ( microvention , aliso viejo , ca , usa ) , or a combination of coils . in the event of a periprocedural thromboembolic complication , tirofiban ( aggrastat , medicure pharma , somerset , nj , usa ) was slowly injected intra - arterially as a loading dose ( 500 mcg/10 ml ) and then administered intravenously , at an initial rate of 0.4 mcg / kg / min for 30 minutes and then continued at 0.1 mcg / kg / min . the clinical outcomes of patients were evaluated during hospitalization after procedure and then re - evaluated at the first time of angiographic control , which was generally performed at 6 - 12 months by dsa . additional controls by mra were then scheduled at 2 years , 3 years , and 5 years . in our institution , the mra sequences ( time - of - flight ) were analyzed by using source images ; maximum - intensity - projection and contrast enhanced mra images were irrelevant due to major artifacts caused by the stents . in cases of unreliable mr imaging findings , dsa was performed . morbidity - mortality rates and angiographic results were retrospectively analyzed at initial and midterm follow - ups . we used the raymond classification scale22 ) to compare the initial and midterm angiographic results . we defined the midterm results as follows : 1 ) a stable result showed no change from the initial results ; 2 ) the improved state demonstrated further occlusion of the remnant neck or residual aneurysm ; 3 ) a minor recurrence revealed no need for retreatment ( aneurysms changed from the class 1 to class 2 ) ; and 4 ) a major recurrence required requiring retreatment ( aneurysms changed from class 1 to class 3 or from class 2 to class 3 ) . a total of 23 patients having 24 mca aneurysms were eligible for evt with the stent assisted coiling technique . treatment techniques , initial and follow - up angiographic outcomes , and treatment related complication after stent assisted coiling of mca aneurysms are shown in table 1 . fifteen aneurysms were treated by the use of a single stent , six aneurysms by a stent - within - a - stent procedure , and two using the y - stent technique . twenty - three aneurysms were successfully treated with a stent - assisted technique ( 95.9% ) and one aneurysm embolization was failed due to an inability to catheterize the inferior divisional branch due to acute angle and complex geometry . in - stent thrombosis was noted in five patients ( 20.8% ) with symptomatic thromboembolism in two patients , and they showed complete or nearly complete resolution of the thrombi after intra - arterial and intravenous administration of glycoprotein iib / iiia inhibitor ( tirofiban , 12.5 to 25 mg ) . two symptomatic patients with dysarthria and left side weakness showed complete recovery without permanent deficit . one aneurysm was treated with single stent technique and achieved complete embolization of the mca bifurcation aneurysm , but the superior divisional branch was occluded due to in - stent thrombosis . another stent was deployed within the stent after 20 minutes following partial recanalization of the occlusion by tirofiban injection and immediate control angiogram showed complete recanalization of the superior divisional branch ( fig . immediate thromboembolic complication was seen in 5 patients ( 20.8% ) during or after procedure showing motor weakness ( grade ii - iv ) , dysarthria , and facial weakness . two symptomatic thromboembolisms were resolved by tirofiban injection and one left side weakness was disappeared after conservative treatment . permanent morbidity was noted in 2 patients ( 8.3% ) with partial aphasia and grade iii motor weakness [ ( modified rankin scale ( mrs ) 1 and 2 ] . periprocedural thromboembolic complication was noted in 3 single stent - assisted coiling , 1 a stent - within - a - stent technique , and 1 y - stent technique . intraprocedural aneurysm rupture occurred in one patient who was treated with a single - stent assisted technique . the rupture was immediately controlled by the insertion of further coils and left no sequelae except for transient post - procedural headaches . postembolization control angiograms revealed complete occlusion in 15 ( 65.2% ) , residual neck in 5 ( 21.7% ) , and a residual aneurysm in 3 ( 13.1% ) patients . clinical follow - up was available in all patients ( mean , 28 months ; range , 5 - 60 months ) . there were no cases of sah and no deaths by the recurrence of the coiled aneurysms . none of the surviving 23 patients had any additional deterioration of functional neurological outcomes ( mrs , 0 - 2 ) . follow - up angiography was available in 17 of the 23 treated aneurysms ( 73.9% ) by using either mra ( n=5 , 29.4% ) or dsa ( n=12 , 70.6% ) at a mean period of 13.2 months ( 6 - 31 months ) . an artifact of the stent disturbed mra interpretation in two cases for which dsa was performed . one major recurrent aneurysm treated by the stent - within - a - stent technique was retreated using a stent - assisted technique without any complications ( fig . fifteen aneurysms were treated by the use of a single stent , six aneurysms by a stent - within - a - stent procedure , and two using the y - stent technique . twenty - three aneurysms were successfully treated with a stent - assisted technique ( 95.9% ) and one aneurysm embolization was failed due to an inability to catheterize the inferior divisional branch due to acute angle and complex geometry . in - stent thrombosis was noted in five patients ( 20.8% ) with symptomatic thromboembolism in two patients , and they showed complete or nearly complete resolution of the thrombi after intra - arterial and intravenous administration of glycoprotein iib / iiia inhibitor ( tirofiban , 12.5 to 25 mg ) . two symptomatic patients with dysarthria and left side weakness showed complete recovery without permanent deficit . one aneurysm was treated with single stent technique and achieved complete embolization of the mca bifurcation aneurysm , but the superior divisional branch was occluded due to in - stent thrombosis . another stent was deployed within the stent after 20 minutes following partial recanalization of the occlusion by tirofiban injection and immediate control angiogram showed complete recanalization of the superior divisional branch ( fig . immediate thromboembolic complication was seen in 5 patients ( 20.8% ) during or after procedure showing motor weakness ( grade ii - iv ) , dysarthria , and facial weakness . two symptomatic thromboembolisms were resolved by tirofiban injection and one left side weakness was disappeared after conservative treatment . permanent morbidity was noted in 2 patients ( 8.3% ) with partial aphasia and grade iii motor weakness [ ( modified rankin scale ( mrs ) 1 and 2 ] . periprocedural thromboembolic complication was noted in 3 single stent - assisted coiling , 1 a stent - within - a - stent technique , and 1 y - stent technique . intraprocedural aneurysm rupture occurred in one patient who was treated with a single - stent assisted technique . the rupture was immediately controlled by the insertion of further coils and left no sequelae except for transient post - procedural headaches . postembolization control angiograms revealed complete occlusion in 15 ( 65.2% ) , residual neck in 5 ( 21.7% ) , and a residual aneurysm in 3 ( 13.1% ) patients . clinical follow - up was available in all patients ( mean , 28 months ; range , 5 - 60 months ) . there were no cases of sah and no deaths by the recurrence of the coiled aneurysms . none of the surviving 23 patients had any additional deterioration of functional neurological outcomes ( mrs , 0 - 2 ) . follow - up angiography was available in 17 of the 23 treated aneurysms ( 73.9% ) by using either mra ( n=5 , 29.4% ) or dsa ( n=12 , 70.6% ) at a mean period of 13.2 months ( 6 - 31 months ) . an artifact of the stent disturbed mra interpretation in two cases for which dsa was performed . one major recurrent aneurysm treated by the stent - within - a - stent technique was retreated using a stent - assisted technique without any complications ( fig . since the advent of the detachable coil , endovascular coil embolization has been an important treatment method for both ruptured and unruptured aneurysms . however , coil embolization of aneurysms with wide neck and complex geometry remains a challenging procedure despite the use of several techniques such as balloon - assisted20,30 ) , double2 ) or multiple microcatheter12 ) , microcatheter protective15 ) , coil protective , and stent - assisted techniques1,3,6,7,17,18,21,23,26,29 ) . ever since the advent of self - expandable intracranial stents for coil embolization of aneurysms , especially closed cell design stent ( enterprise and leo stent , balt co. , montmorency , france ) , stent - assisted embolization has been widely used due to ease of deployment of the stent and high success rates10 ) . recently , many of complex aneurysms is treated by stent - assisted technique rather than balloon - assisted and multiple microcatheter techniques . stent - assisted embolization of the complex mca aneurysms was feasible , safe and durable , and could be considered as an alternative treatment26 ) . however , surgical clipping of mca aneurysms is preferred in many institutions due to relative ease and effectiveness of aneurysm clipping . the initial complete or near - complete embolization rate in our series ( 65.2% ) was similar to other studies ( 67 - 71%)6,26,29 ) . we performed the jailing technique in 17 aneurysms and repositioning of the microcatheter through the strut of the stent in the remnant neck or aneurysm during procedure if the jailed microcatheter was kickback in the parent artery . a stent - within - a - stent technique in six aneurysms allowed more compact packing and efficient flow diversion than single stent - assisted embolization . flow diverter might be used , especially in the m1 aneurysm without branching parent vessels , but we could not use them because those stents were not available in our country . in the present study , in - stent thrombosis ( 20.8% ) and the symptomatic thromboembolic complication rate ( 20.8% ) were high despite the use of dual antiplatelet therapy . we did n't clopidogrel and aspirin resistance test and high thromboembolic complication rate may be antiplatelet resistance . we also think that our initial high complication rate may arise from more complex procedures such as a stent - within - a - stent or y - stent technique than single stent - assisted technique . the permanent morbidity and mortality rates were decreased as result of complete thrombolysis by immediate tirofiban injection and leaved two mild disabilities ( mrs 1 and 2 ) . however , this morbidity - mortality rate ( 8.3% ) was higher than those of other series ( 0 - 12% ) ( table 2 ) and may not be acceptable for treatment method . during recent 3 years , 10 aneurysms was treated and showed no permanent morbidity and one immediate symptomatic complication . in this result , experience of neurointerventionist and careful case selection are important factors for reducing complication rate of stent - assisted coil embolization of the mca aneurysms . the recurrence rate for stent - assisted coil embolization of the aneurysms was relatively high in other studies ( non - stenting ) , ranging from 14.6% to 29%4,11,27 ) , and our results were either comparable ( 11.8% ) with previous stent - assisted coil embolization studies ( table 2 ) . coil embolization with a stent - within - a - stent technique is a feasible and reconstructive treatment option for ruptured dissecting aneurysms25 ) and effectively prevents rebleeding and regrowth of ruptured blood blister - like aneurysms without requiring sacrifice of the internal carotid artery14 ) . therefore , we expected high packing density and lower recurrence rates for large and wide - necked aneurysms while preserving the parent artery , especially ultra - wide necked circumferential mca aneurysms . in addition to the neck covering role of the stent - assisted technique during embolization , advantages of a stent - within - a - stent technique included prevention of possible protrusion of the coils into the parent artery after first stenting , coil insertion of a very wide - necked circumferential aneurysms due to poor discrimination of the parent artery and neck of the aneurysm due to complex anatomic geometry . although a down - the - barrel view may help to differentiate the parent artery lumen from coils8 ) , it is not always available as a working projection . another advantage is prevention of the recurrence of incomplete embolization of the aneurysm due to more effective flow diversion than the single stent , resultant aneurysmal thrombosis , and eventual endothelialization around the neck5,16,28 ) . finally , blood flow may be compromised due to in - stent thrombosis or coil protrusion within the parent vessel during coiling , so a second stent can easily restore blood flow through the stented parent artery if tirofiban injection could not recanalized sufficiently ( fig . lawson et al.13 ) reported that stent - assisted coiling causes the progression of occlusion and complete thrombosis of incompletely coiled aneurysms , possibly by a flow remodeling effect , and is therefore associated with lower rates of aneurysm recurrence . there was also a larger series ( 216 aneurysms ) that show a statistically significant lower recurrence rate of stenting ( 14.9% ) compare to coiling ( 33.5%)21 ) . hwang et al.9 ) insisted their study did not show that additional hemodynamic and biologic effects of stents designed for neck remodeling were sufficient to enhance progressive occlusion and to prevent the recanalization of unruptured aneurysms . their findings also suggested that stent placement provides no better long - term angiographic outcomes for unruptured aneurysms with an unfavorable configuration for coiling as compared with nonstent - assisted ( multiple microcatheter technique or balloon - assisted technique ) coil embolization groups . we believe that selection bias between the stenting and non - stenting groups in each study that were non - randomized and retrospective study , caused the discrepancies in results . we also believe that the most important factor for lower recanalization rate was complete embolization and compact packing of the aneurysms irrespective of the techniques used . another limitation was that we included no comparable non - stenting group or surgical clipping group with same geometry of the aneurysms because unruptured mcas are not treated by surgical clipping at our hospital . our limited number of angiographic follow up cases and limited follow up times were another major limitation . mca aneurysms with wide neck or fusiform could be treated by stent - assisted coiling with more than one stent , and showed good initial angiographic outcomes and lower recanalization rate during mid - term follow - up by effective flow diversion . our results warrant further study with a longer follow - up period in a larger sample .

ferromagnetic thin films of heusler compounds are highly relevant for spintronic applications owing to their predicted half - metallicity , that is , 100% spin polarization at the fermi energy . however , experimental evidence for this property is scarce . here we investigate epitaxial thin films of the compound co2mnsi in situ by ultraviolet - photoemission spectroscopy , taking advantage of a novel multi - channel spin filter . by this surface sensitive method , an exceptionally large spin polarization of ( ) % at room temperature is observed directly . as a more bulk sensitive method , additional ex situ spin - integrated high energy x - ray photoemission spectroscopy experiments are performed . all experimental results are compared with advanced band structure and photoemission calculations which include surface effects . excellent agreement is obtained with calculations , which show a highly spin polarized bulk - like surface resonance ingrained in a half metallic bulk band structure .

co2mnsi samples were prepared and investigated completely in situ in an ultrahigh vacuum cluster consisting of sputtering chambers , an molecular beam epitaxy ( mbe ) chamber , and a srups chamber equipped with a he gas discharge lamp ( h=21.2 ev ) and a hemispherical energy analyzer with multi - channel spin filter27 ( energy resolution 400 mev , sherman function s=0.420.05 ( ref . first , an epitaxial buffer layer of the heusler compound co2mnga ( 30 nm ) was grown on the mgo(100 ) substrate by radio frequency ( rf)-sputtering at room temperature . by an optimized additional annealing process at 550 c l21 order is obtained as shown by high energy electron diffraction ( rheed ) and x - ray diffraction ( xrd ) . . induced by the buffer layer the co2mnsi thin films show already some degree of l21 surface order as deposited . by additional annealing the order is improved as demonstrated for the film surface by rheed ( fig . a low annealing temperature of ta=300c results in a significantly increased intensity of the characteristic rheed l21 superstructure peaks . however , by xrd no ( 111 ) peak , which is indicative for l21 order , is observed for ta<400 c . this suggests that l21 order is present at the film surface , but not in the bulk of the thin film . for ta400 c the ( 111 ) peak appears in xrd . for ta500 c some ga from the buffer layer is observed by core - level haxpes to have diffused to the co2mnsi surface . the magnetic moments of all samples amount to 5 b per formula unit at 4 k and is reduced by 3% at room temperature , in agreement with theoretical predictions and experimental values measured on bulk samples29 . figure 2 shows in situ ups spectra of co2mnsi thin films annealed at different temperatures ta without spin analysis . the large acceptance angle of the spectrometer ( 10 ) and applied sample bias voltage of 10 v result in k|| values which cover the complete brillouin zone . the spectra of all samples are almost identical , only the broad hump at eef=2,900 mev vanishes and the peak at eef=1,150 mev is slightly broadened for the deposited and the ta=550 c sample . however , by spin analysis clear differences between the samples are revealed . figure 3 shows the spin polarization of mgo / co2mnga(30 nm)/co2mnsi(70 nm ) thin films annealed at different temperatures ta as measured by srups . a huge room temperature spin polarization of 9093% at the fermi energy at room temperature was obtained for samples annealed between 300 c and 450 c . in combination with the ups calculations discussed below , these exceptionally high values are the first direct observation of half - metallicity in the surface region of any heusler compound , which provide strong evidence for 100% spin polarization in the bulk of the thin films . with lower annealing temperatures the spin polarization is reduced at ef and slightly increased at higher binding energies , which can be explained by an energy broadening of the electronic states owing to reduced structural order . with higher annealing temperatures ups is a surface sensitive method and thus the results can not be directly associated with electronic bulk band structure properties . however , as will be shown below , band structure based calculations of photoemission spectra provide this link . as additional experimental input for such calculations , a comparison of spin - integrated ex situ haxpes with a photon energy of 6 kev of alox capped ( oxidation protection ) co2mnsi thin films and spin - integrated in situ ups ( uncapped films ) was carried out . owing to the increased information depth of haxpes , true surface states are typically not observed by this method . as shown in fig . 4 , the in situ spin - integrated ups and the haxpes results fundamentally agree although the information depth of both experiments varies from 2 nm to 20 nm . this provides evidence that true surface states like shockley or tamm states , which are mainly located at the first atomic layer30 , do not contribute to the ups data . we calculated the spin resolved bulk dos of co2mnsi using the spin polarized relativistic korringa kohn rostoker ( spr - kkr ) green function method implemented in the munich spr - kkr band structure programme package employing the perdew ernzerhof functional32 shifts the upper edge to higher energies , but leaves the lower edge almost unchanged . for a comparison of our experimental data with ups- and haxpes calculations this electronic structure provides the basis for a one - step model of photoemission , which includes all matrix - element effects , multiple scattering in the initial and final states33 , and all surface - related effects in the excitation process . we used a recently developed relativistic generalization for excitation energies ranging from about 10 ev to more than 10 kev ( ref . 34 ) realized in the full spin - density matrix formulation for the photocurrent35 . in fig . 4 the calculations and the experimental spin - integrated ups and haxpes results are compared . nearly quantitative agreement for both , uv and hard x - ray photon energies , is obtained . particularly with regard to the small dos just below the fermi energy the agreement of the calculations with the high ups and haxpes intensities in this energy range is remarkable and is traced back to a bulk - like surface resonance as will be discussed below . the obtained agreement between the spin - integrated ups / haxpes experiments and calculations based on a half metallic bulk band structure represents already evidence for half - metallicity . additional strong evidence is provided by the analysis of the srups data . for the surface region we can estimate the position of the lower band edge of the minority gap directly from the experimental data by taking the maximum of the derivative of the minority spin intensity with respect to the energy , which is found at eef 500 mev . from previous surface sensitive x - ray magnetic circular dichroism experiments we estimated the position of the upper band edge to be at eef+400 mev ( ref . 5 the highest experimentally obtained spin polarization is shown together with the spin polarization derived directly from the calculated dos , the calculated photoemission asymmetry including all broadening effects considering bulk contributions only , and the calculated photoemission asymmetry including surface - related effects . the correspondence between the dos and calculated pure bulk - like ups spectrum becomes clear , if the influence of intrinsic life time broadening owing to electronic correlations and included experimental energy resolution ( e=400 mev ) is considered . it is obvious that these broadening effects within the bulk calculations reduce the expected ups spin polarization although the dos is half - metallic . however true surface states contribute to the layer - resolved photocurrent with an intensity distribution that is nonzero for the first atomic layer only . consequently , their contribution to the total spectral weight decreases with increasing number of layers generating the photocurrent . thus in general with increasing photon energies the combined effect of energy - dependent cross - sections and larger inelastic mean free path results in a reduced weight of surface state photoemission . however , the situation is very different for co2mnsi , where we identified in our calculations a resonance on the ( 001)-surface , which is embedded in the bulk continuum with a strong coupling to the majority bulk states . in our case this surface resonance extends over the first six atomic layers , which is similar to the case of w(110 ) , where we found a surface resonance revealing a considerable bulk contribution35 as well . the spectral weight of this surface resonance is much larger than that of a true surface state resulting in a significant contribution to the total intensity even at hard x - ray energies . co2mnsi samples were prepared and investigated completely in situ in an ultrahigh vacuum cluster consisting of sputtering chambers , an molecular beam epitaxy ( mbe ) chamber , and a srups chamber equipped with a he gas discharge lamp ( h=21.2 ev ) and a hemispherical energy analyzer with multi - channel spin filter27 ( energy resolution 400 mev , sherman function s=0.420.05 ( ref . first , an epitaxial buffer layer of the heusler compound co2mnga ( 30 nm ) was grown on the mgo(100 ) substrate by radio frequency ( rf)-sputtering at room temperature . by an optimized additional annealing process at 550 c l21 order is obtained as shown by high energy electron diffraction ( rheed ) and x - ray diffraction ( xrd ) . . induced by the buffer layer the co2mnsi thin films show already some degree of l21 surface order as deposited . by additional annealing the order is improved as demonstrated for the film surface by rheed ( fig . a low annealing temperature of ta=300c results in a significantly increased intensity of the characteristic rheed l21 superstructure peaks . however , by xrd no ( 111 ) peak , which is indicative for l21 order , is observed for ta<400 c . this suggests that l21 order is present at the film surface , but not in the bulk of the thin film . for ta400 c the ( 111 ) peak appears in xrd . for ta500 c some ga from the buffer layer is observed by core - level haxpes to have diffused to the co2mnsi surface . the magnetic moments of all samples amount to 5 b per formula unit at 4 k and is reduced by 3% at room temperature , in agreement with theoretical predictions and experimental values measured on bulk samples29 . figure 2 shows in situ ups spectra of co2mnsi thin films annealed at different temperatures ta without spin analysis . the large acceptance angle of the spectrometer ( 10 ) and applied sample bias voltage of 10 v result in k|| values which cover the complete brillouin zone . the spectra of all samples are almost identical , only the broad hump at eef=2,900 mev vanishes and the peak at eef=1,150 mev is slightly broadened for the deposited and the ta=550 c sample . however , by spin analysis clear differences between the samples are revealed . figure 3 shows the spin polarization of mgo / co2mnga(30 nm)/co2mnsi(70 nm ) thin films annealed at different temperatures ta as measured by srups . a huge room temperature spin polarization of 9093% at the fermi energy at room temperature was obtained for samples annealed between 300 c and 450 c . in combination with the ups calculations discussed below , these exceptionally high values are the first direct observation of half - metallicity in the surface region of any heusler compound , which provide strong evidence for 100% spin polarization in the bulk of the thin films . with lower annealing temperatures the spin polarization is reduced at ef and slightly increased at higher binding energies , which can be explained by an energy broadening of the electronic states owing to reduced structural order . with higher annealing temperatures ups is a surface sensitive method and thus the results can not be directly associated with electronic bulk band structure properties . however , as will be shown below , band structure based calculations of photoemission spectra provide this link . as additional experimental input for such calculations , a comparison of spin - integrated ex situ haxpes with a photon energy of 6 kev of alox capped ( oxidation protection ) co2mnsi thin films and spin - integrated in situ ups ( uncapped films ) was carried out . owing to the increased information depth of haxpes , true surface states are typically not observed by this method . as shown in fig . 4 , the in situ spin - integrated ups and the haxpes results fundamentally agree although the information depth of both experiments varies from 2 nm to 20 nm . this provides evidence that true surface states like shockley or tamm states , which are mainly located at the first atomic layer30 , do not contribute to the ups data . we calculated the spin resolved bulk dos of co2mnsi using the spin polarized relativistic korringa kohn rostoker ( spr - kkr ) green function method implemented in the munich spr - kkr band structure programme package employing the perdew ernzerhof functional32 shifts the upper edge to higher energies , but leaves the lower edge almost unchanged . for a comparison of our experimental data with ups- and haxpes calculations this electronic structure provides the basis for a one - step model of photoemission , which includes all matrix - element effects , multiple scattering in the initial and final states33 , and all surface - related effects in the excitation process . we used a recently developed relativistic generalization for excitation energies ranging from about 10 ev to more than 10 kev ( ref . 34 ) realized in the full spin - density matrix formulation for the photocurrent35 . in fig . 4 the calculations and the experimental spin - integrated ups and haxpes results are compared . nearly quantitative agreement for both , uv and hard x - ray photon energies , is obtained . particularly with regard to the small dos just below the fermi energy the agreement of the calculations with the high ups and haxpes intensities in this energy range is remarkable and is traced back to a bulk - like surface resonance as will be discussed below . the obtained agreement between the spin - integrated ups / haxpes experiments and calculations based on a half metallic bulk band structure represents already evidence for half - metallicity . additional strong evidence is provided by the analysis of the srups data . for the surface region we can estimate the position of the lower band edge of the minority gap directly from the experimental data by taking the maximum of the derivative of the minority spin intensity with respect to the energy , which is found at eef 500 mev . from previous surface sensitive x - ray magnetic circular dichroism experiments we estimated the position of the upper band edge to be at eef+400 mev ( ref . 5 the highest experimentally obtained spin polarization is shown together with the spin polarization derived directly from the calculated dos , the calculated photoemission asymmetry including all broadening effects considering bulk contributions only , and the calculated photoemission asymmetry including surface - related effects . the correspondence between the dos and calculated pure bulk - like ups spectrum becomes clear , if the influence of intrinsic life time broadening owing to electronic correlations and included experimental energy resolution ( e=400 mev ) is considered . it is obvious that these broadening effects within the bulk calculations reduce the expected ups spin polarization although the dos is half - metallic . true surface states contribute to the layer - resolved photocurrent with an intensity distribution that is nonzero for the first atomic layer only . consequently , their contribution to the total spectral weight decreases with increasing number of layers generating the photocurrent . thus in general with increasing photon energies the combined effect of energy - dependent cross - sections and larger inelastic mean free path results in a reduced weight of surface state photoemission . however , the situation is very different for co2mnsi , where we identified in our calculations a resonance on the ( 001)-surface , which is embedded in the bulk continuum with a strong coupling to the majority bulk states . in our case this surface resonance extends over the first six atomic layers , which is similar to the case of w(110 ) , where we found a surface resonance revealing a considerable bulk contribution35 as well . the spectral weight of this surface resonance is much larger than that of a true surface state resulting in a significant contribution to the total intensity even at hard x - ray energies . as shown in fig . 5 , the inclusion of the complete surface - related photoexcitation in the ups calculation results in perfect agreement with the experiment . if the surface resonance were not present , half - metallic behaviour would persist but the finite experimental resolution in photoemission would hinder the observation of a high spin polarization . because the surface resonance is strongly coupled to the band structure of the bulk , this provides evidence for the validity of our calculated half metallic bulk band structure of co2mnsi . and , from the spintronics applications point of view it is the room temperature spin polarization in the thin film surface region , which is relevant . in conclusion , investigating optimized thin films of the compound co2mnsi by in situ srups , we were able to demonstrate for the first time half - metallicity in combination with directly measured ( ) % spin polarization at room temperature in the surface region of a heusler thin film . novel band structure and photoemission calculations including all surface - related effects show that the observation of a high spin polarization in a wide energy range below the fermi energy is related to a stable surface resonance in the majority band of co2mnsi extending deep into the bulk of the material . our results show that careful thin film preparation can indeed result in a high spin polarization with a sufficient degree of stability in a surface region of several atomic layers . in particular it shows that the observed tunnelling magnetoresistance values are not limited by the intrinsic spin polarization of the heusler alloy and that potentially much larger values can be obtained by carefully optimized growth . fundamentally our observation paves the way for most powerful future spintronic devices on the basis of heusler materials . m.j . initiated and coordinated the project and wrote the paper . a.k . and m.j

kawasaki disease is a generalized systemic vasculitis of unknown etiology involving medium and small size blood vessels throughout the body , virtually always involving the coronaries . in many part of this world , it is more common than rheumatic fever and viral myocarditis . here , we are reporting 5 cases with history suggestive of kawasaki disease in the early life , presenting with different coronary artery abnormalities . one of the patients had left main coronary artery cut - off , and the remaining 4 had some form of coronary artery aneurysms . 3 patients were given medical management , 1 patient had coronary bypass grafting , and the remaining 1 was planned for surgical correction . all the patients were stable on subsequent follow - ups .

mucocutaneous lymph node syndrome , which had been affecting infants and young children in japan . diagnosis requires fever and 4 out of 5 principal clinical features ( conjunctivitis ; erythematous lips , oral cavity ; palms and soles ; polymorphous exanthema of the body trunk ; swelling of the cervical lymph nodes ) or if fewer than 4 , patients should also have documented coronary artery disease diagnosed by echocardiography or coronary angiography . cardiac , particularly coronary artery complications are well - known fact in this syndrome . here , we are reporting 5 cases [ table 1 ] with past history of kawasaki disease presenting with coronary artery abnormalities . 7-year - old boy with documented history of kawasaki disease at the age of 1 year , treated conservatively without using intra - venous immunoglobulin ( ivig ) , presented to us with shortness of breath ( nyha class 3 ) . echocardiography revealed feature suggestive of dilated cardiomyopathy with left ventricular ejection fraction ( lvef ) of 30% . angiogram [ figure 1 ] revealed normal right coronary artery ( rca ) and left main coronary artery ( lmca ) cut - off at mid part with collateral filling from right coronary . further evaluation by cardiac stress testing revealed presence of provocable ischemia . after stabilization of heart failure , he was referred to cardiothoracic surgery for coronary artery bypass grafting ( cabg ) . non selective coronary angiogram showing normal rca and lmca cut - off from mid part with collateral filling from right system 11-year - old boy with history of kawasaki disease at 3 years of age treated without any ivig presented with non - specific chest discomfort . coronary angiography revealed aneurysm of proximal rca with internal diameter of 9.2 mm [ figure 2a ] . he was started with aspirin 5 mg / kg body weight and warfarin keeping inr between 2 to 2.5 . on more than 1 year follow - up ( a ) coronary angiogram showing aneurism of osteo - proximal rca without any feature of stenosis of lumen . ( b ) coronary angiogram showing osteo - proximal lmca aneurism involving upto bifurcation , without any flow limiting obstruction of lumens . ( c ) coronary angiogram showing aneurismal dilatation of lcx prior to 1st obtuse marginal ( om 1 ) branch followed by severe stenosis of lcx and om 1 15-year - old boy presented with exertional chest discomfort and occasional palpitation . he had a history of kawasaki disease at the age of 1.5 years . coronary angiogram revealed giant aneurysm of lmca with maximum diameter of 12 mm [ figure 2b ] . he was similarly put on oral aspirin and anti - coagulation therapy and keeping well on 6 months follow - up . 19-year - old male presented to us with exertional chest pain and dyspnea for last 6 months . he had a history of fever for 3 weeks - duration at the age of 4 years associated with conjunctival redness and skin rash over trunk . coronary angiography showed aneurysm of left circumflex ( lcx ) artery with maximum internal diameter measuring 8.8 mm , followed by severe stenosis of lcx and 1 obtuse marginal branch [ figure 2c ] . though no documents were available , his childhood exanthem was postulated to be kawasaki disease . on dobutamine stress echo he was kept on oral anti - coagulation , aspirin , and enalapril along with other anti - anginal medications and later referred for surgical correction in the form of aneurysm closure and bypass grafting to coronaries distal to stenosis . 6-year - old boy with history of kawasaki disease at the age of 3 years was complaining of occasional chest tightness . he had dilatation of lmca in the initial echocardiography , which persisted on serial evaluations . a 64 slice ct coronary angiography done this time revealed fusiform dilatation ( 5 mm diameter ) of lmca at its trifurcation up to ostio - proximal segment of left anterior descending ( lad ) artery without any plaque or stenosis [ figure 3a and b ] . he was continued with oral aspirin 5 mg/ kg body weight and was asymptomatic thereafter . ( a ) and ( b ) 64 slice ct coronary angiography showing fusiform dilatation ( 5 mm ) of lmca at its trifurcation upto osteo - proximal lad without any significant stenosis . 7-year - old boy with documented history of kawasaki disease at the age of 1 year , treated conservatively without using intra - venous immunoglobulin ( ivig ) , presented to us with shortness of breath ( nyha class 3 ) . echocardiography revealed feature suggestive of dilated cardiomyopathy with left ventricular ejection fraction ( lvef ) of 30% . angiogram [ figure 1 ] revealed normal right coronary artery ( rca ) and left main coronary artery ( lmca ) cut - off at mid part with collateral filling from right coronary . further evaluation by cardiac stress testing revealed presence of provocable ischemia . after stabilization of heart failure , he was referred to cardiothoracic surgery for coronary artery bypass grafting ( cabg ) . non selective coronary angiogram showing normal rca and lmca cut - off from mid part with collateral filling from right system 11-year - old boy with history of kawasaki disease at 3 years of age treated without any ivig presented with non - specific chest discomfort . coronary angiography revealed aneurysm of proximal rca with internal diameter of 9.2 mm [ figure 2a ] . he was started with aspirin 5 mg / kg body weight and warfarin keeping inr between 2 to 2.5 . on more than 1 year follow - up , he is doing fine with these medicines . ( a ) coronary angiogram showing aneurism of osteo - proximal rca without any feature of stenosis of lumen . ( b ) coronary angiogram showing osteo - proximal lmca aneurism involving upto bifurcation , without any flow limiting obstruction of lumens . ( c ) coronary angiogram showing aneurismal dilatation of lcx prior to 1st obtuse marginal ( om 1 ) branch followed by severe stenosis of lcx and om 1 coronary angiogram revealed giant aneurysm of lmca with maximum diameter of 12 mm [ figure 2b ] . he was similarly put on oral aspirin and anti - coagulation therapy and keeping well on 6 months follow - up . 19-year - old male presented to us with exertional chest pain and dyspnea for last 6 months . he had a history of fever for 3 weeks - duration at the age of 4 years associated with conjunctival redness and skin rash over trunk . coronary angiography showed aneurysm of left circumflex ( lcx ) artery with maximum internal diameter measuring 8.8 mm , followed by severe stenosis of lcx and 1 obtuse marginal branch [ figure 2c ] . though no documents were available , his childhood exanthem was postulated to be kawasaki disease . on dobutamine stress echo he was kept on oral anti - coagulation , aspirin , and enalapril along with other anti - anginal medications and later referred for surgical correction in the form of aneurysm closure and bypass grafting to coronaries distal to stenosis . 6-year - old boy with history of kawasaki disease at the age of 3 years was complaining of occasional chest tightness . he had dilatation of lmca in the initial echocardiography , which persisted on serial evaluations . a 64 slice ct coronary angiography done this time revealed fusiform dilatation ( 5 mm diameter ) of lmca at its trifurcation up to ostio - proximal segment of left anterior descending ( lad ) artery without any plaque or stenosis [ figure 3a and b ] . he was continued with oral aspirin 5 mg/ kg body weight and was asymptomatic thereafter . ( a ) and ( b ) 64 slice ct coronary angiography showing fusiform dilatation ( 5 mm ) of lmca at its trifurcation upto osteo - proximal lad without any significant stenosis . although most commonly found in asian population , particularly in japan , kawasaki disease occurs worldwide . in many areas , it is more common than rheumatic fever and viral myocarditis . there is suggestion that both genetic susceptibility and environmental factors may play a role in kawasaki disease . its rarity in the first few months of life and in adults suggests an agent to which the latter are immune and from which very young infants are protected by passive maternal antibodies . kawasaki disease has 2 phases : an acute phase lasting 1 to 2 weeks , followed by a chronic ( convalescent ) phase . kawasaki disease is a generalized systemic vasculitis involving medium and small size blood vessels throughout the body , virtually always involving the coronaries . progressive stenosis in the disease results from active remodeling with an intimal proliferation and neoangiogenesis ; the intima is markedly thickened and consists of linearly - arranged microvessels , a layer that is rich in smooth muscle cells , and fibrous layers . several growth factors are prominently expressed at the inlet and outlet of aneurysms where they are activated by high shear stress . they display normal findings on electrocardiograms and stress tests and are at low risk of subsequent myocardial infarction or sudden death . sometimes , aneurysms persist and become occlusive , thereby increasing the risk of myocardial infarction or sudden death . the risk factors to predict the presence of coronary aneurysms in kawasaki disease include boys less than 1 year of age , fever lasting longer than 2 weeks , elevated erythrocyte sedimentation rate persisting for more than 4 weeks , and palpable axillary artery aneurysms . the size less than 4 mm in diameter regresses spontaneously within a short time , whereas those larger than 8 mm in diameter are often associated with a stenotic lesion . factors associated with regression of aneurysm in kawasaki disease include age less than 1 year , saccular as opposed to fusiform morphology , and distal location . myocardial infarction can occur early or late after the acute phase ; therefore , patients should be counseled to avoid atherosclerotic risk factors , and the progress of these patients should be followed into adulthood . the longer the aneurysms or stenotic lesions persist , the less likely they are to resolve . massive thrombosis of the aneurysm is seen predominantly in the right coronary artery , usually within 1 year after disease onset . progressive localized stenosis at the aneurysm inlet or outlet is seen predominantly in the left coronary artery , again usually within 1 year . our patients presented with coronary complications 3 to 14 years after the initial attack of kawasaki disease . many a time , the initial diagnosis is either missed or not documentable as is the case in our 4 patient . the laboratory findings of incomplete cases appear to be similar to those of classic cases . therefore , although laboratory findings in kawasaki disease are non - diagnostic , they may prove useful in heightening or reducing the suspicion of incomplete kawasaki disease . the goal in treating kawasaki disease is to control acute inflammation and to prevent serious cardiovascular complications , such as coronary artery disease . during the acute phase of illness , aspirin is administered at 80 to 100 mg / kg per day in 4 divided doses together with ivig 2 g/ kg in a single infusion . high dose aspirin continued until day 14 of illness or 48 to 72 hours after fever cessation . when high - dose aspirin is discontinued , clinicians begin low - dose aspirin ( 3 to 5 mg / kg per day ) and maintain it until the patient shows no evidence of coronary changes by 6 to 8 weeks after the onset of illness . for children who develop coronary abnormalities other forms of treatment including glucocorticoids , pentoxyfylline , plasma exchange , abciximab , infliximab , cyclophosphamide etc . the most common anti - thrombotic regimen for patients with giant aneurysms is low - dose aspirin together with warfarin , maintaining an international normalized ratio ( inr ) of 2.0 to 2.5 . the treatment of acute coronary occlusion in patients with kawasaki disease should target multiple steps in the coagulation cascade . because no randomized controlled trials have been performed in children , the treatment of infants and children with coronary thrombosis should be done similar to adults . attempts at excision or plication of the coronary artery aneurysm have not been successful and have caused deaths . surgical management in kawasaki disease coronary revascularization is recommended for patients with giant or multiple coronary artery aneurysms or significant stenosis . overall mortality is low for such operations , and graft target sites are easily accessible since most coronary artery lesions occur in proximal segments with little distal involvement . in situ arterial grafts the indications for coronary bypass graft procedures in children have not been established in clinical trials , but such surgery should be considered when reversible ischemia is present on stress - imaging test results , the myocardium to be perfused through the graft is still viable , and no appreciable lesions are present in the artery distal to the planned graft site . according to one older panel of experts , surgical revascularization may be considered under the following conditions : severe occlusion of the main trunk of the lmca , severe occlusion of > 1 major coronary arteries , severe occlusion in the proximal segment of the lad , collateral coronary arteries in jeopardy , or all of the above . along with the technical advancement , more and more cases of kawasaki diseases are going to be diagnosed . therefore , we shall need to follow up more such cases and upgrade our knowledge regarding this condition . as coronary artery sequelae of kawasaki disease can be a cause of ischemic heart disease even in adults , heightened awareness of this possibility is required for adults with coronary lesions but without coronary risk factors .

information about the genomic coordinates and the sequence of experimentally identified transcription factor binding sites is found scattered under a variety of diverse formats . the availability of standard collections of such high - quality data is important to design , evaluate and improve novel computational approaches to identify binding motifs on promoter sequences from related genes . abs ( ) is a public database of known binding sites identified in promoters of orthologous vertebrate genes that have been manually curated from bibliography . we have annotated 650 experimental binding sites from 68 transcription factors and 100 orthologous target genes in human , mouse , rat or chicken genome sequences . computational predictions and promoter alignment information are also provided for each entry . a simple and easy - to - use web interface facilitates data retrieval allowing different views of the information . in addition , the release 1.0 of abs includes a customizable generator of artificial datasets based on the known sites contained in the collection and an evaluation tool to aid during the training and the assessment of motif - finding programs .

expression of genes is regulated at many different levels , transcription of dna being one of the most critical stages . specific configurations of transcription factors ( tfs ) that interact with gene promoter regions are recruited to activate or modulate the production of a given transcript . many of these tfs possess the ability to recognize a small set of genomic sequence footprints called tf - binding sites ( tfbss ) . these motifs are typically 615 bp long and in some cases , they show a high degree of variability . in addition because of these flexible binding rules , computational methods for the identification of regulatory elements in a promoter sequence tend to produce an overwhelming amount of false positives . however , the identification of conserved regulatory elements present in orthologous gene promoters ( also called phylogenetic footprinting ) has proved to be more effective to characterize such sequences ( 13 ) . in fact , the ever - growing availability of more genomes and the constant improvement of bioinformatics algorithms hold great promise for unveiling the overall network of gene interactions of each organism ( 4 ) . typically , computational methods to detect regulatory elements use their own training set of experimental annotated tfbss . these annotations are usually collected from bibliography or from general repositories of gene regulation information , such as jaspar ( 5 ) and transfac ( 6 ) . however , each program establishes different criteria and formats to retrieve and display the data that forms the final training set , which makes the comparison between different methods very difficult . the construction of a good benchmark to evaluate the accuracy of several pattern discovery methods is therefore not a trivial procedure ( 7 ) . although important efforts are being carried out to standardize the construction of collections of promoter regions ( 8) or the presentation of experimental data ( 9 ) , there is a clear necessity to provide stable and common datasets for future algorithmic developments . in this direction , we present here the release 1.0 of the abs database constructed from literature annotations that have been experimentally verified in human , mouse , rat or chicken . we have gathered from the literature a collection of experimentally validated binding sites that are conserved in at least two orthologous vertebrate promoters . the compiled data are suitable for training both classical pattern discovery programs and new emerging comparative methods . flat files accomplishing the gff standard format were used to store and query the information . the genbank accession number of the sequences in each bibliographical reference was utilized to retrieve the promoters . such sequences were mapped on to the corresponding refseq annotations to ensure we were retrieving the actual promoter . the dbtss database ( 10 ) was finally used to refine the annotation of the tsss . since it is the region in which most experimental studies have been focused on , we considered the sequence 500 bp immediately upstream the annotated tss , as the promoter region in this first release . for each annotated promoter , we only included experimentally tested sites in this proximal region whose motifs were correctly identified in at least two species , i.e. orthologous sites . every known binding site was mapped on to the corresponding promoter sequence by blastn ( 11 ) . those matches that exhibited < 80% of identity between the sequence of the original site and the mapped motif in the promoter region were rejected . we computed blastn ( 11 ) , clustalw ( 12 ) , avid ( 13 ) and lagan ( 14 ) alignments of the orthologous promoters from each gene . moreover , we produced a dotplot of word matches with emboss ( 15 ) to visualize unusually conserved regions . for comparative assessments , computational predictions using the jaspar ( 5 ) , transfac ( 6 ) and promo ( 16 ) collections of position weight matrices were calculated . a very restrictive threshold of 0.85 was used to remove those predicted tfbss whose score was below this value , creating a first group of more reliable predictions . a second group of predictions was produced using a more flexible threshold of 0.70 ( see the abs website for further information about the scoring method ) . release 1.0 of abs database contains 100 annotated orthologous genes , each entry corresponding to two or more species . there is a clear predominance of human and mouse annotations : 73 entries contain at least annotations for human mouse orthologs . a total of 650 experimental binding sites from 68 different tfs are associated with abs entries , covering 8624 nt . in average , three tfbss per sequence have been mapped on to the promoters with an average length of 13.3 nt per site . the tfs that appear more frequently are tbp ( 14.6% of sites ) , sp1 ( 13.6% ) and cebp ( 5.6% ) . those tfs are known to be part of the core of many eukaryotic promoters ( see the abs documentation for further details about the contents of the database ) . the abs database can be accessed through a simple cgi / perl - based web interface at . the following functionalities are implemented in the current release ( see figure 1 ) : for each gene in the collection , show the orthologous promoters and a list of experimentally verified tfbss annotated on the corresponding sequence . promoter sequence alignments , computational predictions , dotplots and cross - references to other well - known databases , such as genbank , entrez gene and pubmed , are also provided for each annotation.retrieve all of the binding motifs associated with a given tf , filtering by species . moreover , a global alignment of the motifs is provided and the corresponding sequence logo representation is displayed by using weblogo ( 17 ) . this information could be used to produce new profiles for subsequent detection of this tf in other promoters.retrieve all of the promoter sequences in which at least one binding site for a given tf was annotated . these sequences and the associated motifs could be used to generate datasets based on known sites to train motif - finding programs.the gene catalogue , the promoter sequences , the collection of annotations , the sequence alignments and the computational predictions are also individually distributed in several flat files . for each gene in the collection , show the orthologous promoters and a list of experimentally verified tfbss annotated on the corresponding sequence . promoter sequence alignments , computational predictions , dotplots and cross - references to other well - known databases , such as genbank , entrez gene and pubmed , are also provided for each annotation . retrieve all of the binding motifs associated with a given tf , filtering by species . moreover , a global alignment of the motifs is provided and the corresponding sequence logo representation is displayed by using weblogo ( 17 ) . this information could be used to produce new profiles for subsequent detection of this tf in other promoters . retrieve all of the promoter sequences in which at least one binding site for a given tf was annotated . these sequences and the associated motifs could be used to generate datasets based on known sites to train motif - finding programs . the gene catalogue , the promoter sequences , the collection of annotations , the sequence alignments and the computational predictions are also individually distributed in several flat files . the abs database aims to become a platform to evaluate new algorithms for the discovery of novel regulatory elements in a set of related gene promoters ( e.g. orthologous promoters or co - regulated genes from microarray experiments ) . in addition to the data retrieval functions , two on - line applications are available to perform the benchmarking of such algorithms ( see figure 2 ) : constructor is a web server to produce synthetic datasets based on the abs annotations . the design of the benchmark is highly flexible allowing to customize the number of sequences , their length , the background nucleotide distribution , the number of motifs to plant on them , the probability to plant a motif , the species and the tfs for which the associated motifs will be randomly selected from the known sites collection . the output consists of the artificial sequences with the embedded motifs , the list of motifs and a graphical representation of the occurrences in the sequences produced with the program gff2ps ( 18).evaluator is a web server to determine the accuracy of a set of predicted motifs in several sequences using a list of known binding sites as a reference set . a complete accuracy assessment at both nucleotide and site levels is computed using the standard measures in the field ( 7,19 ) . the design of the benchmark is highly flexible allowing to customize the number of sequences , their length , the background nucleotide distribution , the number of motifs to plant on them , the probability to plant a motif , the species and the tfs for which the associated motifs will be randomly selected from the known sites collection . the output consists of the artificial sequences with the embedded motifs , the list of motifs and a graphical representation of the occurrences in the sequences produced with the program gff2ps ( 18 ) . evaluator is a web server to determine the accuracy of a set of predicted motifs in several sequences using a list of known binding sites as a reference set . a complete accuracy assessment at both nucleotide and site levels is computed using the standard measures in the field ( 7,19 ) . the abs database can be accessed through a simple cgi / perl - based web interface at . the following functionalities are implemented in the current release ( see figure 1 ) : for each gene in the collection , show the orthologous promoters and a list of experimentally verified tfbss annotated on the corresponding sequence . promoter sequence alignments , computational predictions , dotplots and cross - references to other well - known databases , such as genbank , entrez gene and pubmed , are also provided for each annotation.retrieve all of the binding motifs associated with a given tf , filtering by species . moreover , a global alignment of the motifs is provided and the corresponding sequence logo representation is displayed by using weblogo ( 17 ) . this information could be used to produce new profiles for subsequent detection of this tf in other promoters.retrieve all of the promoter sequences in which at least one binding site for a given tf was annotated . these sequences and the associated motifs could be used to generate datasets based on known sites to train motif - finding programs.the gene catalogue , the promoter sequences , the collection of annotations , the sequence alignments and the computational predictions are also individually distributed in several flat files . for each gene in the collection , show the orthologous promoters and a list of experimentally verified tfbss annotated on the corresponding sequence . promoter sequence alignments , computational predictions , dotplots and cross - references to other well - known databases , such as genbank , entrez gene and pubmed , are also provided for each annotation . retrieve all of the binding motifs associated with a given tf , filtering by species . moreover , a global alignment of the motifs is provided and the corresponding sequence logo representation is displayed by using weblogo ( 17 ) . this information could be used to produce new profiles for subsequent detection of this tf in other promoters . retrieve all of the promoter sequences in which at least one binding site for a given tf was annotated . these sequences and the associated motifs could be used to generate datasets based on known sites to train motif - finding programs . the gene catalogue , the promoter sequences , the collection of annotations , the sequence alignments and the computational predictions are also individually distributed in several flat files . the abs database aims to become a platform to evaluate new algorithms for the discovery of novel regulatory elements in a set of related gene promoters ( e.g. orthologous promoters or co - regulated genes from microarray experiments ) . in addition to the data retrieval functions , two on - line applications are available to perform the benchmarking of such algorithms ( see figure 2 ) : constructor is a web server to produce synthetic datasets based on the abs annotations . the design of the benchmark is highly flexible allowing to customize the number of sequences , their length , the background nucleotide distribution , the number of motifs to plant on them , the probability to plant a motif , the species and the tfs for which the associated motifs will be randomly selected from the known sites collection . the output consists of the artificial sequences with the embedded motifs , the list of motifs and a graphical representation of the occurrences in the sequences produced with the program gff2ps ( 18).evaluator is a web server to determine the accuracy of a set of predicted motifs in several sequences using a list of known binding sites as a reference set . a complete accuracy assessment at both nucleotide and site levels is computed using the standard measures in the field ( 7,19 ) . the design of the benchmark is highly flexible allowing to customize the number of sequences , their length , the background nucleotide distribution , the number of motifs to plant on them , the probability to plant a motif , the species and the tfs for which the associated motifs will be randomly selected from the known sites collection . the output consists of the artificial sequences with the embedded motifs , the list of motifs and a graphical representation of the occurrences in the sequences produced with the program gff2ps ( 18 ) . evaluator is a web server to determine the accuracy of a set of predicted motifs in several sequences using a list of known binding sites as a reference set . a complete accuracy assessment at both nucleotide and site levels is computed using the standard measures in the field ( 7,19 ) . the abs database has been developed to fill the existing gap in the availability of consistent datasets to train and compare different pattern discovery programs . the lack of standard collections of tfbss is specially serious in the case of phylogenetic footprinting data . the collection described here contains 650 experimental tfbss identified in human , mouse , rat and chicken genes . in addition , two web applications ( constructor and evaluator ) are included in this first release to facilitate the development of new motif - finding programs using the abs annotations . in the next release , we plan to increase the number of annotations adding known sites in regulatory regions different from the proximal promoter and eventually incorporate binding motifs from other species . examples of the abs data retrieval system showing the annotation of a gene , the set of binding motifs from a given tf in human and mouse and the extraction of the promoter sequences containing such annotations . protocol to evaluate the accuracy of an external motif - finding program on a synthetic dataset generated by planting motifs from abs in randomly generated sequences .

introduction and objectivesa multidimensional self - report questionnaire to evaluate job - related stress factors is presented . the questionnaire , called maugeri stress index reduced form ( masi - r ) , aims to assess the impact of job strain on a team or on a single worker by considering four domains : wellness , resilience , perception of social support , and reactions to stressful situations.material and methodsthe reliability of a first longer version ( 47 items ) of the questionnaire was evaluated by an internal consistency analysis and a confirmatory factor analysis . an item reduction procedure was implemented to obtain a short form of the instrument , and the psychometric properties of the resulting instrument were evaluated using the rasch measurement model.resultsa total of 14 items from the initial pool were deleted because they were not productive for measurement . the analysis of internal consistency led to the exclusion of eight items , while the analysis performed using structural equation models led to the exclusion of another six items . according to the rasch model , item properties and the reliability of the instruments appear good , especially for the scales for wellness and resilience . in contrast , the scales for perception of social support and negative coping styles show a lower internal consistency.conclusionsthe maugeri stress index reduced form provides a reliable and valid measure , useful for early identification of stress levels in workers or in a team along the eustress vadistress continuum .

it is well known that job stress is responsible for poor work performance , high absenteeism , less work productivity , and several diseases.15 recently , results from thirteen independent cohort studies in europe indicated that job strain is responsible for coronary heart disease , as are lifestyle and orthodox risk factors.6 the population s attributable risk for job strain was 3.4% , which is lower than that for smoking habits ( 36% ) , abdominal obesity ( 20% ) , and physical inactivity ( 12% ) . however , the interheart study7 showed that work stress doubled the risk of coronary heart disease . in finland , the increase in job strain was associated with an increase in the risk of requiring a disability pension . moreover , as regarding men , a positive association between cardiovascular diseases and an increased risk of disability pension was found.8 mental health caused disabling conditions in 9% of the population in the uk , and estimations for the 20012002 prevalence of self - reported illness caused or made worse by work classified stress , depression , or anxiety as the second most commonly reported illness.9 within this context , in 2010 , in italy , a governmental commission published several indications for the evaluation of stress . the commission identified a preliminary phase and a second phase for in - depth examination . instruments for self - evaluation such as psychometric tests are recommended during the second phase . in this context of changes in the cultural and legislative background , the authors have taken the opportunity to contribute to the development of models for the identification and assessment of stress , responding to the demand for specific measurement instruments . data from the literature as well as normative requirements suggest the need for methods for the early identification of stress in workers . some instruments have already been developed , such as the job content questionnaire,1012 the work organization assessment questionnaire,13 and the effort reward imbalance questionnaire.14,15 these instruments are considered complementary sources since they are based on different concepts of the work - related stress . they are mainly focused on measuring the job climate , the facets of job organization , or the balance between effort and reward , but an in - depth examination of individual resources and capabilities is lacking . newman and beehr16 underlined that job stress is a situation wherein job - related factors interact with the worker to change his / her psychological and/or physiological condition , forcing a person to deviate from normal functioning . from this viewpoint , the focus is not the stressful situation or the subjective perception , but the interaction between these two components . in agreement with this rationale , we developed a new multidimensional instrument called the maugeri stress index ( masi ) designed to investigate individual resources for coping with stressful situations on the job . the instrument was developed for an in - depth examination , but it is also usable in the preliminary phase and for monitoring homogeneous groups of workers when individual resources play a critical role in coping with the demands arising from organizational changes . this group of workers represents a category particularly at risk for the development of job stress , and for this reason they can be considered a representative sample of the target population . the first version was tested on a restricted sample ( n=329 ) and has already been published with some clinical qualities and some limitations.17 the aims of this study were to develop a shorter form of the questionnaire using an item reduction procedure and to assess the psychometric properties of the resulting instrument using the rasch measurement model . the masi ( figure s1 ) requires participants to express their level of agreement with 51 items on a five - point likert scale ( never , little , enough , much , very much ) . the questionnaire is composed of four main scales including 47 items : wellness ( eleven items ) , resilience ( 20 items ) , perception of social support ( nine items ) , and negative coping styles ( seven items ) . further , a lie scale ( four items ) is included . a high score on the index shows perceived eustress . previously , giorgi et al17 studied the dimensionality of the instrument by using exploratory factor analysis . in the study here presented , we analyzed the internal consistency of the masi and its factorial structure by using structural equation models ( sems).18 therefore , starting with these results , we developed a reduced form of the instrument , the maugeri stress index reduced form ( masi - r ) , which is described in this paper . the psychometric properties of the masi - r are analyzed using the rasch measurement model.19 the scientific and technical committee ( irb ) of the care and research institute of tradate ( varese , italy ) , salvatore maugeri foundation irccs , discussed and approved the study ( 5/5/2015 cod . the sample comprised 1,182 participants ( females : 57.6% ; males : 42.4% ) with a mean age of 45.7 years ( standard deviation = 9.0 ) . other subjects engaged in the study were factory workers ( 15.1% ) , teachers ( 4.6% ) , and managers ( 4.2% ) . of the sample , 93.9% had a permanent work contract and 38.0% were shift workers . all the participants filled in the questionnaire voluntarily after a short motivational presentation made by a psychologist . each participant provided written informed consent for the use of data in anonymous form . participants who omitted at least 25% of responses ( 13 items ) were not considered in the analysis . therefore , seven subjects were removed , and 1,175 units were used for analysis . regarding sem analysis , the remaining missing data were replaced by the mean of the responses for each item . by contrast , for the rasch analysis , only the complete cases were considered , leading to a total sample of 904 subjects . the confirmatory factor analysis was used to test and improve the fit of the data for the hypothesized four - factor model ( see missing data section ) . the confirmatory factor analysis is part of the general methodology of the sem and allows us to test causal relations between latent and observed variables . the analysis was started by building an independence model , assuming uncorrelated latent factors . as a second step , we tested the presence of relations between the latent factors , performing a model comparison . step by step , we imposed covariance relations among factors , looking for an improvement in the fit . a similar methodology was applied to select the items in order to reduce their number and to simplify the instrument . to make model comparisons , we used the akaike information criterion ( aic)20 and the bayesian information criterion ( bic)21 to identify which model was better.22,23 since the best model has the lowest indices , we compared the information criteria of a complex model with those of the simpler one , obtaining two differences : aic and bic . a negative difference indicates that the complex model is better than the simpler one ; a difference of bic 2 is generally accepted as positive evidence for the difference between two models.24 in contrast to the aic , the bic penalizes greatly complex models : a very complex model would have to improve the fit substantially in order for bic to choose it as the best . the item properties were analyzed using the partial credit model,25 a polytomous formulation of the rasch model . the rasch model is a well - known one - parameter logistic model , commonly applied for the evaluation of the properties of a unidimensional psychometric test with an ordinal response scale . it assumes that the probability of a person producing a positive outcome to an item depends on the distance between the person s ability and the item s difficulty . in contrast to the classical test theory and the sem approach , the rasch model is centered on the study of items and their properties . the model tests the calibration of the instrument on a population , putting persons abilities and item difficulties into the same scale . it can distinguish between the easiest and hardest items and evaluate if the instrument leads to a reliable measure of the latent trait . the scales of the masi were studied separately , fitting one partial credit model for each scale . the reliability was evaluated using the separation index g for persons ( gp ) and items ( gi ) and the person separation reliability r , which corresponds to cronbach s .26 a high variability in persons abilities and in item difficulties is necessary so that the measurement is reliable . gp quantifies the number of strata of abilities that the instrument can separate and identify as different ; a low value ( gp < 2 ) is a sign that the instrument may not be sensitive enough to distinguish between individuals with high and low performance abilities.27 by contrast , a low gi value ( gi <3 ) indicates that either the variance of item difficulties is too small or the sample is not large enough . item properties have been evaluated using two popular indices:27 infit ( information - weighted fit ) and outfit ( outlier - sensitive fit ) . both indices are calculated based on the mean square of standardized residuals for items , but outfit is more sensitive to outliers than infit . acceptable values range between 0.6 and 1.4;28 lower values indicate underfit ( ie , the presence of unexplained variance ) and higher values indicate overfit ( ie , redundancy in the set of items ) . the unidimensionality of each scale was assessed by evaluating the strength of correlations between residuals and the results of the principal component analysis performed on the residuals.29 since the rasch analysis extracts the first , and theoretically , the only latent component within each scale , residuals should be constituted only by random noise . a simple rule of thumb27 is that if the residuals are constituted by pure random noise , the eigenvalue of the first extracted component must be less than 2 ( ie , a strength of less than two items ) . furthermore , we used the martin - lf test,30 which splits the scale into two clusters of items on the basis of the median difficulty and performs a likelihood ratio test . analyses were performed in the r environment,31 using the packages lavaan 0.51132 for the confirmatory factor analysis and erm 0.15033 for the rasch analysis . the sample comprised 1,182 participants ( females : 57.6% ; males : 42.4% ) with a mean age of 45.7 years ( standard deviation = 9.0 ) . other subjects engaged in the study were factory workers ( 15.1% ) , teachers ( 4.6% ) , and managers ( 4.2% ) . of the sample , all the participants filled in the questionnaire voluntarily after a short motivational presentation made by a psychologist . each participant provided written informed consent for the use of data in anonymous form . participants who omitted at least 25% of responses ( 13 items ) were not considered in the analysis . therefore , seven subjects were removed , and 1,175 units were used for analysis . regarding sem analysis , the remaining missing data were replaced by the mean of the responses for each item . by contrast , for the rasch analysis , only the complete cases were considered , leading to a total sample of 904 subjects . analyses were conducted to study the reliability of the masi and its factorial structure . the confirmatory factor analysis was used to test and improve the fit of the data for the hypothesized four - factor model ( see missing data section ) . the confirmatory factor analysis is part of the general methodology of the sem and allows us to test causal relations between latent and observed variables . the analysis was started by building an independence model , assuming uncorrelated latent factors . as a second step , we tested the presence of relations between the latent factors , performing a model comparison . step by step , we imposed covariance relations among factors , looking for an improvement in the fit . a similar methodology was applied to select the items in order to reduce their number and to simplify the instrument . to make model comparisons , we used the akaike information criterion ( aic)20 and the bayesian information criterion ( bic)21 to identify which model was better.22,23 since the best model has the lowest indices , we compared the information criteria of a complex model with those of the simpler one , obtaining two differences : aic and bic . a negative difference indicates that the complex model is better than the simpler one ; a difference of bic 2 is generally accepted as positive evidence for the difference between two models.24 in contrast to the aic , the bic penalizes greatly complex models : a very complex model would have to improve the fit substantially in order for bic to choose it as the best . the item properties were analyzed using the partial credit model,25 a polytomous formulation of the rasch model . the rasch model is a well - known one - parameter logistic model , commonly applied for the evaluation of the properties of a unidimensional psychometric test with an ordinal response scale . it assumes that the probability of a person producing a positive outcome to an item depends on the distance between the person s ability and the item s difficulty . in contrast to the classical test theory and the sem approach , the rasch model is centered on the study of items and their properties . the model tests the calibration of the instrument on a population , putting persons abilities and item difficulties into the same scale . it can distinguish between the easiest and hardest items and evaluate if the instrument leads to a reliable measure of the latent trait . the scales of the masi were studied separately , fitting one partial credit model for each scale . the reliability was evaluated using the separation index g for persons ( gp ) and items ( gi ) and the person separation reliability r , which corresponds to cronbach s .26 a high variability in persons abilities and in item difficulties is necessary so that the measurement is reliable . gp quantifies the number of strata of abilities that the instrument can separate and identify as different ; a low value ( gp < 2 ) is a sign that the instrument may not be sensitive enough to distinguish between individuals with high and low performance abilities.27 by contrast , a low gi value ( gi <3 ) indicates that either the variance of item difficulties is too small or the sample is not large enough . item properties have been evaluated using two popular indices:27 infit ( information - weighted fit ) and outfit ( outlier - sensitive fit ) . both indices are calculated based on the mean square of standardized residuals for items , but outfit is more sensitive to outliers than infit . acceptable values range between 0.6 and 1.4;28 lower values indicate underfit ( ie , the presence of unexplained variance ) and higher values indicate overfit ( ie , redundancy in the set of items ) . the unidimensionality of each scale was assessed by evaluating the strength of correlations between residuals and the results of the principal component analysis performed on the residuals.29 since the rasch analysis extracts the first , and theoretically , the only latent component within each scale , residuals should be constituted only by random noise . a simple rule of thumb27 is that if the residuals are constituted by pure random noise , the eigenvalue of the first extracted component must be less than 2 ( ie , a strength of less than two items ) . furthermore , we used the martin - lf test,30 which splits the scale into two clusters of items on the basis of the median difficulty and performs a likelihood ratio test . analyses were performed in the r environment,31 using the packages lavaan 0.51132 for the confirmatory factor analysis and erm 0.15033 for the rasch analysis . the internal consistency , measured by the cronbach s , appeared good for the scales wellness ( =0.94 ) and resilience ( =0.89 ) . in contrast , for the scales perception of social support ( =0.64 ) and negative coping styles ( =0.65 ) , the values were not so good , although they were acceptable . we considered the correlation between each item and the total score of the respective scale ( calculated excluding the target item ) . within the scale of perception of social support these items were : 4 , 13 , 17 , and 28 ( the content of items are reported in figure s1 ) . whereas the item - total correlation for items 4 and 28 were near 0.3 , the indices for items 13 and 17 were lower than 0.2 . the most problematic item was 13 , because the cronbach s of the respective scale increased if this item was deleted . we observed a medium - sized pearson s correlation between the scales resilience and wellness ( r=0.53 , p<0.001 ) and between resilience and perception of social support ( r=0.45 , p<0.001 ) . the confirmatory factor analysis started with the estimation of the independence model , assuming uncorrelated latent dimensions . all parameters were estimated to be significantly different from zero for p<0.001 . for the wellness scale , the standardized parameters ranged between 0.61 and 0.87 , while the values for the resilience scale were between 0.33 and 0.71 . in the perception of social support scale , the coefficients ranged between 0.11 and 0.66 ( item 13 was the only one that was significant for p<0.01 ) . finally , in the negative coping styles scale , the values ranged between 0.35 and 0.55 . the goodness - of - fit indices highlighted a poor fit ( comparative fit index [ cfi ] = 0.78 , nonnormed fit index [ nnfi ] = 0.77 , root mean squares error of approximation [ rmsea ] = 0.06 ) . therefore , we proceeded to free the covariance relations between the latent dimensions one by one , looking for an improvement . we released , in sequence , the covariance parameters for wellness , resilience , and perception of social support . in the last step , we freed the covariance relations with the negative coping styles scale at each step , the new model was compared with the previous one using the difference in aic and bic . the progressive deletion of constraints in covariance parameters improved the model ( cfi = 0.82 , nnfi = 0.81 , rmsea = 0.06 ) . in order to develop a short form of the masi as a first step , starting from the identified model in the confirmatory factor analysis of the masi section ( correlated factors ) , we deleted selected items one by one , looking for a better internal consistency . the candidate items for deletion were those with an item - total correlation of less than 0.3 and those that , if deleted , caused an increase in the cronbach . we deleted three items from the scale perception of social support ( 13 , 17 , and 4 ) , three items from the scale resilience ( 36 , 8 , and 22 ) , and two items from the negative coping styles scale ( 38 and 15 ) . the resulting model showed a better fit than the starting model ( cfi = 0.86 , nnfi = 0.85 , rmsea = 0.06 ) . as the second step , using sem , we tested the presence of covariance between errors . inspecting modification indices , we identified several candidate parameters to free , concentrating our attention on items with semantic similarities in content . one at a time , aic and bic showed an improvement of the fit for each step ; further , the final model showed better fit indices ( cfi = 0.89 , nnfi = 0.88 , rmsea = 0.05 ) than the starting one . since the semantic content of several items was very similar , we kept only one item from each pair , considering the relevance of the item in a clinical perspective as driving principle for the choice . competing items are reported in table 2 , with bold items being included in the final model . in one case ( 6s and 11s ) , we deleted both items , and in two cases , ( items 33 and 25 and items 29 and 39 ) we kept both items . the final model considered item 37 , marked in bold in figure s1 , which constitutes the short version of the tool , called masi - r . figure 1 is a graphical description of the model with estimated parameters ; fit indices indicate an acceptable fit with the data ( cfi = 0.88 , nnfi = 0.87 ) and a good error of approximation of the model ( rmsea = 0.05 ) . preliminarily , we evaluated the possible presence of a differential item functioning on the scales . using the regression approach , we tested the invariance of item scores across several clusters of subjects , taking into account the sex of participants , their age , the presence / absence of job shifts , and the length of service . for each scale , f - tests sometimes showed significant results at the 5% level , but the effect sizes were always very low ( < 0.05 ) . since the effect of these variables on the total scores was not noticeable , they were not considered significant . regarding the issue of reliability , the results of the analyses on the masi - r were similar to the results of the analyses on the masi . the person reliability appeared good for the scales wellness ( r=0.89 ) and resilience ( r=0.89 ) and were acceptable for the scales perception of social support ( r=0.70 ) and negative coping styles ( r=0.50 ) . in the two latter scales , the persons separation indices seemed critical : gp = 1.52 for perception of social support and gp = 0.99 for negative coping styles . in contrast , for the wellnesss ( gp = 2.87 ) and resilience ( gp = 2.79 ) scales , the persons separation indices were more adequate . both infit and outfit were always good ( table 3 ) and neither underfit nor overfit affected the items . figure 2 depicts the calibration of the instrument , comparing the distribution of the persons abilities and item difficulties along the logit scale . for each panel , the scores on the left side represent persons with high stress ( ie , low ability ) , while the right side represents persons with low stress ( ie , high ability ) . the matched distributions of persons abilities and item difficulties show that difficulties cover the abilities quite well . this is confirmed by the separation indices gi , for which the values are always good ( wellness : 15.10 , resilience : 6.80 , perception of social support : 11.01 , negative coping styles : 4.55 ) , showing that the variances of item difficulties are adequate for measuring latent dimensions because they cover the entire spectrum of each trait . finally , we tested the actual unidimensionality of the masi - r scales . for each one , all of the correlations between residuals were lower than 0.4 ( rarely did they exceed 0.3 ) . further , the eigenvalues estimated by the principal component analyses were always lower than 2 . moreover , the martin - lf test confirmed that no other components appeared to be present within the four scales : values were never significant . the internal consistency , measured by the cronbach s , appeared good for the scales wellness ( =0.94 ) and resilience ( =0.89 ) . in contrast , for the scales perception of social support ( =0.64 ) and negative coping styles ( =0.65 ) , the values were not so good , although they were acceptable . we considered the correlation between each item and the total score of the respective scale ( calculated excluding the target item ) . within the scale of perception of social support these items were : 4 , 13 , 17 , and 28 ( the content of items are reported in figure s1 ) . whereas the item - total correlation for items 4 and 28 were near 0.3 , the indices for items 13 and 17 were lower than 0.2 . the most problematic item was 13 , because the cronbach s of the respective scale increased if this item was deleted . we observed a medium - sized pearson s correlation between the scales resilience and wellness ( r=0.53 , p<0.001 ) and between resilience and perception of social support ( r=0.45 , p<0.001 ) . the confirmatory factor analysis started with the estimation of the independence model , assuming uncorrelated latent dimensions . for the wellness scale , the standardized parameters ranged between 0.61 and 0.87 , while the values for the resilience scale were between 0.33 and 0.71 . in the perception of social support scale , the coefficients ranged between 0.11 and 0.66 ( item 13 was the only one that was significant for p<0.01 ) . finally , in the negative coping styles scale , the values ranged between 0.35 and 0.55 . the goodness - of - fit indices highlighted a poor fit ( comparative fit index [ cfi ] = 0.78 , nonnormed fit index [ nnfi ] = 0.77 , root mean squares error of approximation [ rmsea ] = 0.06 ) . therefore , we proceeded to free the covariance relations between the latent dimensions one by one , looking for an improvement . we released , in sequence , the covariance parameters for wellness , resilience , and perception of social support . in the last step , we freed the covariance relations with the negative coping styles scale at each step , the new model was compared with the previous one using the difference in aic and bic . the progressive deletion of constraints in covariance parameters improved the model ( cfi = 0.82 , nnfi = 0.81 , rmsea = 0.06 ) . in order to develop a short form of the masi , we implemented an item selection procedure to reduce the length of the questionnaire . as a first step , starting from the identified model in the confirmatory factor analysis of the masi section ( correlated factors ) , we deleted selected items one by one , looking for a better internal consistency . the candidate items for deletion were those with an item - total correlation of less than 0.3 and those that , if deleted , caused an increase in the cronbach . we deleted three items from the scale perception of social support ( 13 , 17 , and 4 ) , three items from the scale resilience ( 36 , 8 , and 22 ) , and two items from the negative coping styles scale ( 38 and 15 ) . the resulting model showed a better fit than the starting model ( cfi = 0.86 , nnfi = 0.85 , rmsea = 0.06 ) . as the second step , using sem , we tested the presence of covariance between errors . inspecting modification indices , we identified several candidate parameters to free , concentrating our attention on items with semantic similarities in content . one at a time , aic and bic showed an improvement of the fit for each step ; further , the final model showed better fit indices ( cfi = 0.89 , nnfi = 0.88 , rmsea = 0.05 ) than the starting one . since the semantic content of several items was very similar , we kept only one item from each pair , considering the relevance of the item in a clinical perspective as driving principle for the choice . competing items are reported in table 2 , with bold items being included in the final model . in one case ( 6s and 11s ) , we deleted both items , and in two cases , ( items 33 and 25 and items 29 and 39 ) we kept both items . the final model considered item 37 , marked in bold in figure s1 , which constitutes the short version of the tool , called masi - r . figure 1 is a graphical description of the model with estimated parameters ; fit indices indicate an acceptable fit with the data ( cfi = 0.88 , nnfi = 0.87 ) and a good error of approximation of the model ( rmsea = 0.05 ) . preliminarily , we evaluated the possible presence of a differential item functioning on the scales . using the regression approach , we tested the invariance of item scores across several clusters of subjects , taking into account the sex of participants , their age , the presence / absence of job shifts , and the length of service . for each scale , f - tests sometimes showed significant results at the 5% level , but the effect sizes were always very low ( < 0.05 ) . since the effect of these variables on the total scores was not noticeable , they were not considered significant . regarding the issue of reliability , the results of the analyses on the masi - r were similar to the results of the analyses on the masi . the person reliability appeared good for the scales wellness ( r=0.89 ) and resilience ( r=0.89 ) and were acceptable for the scales perception of social support ( r=0.70 ) and negative coping styles ( r=0.50 ) . in the two latter scales , the persons separation indices seemed critical : gp = 1.52 for perception of social support and gp = 0.99 for negative coping styles . in contrast , for the wellnesss ( gp = 2.87 ) and resilience ( gp = 2.79 ) scales , the persons separation indices were more adequate . both infit and outfit were always good ( table 3 ) and neither underfit nor overfit affected the items . figure 2 depicts the calibration of the instrument , comparing the distribution of the persons abilities and item difficulties along the logit scale . for each panel , the scores on the left side represent persons with high stress ( ie , low ability ) , while the right side represents persons with low stress ( ie , high ability ) . the matched distributions of persons abilities and item difficulties show that difficulties cover the abilities quite well . this is confirmed by the separation indices gi , for which the values are always good ( wellness : 15.10 , resilience : 6.80 , perception of social support : 11.01 , negative coping styles : 4.55 ) , showing that the variances of item difficulties are adequate for measuring latent dimensions because they cover the entire spectrum of each trait . finally , we tested the actual unidimensionality of the masi - r scales . for each one , all of the correlations between residuals were lower than 0.4 ( rarely did they exceed 0.3 ) . further , the eigenvalues estimated by the principal component analyses were always lower than 2 . moreover , the martin - lf test confirmed that no other components appeared to be present within the four scales : values were never significant . this paper presents the masi - r , a new psychometric instrument that aims to screen for job stress . the instrument is structured as a self - report questionnaire that is relatively short but psychometrically comprehensive . the masi - r allows us to obtain a reliable measure of exposure to workplace stress and its impact on workers . using four correlated subscales ( wellness , resilience , perception of social support , negative copying styles ) and a lie index , the questionnaire measures several dimensions that determine the total index . the total index can be obtained as the sum of responses ( coded from 1 to 5 ) . when the response pattern presents missing items , these values can be estimated by the mean of the provided responses . the masi - r originates from a reduction of the original longer masi , whose scales include an overall pool of 47 items . using an item selection procedure , we selected and removed those items that did not adequately contribute to the measurement . the structure of the instrument was further improved using an sem . the rasch model indicated good item properties with neither underfit , overfit , nor differential item functioning between population strata . furthermore , item difficulties covered the persons abilities quite well . the first one refers to a general condition of wellness , serenity , and satisfaction ; it is defined by the absence of negative symptoms and by the presence of optimism regarding the work experience . the resilience scale evaluates skills such as tenacity , flexibility , and the ability to deal with problems on the job . the other two scales have a restricted number of items . perception of social support explores a component that plays a fundamental role in stress and burnout , consisting of the perception of adequate support and assistance from colleagues and superiors.34 by contrast , the negative coping styles scale investigates the presence of social isolation , behavioral disengagement , anxiety , or irritability in reaction to stressor situations . for both scales this was probably due to the small number of items included in the scales , but it might also indicate a restricted ability range within the selected sample.27 the masi - r assumes stress as a general and unidimensional latent factor that shows itself in a broad spectrum of facets . these facets are clustered in the four identified components . according to this point of view , the correlation between the four latent traits suggests that the number of items could be further reduced and the total score could be used as a uniform one - factorial measure . recently , a study regarding the discriminant validity of the masi - r was published35 and the results show that workers exposed to workplace mobbing showed higher work stress levels compared to the control group in all aspects measured . in summary , using only 37 items , the masi - r can measure the perception of wellness and support , the resilience capability , and the behavioral reactions of individuals coping with stressful situations in the workplace . further , unlike other instruments , the masi - r provides a lie index capable of identifying cheating responders , a phenomenon that can occur in workplace contexts . it is focused on the relation between the worker and stressful situations on the job , and it could be used to drive interventions centered on the interaction between the work environment and individual perceptions and resources . list of the items included into the masi , the full version of the questionnaire . notes : only the bold items are included in the masi - r ( 33 items ) . items from the lie control scale ( 4 items ) are marked with an asterisk .

objectivefour combinations of five neutral sequence changes at rs713040 , rs10768683 , rs7480526 , rs7946748 , and rs1609812 occurring in the human beta globin gene defined as frameworks have been reported in beta globin gene . here we report for the frequency of these frameworks in thalassemia major patients of north iran.methodsbeta globin gene frameworks of 46 thalassemia major patients of north iran were determined using denaturing gradient gel electrophoresis.findingsall these frameworks called framework 1 , 2 , 3 , 3a were present at the frequency of 23.9% , 45.7% , 6.5% and 23.9% respectively.conclusionthese frameworks may be used for tracking mutant alleles in prenatal diagnosis programs .

detection of beta globin gene lesions in thalassemic patients and carriers is based on screening for the mutations most often found in the relevant ethnic group . but in circumstances in which an unknown mutation is encountered , another difficulty arises when at - risk couples present at a late stage of pregnancy with an unusual beta globin mutation asking for prenatal diagnosis . since the nature of the parental beta thalassemia alleles must be determined before fetal sampling , it is fundamental to find either parental specific mutations or dna polymorphisms in beta globin gene cluster for linkage studies . in some instances base changes have been identified as restriction fragment length polymorphisms ( rflps ) near or within beta globin gene[6 , 7 ] . rflps are detected by determining the presence or absence of a restriction enzyme cleavage site within a dna fragment . this concerns rs713040 or codon2 ( c - t ) recognized by hgiai , rs10768683 or ivs2 - 16 ( c - g ) recognized by avaii and rs1609812 or ivs2 - 666 recognized by sspi[8 , 9].oas there are hot spots for recombination upstream of beta globin gene[10 , 11 ] and few rflps within beta globin gene , it is wiser to study either polymorphism in both sides of beta globin gene or look for polymorphisms within beta globin gene for prenatal diagnosis . moreover in many instances rflps are not informative due to the low degree of polymorphism and each restriction site 's study necessitates a separate pcr reaction . the combined pcr and denaturing gradient gel electrophoresis ( dgge ) can be used to detect the presence of uncommon mutations or natural polymorphisms within beta globin genes[12 , 13 ] . this method permits the separation of strands of small dna fragments ( 200 - 700bp ) according to the nature of their sequence . it involves the electrophoresis of double stranded dna fragments through an acrylamide gel containing a linear gradient of dna denaturants , such as formamide and urea . using this method , 4 different beta globin gene frameworks called fw1 , fw2 , fw3 and fw3a representing combinations of 5 natural sequence changes at exon one ( one site ) and intron two ( 4 sites ) of beta globin can be distinguished upon a single pcr reaction . these five neutral sequence changes occur in the human beta globin gene in codon 2 , ivs 2 ( nt 16 , 74 , 81 and 666 ) . the five neutral sequence changes at codon 2 c / t , ivs 2 nt 16 c / g , nt 74 g / t , nt 81 c / t , nt 666 t / c occurring in the human beta globin gene define four frameworks called : 1 , 2 , 3 and 3a ( table 1 ) that differ from each other by at least one nucleotide . to detect these polymorphisms , we analyzed the dgge pattern of the fragment corresponding to nucleotides + 188 to + 621 of beta globin and containing ivs2 ( nt 16 , 74 , 81 ) and report the frequency of these frameworks among 92 beta globin alleles belonging to 46 thalassemia major patients for the first time in northern iran . blood samples were collected from 46 unrelated regularly transfused thalassemic children of mazandaran province , iran . for each sample , dna was obtained from 0.5 ml blood after alkaline lysis of white blood cells . all patients were compound heterozygote with one or both mutations being unknown or present at a frequency lower than 2% in the population studied . dna amplification : pcr was performed using a techne progene thermal cycler ( uk ) . the primer sequences and pcr conditions are as described in ghanem et al , for fragment g amplification . a 474 bp dna fragment corresponding to nucleotides + 188 to + 621 of beta globin gene was amplified using forward 5'ctgggcatgtggagacagag3 primer containing an additional 5'gc rich oligomer which creates a high temperature melting domain : 5'gcgggcggggcggggcgcggcgggcggggc called gc clamp and a reverse 5'cactgatgcaatcattcgtc3 primer ( bioneer company , south korea ) . beta globin gene representation : a portion of beta globin gene spanning from nucleotide + 188 to + 621 and covering exon1 and 2 was amplified using pcr . the position of the 5 single nucleotide polymorphisms ( one in exon 1 and four in intron 2 ) is also presented . three polymorphic nucleotides ivs2(nt 16,74,81 ) are present in the amplicon , and their association defines the beta globin frameworks . dgge was accomplished according to ghanem et al : 10 l of pcr product was subjected to electrophoresis at 160 v for 5 hours in a 6.5% polyacrylamide gel containing a linearly increasing denaturant gradient of 10% - 60% in a water bath at 60c . after migration the gel was stained by ethidium bromide and the position of different bands visualized under 312 nm uv wavelength . as dgge is based on the principle that two double - stranded dna fragments of the same size , but differing in sequence , melt at different points in a denaturing gradient and can be distinguished by differential migration , a uniform temperature with a linear denaturant gradient formed by urea and formamide creates the denaturing environment in dgge . dgge analysis of amplicons belonging to 46 thalassemia major patients revealed that framework 2 is the most abundant among mutant alleles analyzed and represents 45.7% of the cases . frameworks 1 and 3a represent each 23.9% of the cases and framework 3 is the less frequent , namely 6.5% . table 2 shows the frequency of each framework in the population studied and fig 2 shows dgge patterns for individuals with different combinations of these frameworks . as expected , in individuals homozygous for a particular framework , heteroduplex can not form upon renaturation of the amplicon 's strand and only homoduplexes corresponding to complete matching between the two complementary strands of amplicons are possible and consequently a single band is visible upon electrophoresis in a gradient gel and under denaturing conditions ( fig . 4 bands are detectable under similar conditions : 2 bands migrating faster and representing homoduplexes corresponding to each framework and 2 other bands which migrate slowly comparing to homoduplexes due to mismatches between complementary dna strands belonging to different frameworks . each framework combination has its particular pattern of heteroduplexes and homoduplexes migration as nuclotidic sequences and consequently dna migration pattern under denaturing gradient gel electrophoresis varies in each framework . the higher the number of g and c in the polymorphic sites of the amplican , the faster is the migration of homoduplexes . thus , amplicons corresponding to framework 1 which contains ivs2 nucleotide polymorphisms c , g and c respectively at positions 16 , 74 and 81 migrate faster than amplicons corresponding to framework 3a with nucleotides g , t and c and framework 2 with nucleotides c , t and c at those polymorphic positions . amplicons corresponding to framework 3 with nucleotides g , t and t at the above mentioned positions are the slowest ( fig . 2 ) . a : schematic representation of relative migration pattern expected for different frameworks homoduplex after denaturing gradient gel electrophoresis . lanes 1 , 3 : 2/3a lane 2 : 1/2 lane 4 : 1/3a lane 5 : 1/3 lane 6 : 2/2 . in each lane , lower bands migrate faster and thus correspond to homoduplexes while upper bands correspond to heteroduplexes . although extensive studies were performed for molecular characterization of beta globin mutations in northern iran[1517 ] , but as in any prenatal diagnosis program , new or undefined mutations may be encountered . different restriction site polymorphisms have been described near beta globin gene[6 , 7 ] , but few of them are intragenic markers[8 , 9 ] . here we report for the first time polymorphisms for five intragenic markers in north iran using dgge analysis . , we have determined the frequency of these 4 different frameworks among mutant beta globin alleles present in northern iran . their frequency vary between 45.7% for the most abundant ( framework 2 ) to 6.5% for the less abundant ( framework 3 ) . dgge analysis of beta globin gene was also performed in other populations . in india where framework 3a is the most prevalent with a frequency of 66.5% followed by framework 1 with a frequency of 25.2% , frameworks 2 and 3 represent 5.8% and 2.5% respectively of the 206 mutant alleles studied . another study accomplished in north thailand showed that the frequency of fw1 and fw3a was 73.3% and 26.7% respectively in 30 mutant alleles studied . similar frequencies were also reported in a limited number of mutant alleles analyzed in korean population . the difference in framework distribution and their frequencies among population studied reflect the differences in both mutation spectrum and the degree of heterogeneity within a population . indeed framework 2 was the most prevalent in north iran with a frequency of 45.7% while it was absent in east asians and rather rare in indian population studied . as ivsii-1(g - a ) mutation is the most prevalent mutation in north iran with a frequency of around 60%[15 , 16 ] and compound heterozygotes presenting ivsii-1(g - a ) alleles included in this study were also possessing the framework 2 ( data not presented ) , ivsii-1(g - a ) may be associated to framework 2 . this could explain the difference in framework distribution and frequencies observed in iranian and indian or east asian populations where ivsii-1(g - a ) was not reported . although direct mutation detection is routinely performed either by using arms pcr or reverse dot blot analysis or sequencing analysis in molecular diagnosis of thalassemia , this indirect method which necessitates segregation analysis may be used either in cases where unknown mutations are encountered or as a complementary method to confirm the genotype . many diagnostic laboratories apply traditional haplotype analysis by co - segregation studies within a family either in order to detect an unknown mutation for carrier screening or prenatal diagnosis in families with a previous history of disease or as a complementary analysis to direct mutation methods in prenatal diagnosis to confirm the genotype of the fetus . however , most polymorphic sites studied routinely using restriction enzymes are either located outside the gene which may be subject to errors in genotype determination at beta globin gene locus in rare cases of recombination due to the presence of a hotspot for recombination located upstream of beta globin gene[10 , 11 ] or may be located within the beta globin gene with a low degree of polymorphism and therefore non - informative in many families . the frameworks characterized in the present study represent a combination of 5 single nucleotide polymorphisms all located within the beta globin gene which abolishes errors due to recombination . moreover a single pcr reaction allows the simultaneous evaluation of the genotype in all 5 intragenic polymorphic sites determining each specific framework , while in segregation analysis using rflp method , genotyping of each polymorphic site necessitates a separate pcr reaction followed by enzymatic digestion . regarding their abundance these polymorphisms could be studied either as frameworks or as single polymorphic sites using restriction endonuclease analysis : alw44 for codon 2 , avaii for ivs2 - 16 , maeiii for ivs2 - 81 and sspi for ivs2 - 666 as intragenic markers for detection of beta thalassemia by linkage analysis . polymorphism at ivs2 nt666 was studied separately in north iran as the t nucleotide at this position is recognized by sspi . our previous findings show that ivs2 nt 666(t ) is present in about 85.7% of mutant alleles . this nucleotide is present in frameworks 1 and 2 which together represent about 70% of studied mutant alleles . as table 1 indicates the c allele at codon 2 is in the same linkage phase than ivs2 nt 16(c ) and ivs2 nt 666(t ) . the two former polymorphisms can also be distinguished by restriction analysis using alw44 and avaii respectively . thus as alw44 is commercially less expensive than avaii , we recommend the use of the former enzyme in segregation analysis using rflp . all 4 defined frameworks were present in beta thalassemia chromosomes at a frequency higher than 5% and consequently may be useful for tracking mutant alleles in prenatal diagnosis programs .

backgroundtendinopathy is an overuse tendon injury that occurs in loaded tendons and results in pain and functional impairment . although many treatments for painful tendons are described , the scientific evidence for most of the conservative and surgical treatments is not always conclusive.objectivesthis study was designed to evaluate the efficacy of 3 different interventions in patients with achilles tendinopathy . the interventions include the combination of 2 physical therapy programs ( eccentric training [ ec ] or passive stretching [ ps ] ) with a supplement containing mucopolisaccharides . the efficacy of the interventions was evaluated depending on the stage of the disease.methodsfifty-nine patients were randomly assigned to 1 of 3 treatment groups , and classified according to the disease stage : reactive versus degenerative tendinopathy . treatment groups were ec ; ec + a dietary supplement containing mucopolisaccharides , type i collagen , and vitamin c ( mcvc ) ; and a passive stretching program + mcvc . patients were evaluated at baseline , 6 weeks , and 12 weeks with the victorian institute of sports assessment - achilles questionnaire for function , a visual analog scale for pain , and ultrasound characterization for the evolution of tendon structure.resultsa significant improvement in victorian institute of sports assessment - achilles questionnaire score , pain at rest , and pain during activity were detected in all 3 treatment groups at 6 and 12 weeks follow - up when compared with baseline . in patients with reactive tendinopathy , the reduction in pain at rest was greater in the groups who took the supplemental mcvc than in the ec alone group ( p < 0.05).conclusionsmcvc seems to be therapeutically useful for management of tendinopathies , providing some additional benefit to physical therapy . this is especially evident in early stages of the disease , when the tendon does not present severe matrix and vascular changes.clinicaltrials.gov identifiernct01691716 .

tendinopathy is an overuse tendon injury that occurs in loaded tendons and results in pain and functional impairment , a major problem in sports and occupational medicine . characteristic changes in tendon structure include loss of type i collagen , increase in proteoglycan content , and neovascularization . this has been termed failed healing response and results in the tendon being less capable of sustaining repeated tensile load . recently , a new pathology model has been accepted to explain the clinical course of tendinopathy . the cook and purdam model states there is a continuum of tendon pathology that , for easier clinical use , has been divided into 2 clear groups : reactive tendinopathy / early tendon disrepair and degenerative tendinopathy / late tendon disrepair . because the same intervention to all presentations of tendinopathy is unlikely to show efficacy , applying this model might be useful to rationally place specific treatments through the evolution of the pathology . although many treatments are described for painful tendons , the scientific evidence for most of the conservative and surgical treatments is not always conclusive . eccentric exercise has evidential basis obtained from randomized controlled trials and is currently included in most treatment algorithms.4 , 5 it involves active lengthening of the muscle tendon unit and short - term improvements in tendon pain6 , 7 , 8 and normalization of tendon structure have been demonstrated . nevertheless , why eccentric exercise reduces pain in tendinopathy and what is the correct intensity , speed , load , and frequency remains unclear.2 , 10 other therapeutic options that have shown some positive outcomes for tendinopathies treatment include shockwaves , sclerosing polidocanol injections , topical nitroglycerin therapy , and cooling therapies . costicosteoid injections have also shown very good outcomes in the short term but should be avoided due to the cortisone s negative influence on the tendon structure . a composition containing mucopolysaccharides , type i collagen , and vitamin c ( mcvc ) demonstrated efficacy both in in vitro models and human studies.13 , 14 , 15 mcvc induces changes in tenocytes metabolism in vitro , thereby helping to preserve the structure of the extracellular matrix . results indicate that this formulation promotes the endogenous synthesis of collagen type i , avoiding the accumulation of collagen type iii and aggrecan , and therefore may interfere with the degeneration of tendon tissue . these findings suggest that mcvc may improve tendinopathy symptomatology , reducing discomfort and improving tendon function . indeed , in 3 studies in humans , 2 observational and 1 controlled , the consumption of the supplement might be associated with clinical and structural improvements in the recovery of tendinopathies.13 , 14 , 15 the goal of this trial was to determine whether daily consumption of a supplement containing mcvc , added to an eccentric exercise program , provided some additional benefit in improving symptoms of chronic noninsertional tendinopathy in patients with a gradually evolving painful condition in the midportion of the achilles tendon for at least 3 months . in parallel , the specific effect of mcvc when combined with 2 different physical therapies was compared : eccentric training ( ec ) or passive stretching ( ps ) . finally , whether there is a different response pattern to the aforementioned treatments depending on the pathology stage of the patient ( reactive tendinopathy vs degenerative tendinopathy ) was assessed . this was a nutrition - based , randomized , controlled , multicenter trial to assess the efficacy and safety of a supplement containing mcvc combined with either an ec or ps program , and compared with ec alone . patients were randomly assigned to 1 of 3 treatment groups : ec , combination of ec and mcvc , and combination of ps and mcvc . patients were assigned a randomization number taken from a computer - generated randomization list following the chronological order by which they were included in the study , independent of the stage of their tendinopathy . it was conducted in men and nonpregnant women , aged 18 to 70 years , with painful noninsertional achilles tendinopathy of the midportion for at least 3 months . diagnosis was based on clinical examination showing a painful thickening of the achilles tendon located 2 to 6 cm above the tendon insertion , and confirmed by ultrasonography ; specifically , local thickening of the tendon , irregular tendon structure with hypoechoic areas , and irregular fiber orientation . clinical suspicions of insertional tendinopathy , tendon rupture ( thomson test and palpable gap ) , neural disorder , systemic disease , like gout , spondylarthropaty , rheumatoid arthritis and sarcoidosis , or pregnancy were considered exclusion criteria . patients which already received a previous treatment with eccentric training or prp for the studied injury were also excluded . the stage of the tendinopathy was classified into 2 subgroups according to the cook and purdam pathology model : patients with reactive tendinopathy and patients with degenerative tendinopathy . based on ultrasonographic assessment , reactive tendinopathy is characterized by swollen tendon and diffuse hypoechogenicity occurring between intact collagen structures ( figure 1a ) , whereas degenerative tendinopathy shows increased matrix disorganization represented by hypoechoic regions with few reflections from collagen fascicles and clear neovascularization usually visible on doppler ultrasound ( figure 1b ) . stage of tendinopathy was assessed using imaging criteria . to avoid variations between centers , a single specialist blinded to the study group of the participants evaluated the imaging across all study sites . fifty - nine potentially eligible patients were recruited between september 2012 and june 2013 in 5 spanish sports medicine centers . in each clinical center , a team composed of an orthopedist and a physiotherapist was in charge of the study . a consolidated standards of reporting trials diagram of participant flow through the trial is shown in figure 2 . a total of 58 participants fulfilled the inclusion criteria and consented to take part in the study . the intention to treat ( itt ) population was defined by all the participants who took at least 1 unit of the trial product . the subjects did not receive any financial incentive to participate in the study but received the assigned treatment free of charge . the study was approved by the official clinical research ethical committees of consell catal de lesport , barcelona , spain . patients allocated in the ec training group were instructed to perform a training protocol according to alfredson et al . to guarantee the consistency of the procedures among different research centers , the protocol was agreed upon by all the researchers in prestudy meetings , and its correct execution was confirmed by a physiotherapist every week . the gastrocnemius muscle was eccentrically loaded both with the knee straight and , to maximize the activation of the soleus muscle , also with the knee bent . this protocol was repeated twice per day , 7 days a week , for 12 weeks . patients in the ec + mcvc group consumed 3 capsules per day containing 435 mg mucopolysaccharides , 75 mg collagen type i , and 60 mg vitamin c ( tendoactive ; bioiberica sa , palafolls , spain ) and followed the ec protocol . patients in the ps + mcvc group consumed the dietary supplement and were instructed to perform a personalized protocol of passive stretching that was supervised weekly by a physiotherapist . ps routines were included in the program to guarantee the same weekly monitoring in all the study groups . a standard protocol was applied , including 2 exercises to stretch the gastrocnemius muscle and 2 for the soleus muscle . patients were evaluated at baseline , 6 weeks , and 12 weeks after the beginning of the treatment protocol with the original english version of the validated victorian institute of sports assessment - achilles ( visa - a ) questionnaire . the visa - a questionnaire scores range from 0 to 100 , with higher scores corresponding with less pain and increased activity . every week , pain was evaluated at rest and during activity using a visual analog scale ( vas ) . the evolution of the affected tendons was monitored ultrasonographically on every visit and bilateral thickness was recorded . bilateral thickness was measured in the transversal section of the more thickened cross point at a level of 2 to 6 cm above the tendon insertion . the statistical analysis followed the statistical plan according to the principles specified in the guidelines of the international council for harmonization of statistical principles for clinical trials and of the committee for proprietary medicinal products with the efficacy working party points to consider on multiplicity issues in clinical trials . no interim analyses of efficacy were planned for this study and , therefore , no multiplicity adjustments were needed . the primary efficacy analysis was conducted to evaluate the absolute change from baseline to last visit of the visa - a score , using an ancova model with the baseline value as a covariate and a baseline observation carried forward approach on itt . all secondary efficacy variables were assessed similarly as primary variables using an ancova model with the baseline value as a covariate . treatment effect was also evaluated in 2 subgroups designed according to the cook and purdam pathology model : patients with reactive tendinopathy and patients with degenerative tendinopathy . this was a nutrition - based , randomized , controlled , multicenter trial to assess the efficacy and safety of a supplement containing mcvc combined with either an ec or ps program , and compared with ec alone . patients were randomly assigned to 1 of 3 treatment groups : ec , combination of ec and mcvc , and combination of ps and mcvc . patients were assigned a randomization number taken from a computer - generated randomization list following the chronological order by which they were included in the study , independent of the stage of their tendinopathy . it was conducted in men and nonpregnant women , aged 18 to 70 years , with painful noninsertional achilles tendinopathy of the midportion for at least 3 months . diagnosis was based on clinical examination showing a painful thickening of the achilles tendon located 2 to 6 cm above the tendon insertion , and confirmed by ultrasonography ; specifically , local thickening of the tendon , irregular tendon structure with hypoechoic areas , and irregular fiber orientation . clinical suspicions of insertional tendinopathy , tendon rupture ( thomson test and palpable gap ) , neural disorder , systemic disease , like gout , spondylarthropaty , rheumatoid arthritis and sarcoidosis , or pregnancy were considered exclusion criteria . patients which already received a previous treatment with eccentric training or prp for the studied injury were also excluded . the stage of the tendinopathy was classified into 2 subgroups according to the cook and purdam pathology model : patients with reactive tendinopathy and patients with degenerative tendinopathy . based on ultrasonographic assessment , reactive tendinopathy is characterized by swollen tendon and diffuse hypoechogenicity occurring between intact collagen structures ( figure 1a ) , whereas degenerative tendinopathy shows increased matrix disorganization represented by hypoechoic regions with few reflections from collagen fascicles and clear neovascularization usually visible on doppler ultrasound ( figure 1b ) . stage of tendinopathy was assessed using imaging criteria . to avoid variations between centers , a single specialist blinded to the study group of the participants evaluated the imaging across all study sites . fifty - nine potentially eligible patients were recruited between september 2012 and june 2013 in 5 spanish sports medicine centers . in each clinical center , a team composed of an orthopedist and a physiotherapist was in charge of the study . a consolidated standards of reporting trials diagram of participant flow through the trial is shown in figure 2 . a total of 58 participants fulfilled the inclusion criteria and consented to take part in the study . the intention to treat ( itt ) population was defined by all the participants who took at least 1 unit of the trial product . the subjects did not receive any financial incentive to participate in the study but received the assigned treatment free of charge . the study was approved by the official clinical research ethical committees of consell catal de lesport , barcelona , spain . patients allocated in the ec training group were instructed to perform a training protocol according to alfredson et al . to guarantee the consistency of the procedures among different research centers , the protocol was agreed upon by all the researchers in prestudy meetings , and its correct execution was confirmed by a physiotherapist every week . the gastrocnemius muscle was eccentrically loaded both with the knee straight and , to maximize the activation of the soleus muscle , also with the knee bent . this protocol was repeated twice per day , 7 days a week , for 12 weeks . patients in the ec + mcvc group consumed 3 capsules per day containing 435 mg mucopolysaccharides , 75 mg collagen type i , and 60 mg vitamin c ( tendoactive ; bioiberica sa , palafolls , spain ) and followed the ec protocol . patients in the ps + mcvc group consumed the dietary supplement and were instructed to perform a personalized protocol of passive stretching that was supervised weekly by a physiotherapist . ps routines were included in the program to guarantee the same weekly monitoring in all the study groups . a standard protocol was applied , including 2 exercises to stretch the gastrocnemius muscle and 2 for the soleus muscle . patients were evaluated at baseline , 6 weeks , and 12 weeks after the beginning of the treatment protocol with the original english version of the validated victorian institute of sports assessment - achilles ( visa - a ) questionnaire . the visa - a questionnaire scores range from 0 to 100 , with higher scores corresponding with less pain and increased activity . every week , pain was evaluated at rest and during activity using a visual analog scale ( vas ) . the evolution of the affected tendons was monitored ultrasonographically on every visit and bilateral thickness was recorded . bilateral thickness was measured in the transversal section of the more thickened cross point at a level of 2 to 6 cm above the tendon insertion . patients were evaluated at baseline , 6 weeks , and 12 weeks after the beginning of the treatment protocol with the original english version of the validated victorian institute of sports assessment - achilles ( visa - a ) questionnaire . the visa - a questionnaire scores range from 0 to 100 , with higher scores corresponding with less pain and increased activity . every week , pain was evaluated at rest and during activity using a visual analog scale ( vas ) . the evolution of the affected tendons was monitored ultrasonographically on every visit and bilateral thickness was recorded . bilateral thickness was measured in the transversal section of the more thickened cross point at a level of 2 to 6 cm above the tendon insertion . the statistical analysis followed the statistical plan according to the principles specified in the guidelines of the international council for harmonization of statistical principles for clinical trials and of the committee for proprietary medicinal products with the efficacy working party points to consider on multiplicity issues in clinical trials . no interim analyses of efficacy were planned for this study and , therefore , no multiplicity adjustments were needed . the primary efficacy analysis was conducted to evaluate the absolute change from baseline to last visit of the visa - a score , using an ancova model with the baseline value as a covariate and a baseline observation carried forward approach on itt . all secondary efficacy variables were assessed similarly as primary variables using an ancova model with the baseline value as a covariate . treatment effect was also evaluated in 2 subgroups designed according to the cook and purdam pathology model : patients with reactive tendinopathy and patients with degenerative tendinopathy . three participants were excluded from the randomized population for not having attended the inclusion visit ( 1 in the ps + mcvc group and 2 in the ec + mcvc group ) . fifty - five participants ( 94.83% of those randomized ) entered the itt population ; 23 had reactive tendinopathy and 32 degenerative tendinopathy . a consort diagram of participant flow through the trial is shown in figure 2 . the average ( sd ) age was 38.9 ( 6.6 ) years for the ec group , 43.5 ( 14.5 ) years for the ec + mcvc group , and 40.2 ( 10.6 ) years for the ps + mcvc group . the participants were mostly men : 78% , 82% , and 80% in the ec , ec + mcvc , and ps + mcvc groups , respectively . there were 5 , 9 , and 9 patients with reactive tendinopathy and 15 , 8 , and 9 patients with degenerative tendinopathy in the ec , ec + mcvc , and ps + mcvc groups , respectively . there were no significant differences at baseline in any of the clinical outputs of the study . when analyzing the obtained data regardless of the patient s stage of disease , a significant improvement in visa - a score was detected in all 3 treatment groups at 6 and 12 weeks follow - up , when compared with baseline . there was no between - group effect for the primary outcome of visa - a questionnaire between baseline and the end of the study ( p > 0.1 ) . in all the studied groups , vas - rated pain was reduced both at rest and during activity after the 12-week treatment . differences in pain reduction among treatments were found between the ps + mcvc and the ec groups , being significant at rest : for ps + mcv ( 3.7 [ 0.8 ] cm ) and ec ( 2.7 [ 1.3 ] cm ) ( p < 0.05 ) , and tending toward significance during activity : ps + mcv ( 4.4 [ 1.1 ] cm ) and ec ( 3.5 [ 1.4 ] cm ) ( p = 0.074 ) . although no significant variation was found in the tendon bilateral thickness among treatments ( p > 0.1 ) , reduction from baseline was only detected in the ps + mcvc treatment group ( 0.63 [ 0.73 ] mm ) ( p < 0.05 ) . in patients with reactive tendinopathy , the improvement of the visa - a score at 12 weeks follow - up was 56% for the ps + mcv group , 44% for the ec + mcv group , and 30% for the ec group ( table i ) ( p = 0.154 ) . although no statistically significant results were found , a tendency toward significance was found in the ec and ec + mcvc groups ( p = 0.069 ) . in patients with degenerative tendinopathy , improvement in visa - a score had the same magnitude among treatments ( table ii ) . similarly , pain at rest improved in all 3 groups of treatment in all randomized patients . in patients with reactive tendinopathy , the pain reduction at 12 weeks follow - up was greater for the group ps + mcvc ( 3.815 [ 0.8 ] cm ) compared with the ec group ( 2.80 [ 1.0 ] cm ) ( p < 0.05 ) . for the ec + mcvc group the pain reduction can not be compared in absolute values because the mean pain value at rest was not balanced at baseline ( table i ) . in patients with degenerative tendinopathy , the magnitude of the pain reduction was similar among the study groups ( table ii ) . the evaluation of pain during activity followed the same pattern of response as pain at rest , although differences among treatment groups failed to reach statistical significance , either in reactive or in degenerative tendinopathy . the bilateral thickness of the affected tendon remained constant during the study period in patients in the ec and ec + mcvc groups from the itt population . in contrast , a significant reduction ( 0.63 [ 0.3 ] mm ; p < 0.05 ) was detected in patients in the ps + mcvc group . in patients with degenerative tendinopathy , a significant reduction was registered in the ps + mcvc group ( p < 0.05 ) , whereas no differences were detected in patients with reactive tendinopathy ( table iii ) . in this study , 3 different interventions have been evaluated : ec alone , the combination of ec with a dietary supplement containing mcvc , and an alternative physical therapy such as ps combined with the dietary supplement in patients with achilles tendinopathy . the main goal was to determine the additional benefit of mcvc to a physical therapy program . after 90 days , all 3 treatment groups experienced significant improvements from baseline in visa - a , vas - rated pain at rest , and vas - rated pain during activity . these improvements were not only statistically , but also clinically , significant because the minimum clinically important difference is 9 mm for vas - rated pain and 6.5 points in the visa - a scale.17 , 18 the clinical improvements registered as a result of the ec training agree with those previously described16 , 19 and were expected because ec is currently the gold standard for achilles tendinopathy treatment . the addition of mcvc to ec significantly improved pain , but not function - related outcomes . when patients were divided according to severity of tendinopathy , a statistically significant clinical benefit from mcvc in patients with reactive but not degenerative tendinopathy , as determined by vas - rated pain , was clearly observed . although functionality , assessed with the visa - a test , did not reach a statistically significant improvement it showed a tendency toward significance , although statistical significance was not reached due to low patient number . however , it reached a clinically significant improvement because it increased more than 6.5 points in both mcvc groups compared with controls . according to the model described by cook and purdam , in reactive tendinopathy a homogeneous , noninflammatory cell response to load that leads to metaplastic change in cells and cell proliferation is found . theoretically , this cellular deregulation could be counterbalanced by the effects of mcvc on tenocyte metabolism , as described by shakibaei et al . in degenerative tendinopathy , areas of cell death due to apoptosis , trauma , or tenocyte exhaustion in tendons have been reported . in this stage of the pathology the efficacy of mcvc could be limited by the reduced viability of the tenocytes . in this study , a protocol of ps supervised by a physiotherapist and adjusted personally and supplemented with mcvc was shown to improve pain reduction and function recovery as good as a standard ec in both reactive and degenerative tendinopathy . although the efficacy of the eccentric gastrocnemius muscle training has been widely described , less data about the efficacy of a protocol of ps are available . it has been recently published that isometric exercises could induce pain reduction in patellar tendinopathy . because ps implies a certain degree of isometric contractions , the potential analgesic effect of this intervention could partially explain the clinical findings for this group . to elucidate the contribution of the ps protocol and the mcvc supplement to the clinical improvement , an additional group of patients following the ps program but without the mcvc supplement would have been required . from our study the placebo effect can not be assessed because the groups not receiving the mcvc supplement did not receive an inert capsule . another weakness from the study is the lack of blinding of the therapists treating the patients . although the lack of blinding could potentially interfere with the results , we think that this should not have had a major impact on the patients evaluation and reported outcomes as greater improvements were expected from the ec+mcvc treatment compared to the ps+mcvc group and the obtained data did not match the expectations . regarding the structural changes during the study period , it has been previously described that a program of eccentric exercise is valid in the symptomatic nonoperative management of tendinopathy , probably by promoting collagen fiber cross - linkage formation within the tendon and , thereby , facilitating tendon remodeling . previous studies report increased type i collagen synthesis and reduction of fluid within the tendon after eccentric training offering a possible explanation for the mechanism of tendon healing.24 , 25 nevertheless , actual histologic adaptations after a program of ec or ps remain unclear . in this trial , the stretching program improved tendon structure when compared with baseline , as evidenced by a reduction in tendon thickness in patients with degenerative tendinopathy . during the study period this might be caused by the harshness of ec , which applies some degree of aggression to the tendon structure , and could maintain longer a temporary reactive disorganization of the tendon tissue . eccentric training is not the only kind of exercises that have been used for treating tendinopathies , other exercises like concentric ones have been tested for tendinopathy management . some studies have compared the effectiveness of eccentric vs. concentric training but conflicting results have been obtained ; some studies found that eccentric training was statistically superior to concentric training and others did not find any statistical difference between the treatments.8 , 26 , 27 some protocols combine eccentric and concentric training in the same sessions , like the silbernagel - combined program or the stanish and curwin programs . besides concentric exercises , one study found that isometric muscle contractions reduce pain in patellar tendinopathy . further research on this field is required as the effectiveness on the achilles tendon can not be assessed . due to the existing uncertainty in the programs equivalence , , a dietary supplement containing mcvc seems to present a high safety profile and to be useful for managing tendinopathies , providing some additional benefit to ec , especially in early stages of the disease . in the reactive phase , tendons present a short - term adaptation to overload with thickening and increased stiffness , but without severe matrix and vascular changes . a reactive tendon has the potential to revert to normal if the overload is sufficiently reduced or if there is enough time between loading sessions . in this type of patient , conversely , in degenerative pathology the tendon presents areas of cell death , vessel - filled matrix , matrix breakdown products , and little collagen , and therefore the reversibility of pathologic changes is limited . although a nutrition - based approach is less likely to offer a clinical benefit at this stage , certain clinical improvements were observed in the ps - mcvc group during our study . however , further studies with larger sample sizes are required to confirm the efficacy of mcvc for the dietary management of tendinopathy . the data obtained from this study confirm the therapeutic potential of ec for the management of tendinopathy , both at reactive and degenerative stages . the novelty of this study is that it shows the effects of a new therapeutic approach consisting of personalized stretching routines and dietary supplementation with mcvc . in patients with reactive tendinopathy , supplementation with mcvc further decreased pain levels compared to ec alone , so it could be included in the therapeutic arsenal for tendinopathy . in patients with degenerative tendinopathy , despite the lack of blinding and placebo treatment , the obtained results suggested that for patients with difficulties in performing ec due to advanced age , illness , or extreme tendon injury , a ps program supplemented with mcvc could be an alternative treatment to be taken into account . financial support for this work was provided by bioiberica , sa , palafolls , spain . bioiberica sa is the trademark owner , manufacturer , and distributor of tendoactive , the supplement used in this study . the authors have indicated that they have no other conflicts of interest regarding the content of this article .

prebiotics and probiotics are considered natural alternatives to dietary antibiotics in animal production . plant extracts and yeast cell walls are mannose rich products that can be used as substrate for adhesion of gram - negative bacteria . we assessed whether the structure of these saccharides is relevant to develop their role as prebiotics and therefore , their suitability to be used as alternatives to antibiotics to prevent intestinal infections in pigs . the prebiotic functionality of -galactomannan ( gm ) , mannanoligosaccharide from yeast saccharomyces cerevisiae ( mannan sc ) and monosaccharide d - mannose were studied in porcine intestinal epithelial cells ( ipi-2i ) challenged with salmonella enterica ser . typhimurium . results showed that in vitro challenge with salmonella induces the secretion of proinflammatory cytokine il6 and chemokine cxcl8 compared with control without infection . both gm and mannan sc , attenuate salmonella - induced secretion of il6 and cxcl8 . interestingly , cells treated with d - mannose showed similar levels of proinflammatory il6 and cxcl8 compared with the control of infection . these data suggest that prebiotic role of gm is related to its oligosaccharide structure .

livestock animals are subjected to a huge sort of stress factors , such as environmental conditions , animal management or diets that may disrupt the intestinal ecosystem and increase the risk of pathogenic infections . in the swine industry , transition from the liquid to a solid diet during weaning and post - weaning are critic processes due to an important decrease of feed intake , impairing animal performance , immune function , microbiota and therefore , gut homeostasis . concerning the immune system , these performance stages lead to the end of transference of passive maternal immunity from the sows to the piglets which still have an immature or developing immune system . this means that piglets become highly susceptible to intestinal pathogens such as enterotoxigenic e.coli ( etec ) or salmonella enterica ser . antibiotic growth promoters ( agps ) have been extensively used as dietary additives to minimize the risk of infections , especially during the critical periods of animal production , like weaning . however , after european ban of agps ( ec 1831/2003 ) several studies proposed the use of probiotics and prebiotics as natural alternatives to agps . in that sense , the health benefits of probiotics and prebiotics have been widely accepted as gut microbiota stabilizers because they are believed to avoid pathogenic colonization and therefore improving animal welfare . our group recently published in vitro results that demonstrated the potential of probiotic saccharomyces cerevisiae var boulardii and prebiotic -galactomannan ( gm ) to prevent intestinal infections by e.coli and salmonella in pigs . here , we aimed to extend the knowledge about the prebiotic role of gm against salmonella challenge according to its polysaccharide structure . the porcine small intestine epithelial cell line ipi-2i ( ecacc 93100622 ) was established from the ileum of an adult boar ( slad / d haplotype ) . ipi-2i cells were maintained in dmem glutamax ( invitrogen , spain ) supplemented with 10% fcs ( invitrogen ) , insulin 10 g / ml ( sigma aldrich , saint - quentin , france ) . in all experiments , cells were cultured in 6-well plates ( nunc , labclinics , spain ) to confluence . before the addition of pre / probiotics and/or infection , cells were washed three times and the cell culture was replaced with dmem media supplemented with insulin 10 g / ml ( sigma aldrich ) . cells were used between passages 30 and 70 and periodically tested to avoid mycoplasma contamination ( mycoalert mycoplasma detection kit , lonza ) . to assess the functionality of mannans according to their structure we compared the prebiotic gm ( salmosan , patent wo2009/144070 a2 , licensed to industrial tcnica pecuaria , itpsa , barcelona , spain ) to another mannanoligosaccharide purified from saccharomyces cerevisiae ( mannan sc , sigma ) and to d - mannose monosaccharide ( sigma - aldrich ) in relation to a negative control ( medium ) . prebiotic gm , mannan sc and d - mannose were diluted in dmem glutamax ( 1 mg / ml ) , vortexed and incubated 30 min at 37c . immediately before the salmonella infection , gm , mannan sc and d - mannose were respectively added to each well at 10g / ml . pathogenic salmonella enterica serovar typhimurium ( salmonella ) with antigenic formulae ( 4,12:i:1,2 ) , resistant to ampicillin , chloramphenicol , streptomycin , sulfonamide and tetracycline was provided by dr . ignacio badiola from centre recerca en sanitat animal ( cresa , irta - uab , bellaterra , spain ) . aliquots of salmonella were provided in bacterial cryo - preservers ( technical service consultants , ferrer international , spain ) and stored at -80c until use . before infection iec cells , a single salmonella cryo - preserver was added to 20 ml of luria - bertani media ( lb ) and cultured for 34h at 37c with 180 rpm rotational agitation ( multitron ht , infors ) . for infection , salmonella was used during the exponential growth phase as determined by absorbance at 600 nm . after 3 h of host cell - pathogen assay performed as described before , 75g / ml of gentamycin ( sigma - aldrich ) were added to each well to avoid bacterial overgrowth . cytokine protein determination in the culture supernatant was performed by enzyme - linked immunosorbent assays ( elisas ) . swine il-6 and cxcl8 duoset elisa kits ( r&d systems , vitro sp ) were used according to manufacturer s recommendations . all statistical analyses were performed using glm procedure ( proc glm ) by sas software version 9.1.3 ( sas institute ) . means for protein secretion were considered in a 2 * 3 factorial design ( two infection levels * 3 experimental treatments ) with duncan s post - test for group analysis . superscript letters are used to designate statistical significance : bars with different superscript letters are significantly different ( p < 0.05 ) and bars with same superscript letters indicate no statistical difference ( n.s ) . the porcine small intestine epithelial cell line ipi-2i ( ecacc 93100622 ) was established from the ileum of an adult boar ( slad / d haplotype ) . ipi-2i cells were maintained in dmem glutamax ( invitrogen , spain ) supplemented with 10% fcs ( invitrogen ) , insulin 10 g / ml ( sigma aldrich , saint - quentin , france ) . in all experiments , cells were cultured in 6-well plates ( nunc , labclinics , spain ) to confluence . before the addition of pre / probiotics and/or infection , cells were washed three times and the cell culture was replaced with dmem media supplemented with insulin 10 g / ml ( sigma aldrich ) . cells were used between passages 30 and 70 and periodically tested to avoid mycoplasma contamination ( mycoalert mycoplasma detection kit , lonza ) . to assess the functionality of mannans according to their structure we compared the prebiotic gm ( salmosan , patent wo2009/144070 a2 , licensed to industrial tcnica pecuaria , itpsa , barcelona , spain ) to another mannanoligosaccharide purified from saccharomyces cerevisiae ( mannan sc , sigma ) and to d - mannose monosaccharide ( sigma - aldrich ) in relation to a negative control ( medium ) . prebiotic gm , mannan sc and d - mannose were diluted in dmem glutamax ( 1 mg / ml ) , vortexed and incubated 30 min at 37c . immediately before the salmonella infection , gm , mannan sc and d - mannose were respectively added to each well at 10g / ml . pathogenic salmonella enterica serovar typhimurium ( salmonella ) with antigenic formulae ( 4,12:i:1,2 ) , resistant to ampicillin , chloramphenicol , streptomycin , sulfonamide and tetracycline was provided by dr . ignacio badiola from centre recerca en sanitat animal ( cresa , irta - uab , bellaterra , spain ) . aliquots of salmonella were provided in bacterial cryo - preservers ( technical service consultants , ferrer international , spain ) and stored at -80c until use . before infection iec cells , a single salmonella cryo - preserver was added to 20 ml of luria - bertani media ( lb ) and cultured for 34h at 37c with 180 rpm rotational agitation ( multitron ht , infors ) . for infection , salmonella was used during the exponential growth phase as determined by absorbance at 600 nm . after 3 h of host cell - pathogen assay performed as described before , 75g / ml of gentamycin ( sigma - aldrich ) were added to each well to avoid bacterial overgrowth . cytokine protein determination in the culture supernatant was performed by enzyme - linked immunosorbent assays ( elisas ) . swine il-6 and cxcl8 duoset elisa kits ( r&d systems , vitro sp ) were used according to manufacturer s recommendations . all statistical analyses were performed using glm procedure ( proc glm ) by sas software version 9.1.3 ( sas institute ) . means for protein secretion were considered in a 2 * 3 factorial design ( two infection levels * 3 experimental treatments ) with duncan s post - test for group analysis . the p value 0.05 was considered to be significant . superscript letters are used to designate statistical significance : bars with different superscript letters are significantly different ( p < 0.05 ) and bars with same superscript letters indicate no statistical difference ( n.s ) . many intestinal pathogenic strains of salmonella and e.coli share type i fimbriae , although from different evolutionary origin , which have high affinity for mannose residues of glycoproteins present on cell surface of the host . the recognition of these residues leads to bacterial adherence to enterocyte surface and intestinal colonization by the pathogen . our recent articles showed how probiotic strain of saccaromyces cerevisiae var boulardii ( scb ) and a recently developed prebiotic -galactomannan ( gm ) , derived from fruit seed of the ceratonia silliqua tree , inhibit in vitro adhesion of salmonella and etec to pig intestinal cell line ipi-2i . indeed , both products decreased the overall proinflammatory profile induced by etec or salmonella , with the least expenditure of resources for innate immune response to deal with each pathogen . mannan derived products , such as scb and gm , are widely accepted to prevent salmonella colonization by binding to the adhesin fim h of the type i fimbriae . the interaction of probiotic scb and prebiotic gm was already confirmed in our previous work . however , enterotoxigenic e.coli k88 , which is one on the most important causes of neonatal and post - weaning diarrhea ( pwd ) in piglets , bears type iv fimbriae or pilli . the latter has been described as mannose resistant to erythrocyte agglutination test in the presence of d - mannose ( 5% ) . nevertheless , the k88 adhesins ( ab , ac and ad ) bind to -galactose residues of receptors of syalic glycoprotein and to neutral glycosphingolipids expressed on the surface of the intestinal mucosa . the presence of -galactose in scb and gm may mimic host receptor for k88 adhesin and therefore inhibit etec attachement to cell surface . in addition to the residue composition , prebiotic functionality is determined by the size and molecular structure of the saccharides . here , we evaluated functionality of gm according to their polysaccharide structure . we compared gm to monosaccharide d - mannose and to another mannan oligosaccharide purified from saccharomyces cerevisiae yeast ( mannan sc ) at a final concentration of 10 g / ml . we observed that in vitro challenge with salmonella induces the secretion of proinflammatory cytokine il6 ( 4-fold increase : 224 vs. 897 pg / ml ; figure 1a ) and chemokine cxcl8 ( 2-fold increase : 2645 vs. 5192 pg / ml ; figure 1b ) compared with control without infection . both , gm and mannan sc showed a 40% reduction of salmonella - induced secretion of il6 ( 897 vs. 563 and 585 pg / ml , figure 1a ) . concerning cxcl8 , gm reduce chemokine secretion by 30% ( 5192 vs 3697 pg / ml ; figure 1b ) . however , mannan sc shows a trend to reduce cxcl8 secretion induced by salmonella ( fig . interestingly , the secretion of proinflammatory il6 and cxcl8 in iecs treated with monosaccharide d - mannose was similar to the control of infection ( fig . present data confirms early results shown by oyofo et al . and vil et al . who showed higher efficiency of oligomeric mannoses compared with monomeric d - mannose to inhibit in vivo salmonella infection in other productive species like chickens . in pigs , the addition of a commercial source of mannanoligosaccharides in the diet increased weight gain during the starter period , improved feed efficiency in vivo together with a reduction of enterobacteria counts in the jejunum ( see ref . indeed , mannanoligosaccharide additives have been shown to modify the composition of the gastrointestinal microbiota , resulting in increased populations of bacteroidetes spp and lactobacillus spp after salmonella infection in vivo , leading to a greater compensatory weight gain of pigs ( see ref . cytokine il6 ( a ) and chemokine cxcl8 ( b ) concentration in supernatants from ipi-2i cells ( 1 10 cells / well ) co - cultured for 24 h with salmonella ( moi = 4 ) is decreased by gm and mannan sc ( 10 g / ml ) . regard , our previous in vitro works showed that scb has a higher anti - inflammatory role in etec infections . in contrast , gm is a potent prophylactic agent against salmonella infections . therefore , the preference of the use of each probiotic or prebiotic additive as a dietary alternative to agps , should be determined , if possible , according to the specific conditions of each farm . this means the prevalence of etec or salmonella of each productive facility or region . in that sense , considering that etec infections often occur during weaning , while salmonella usually colonizes post - weaning and finishing growing pigs , to enrich diets with scb at early stages to prevent etec infections and with gm to reduce salmonella prevalence at final growing stages should be considered . in addition , we hypothesize that the prebiotic gm may enhance probiotic scb survival and colonization of the intestinal tract improving host health . future studies should determine the suitability to combine gm and scb to become a symbiotic additive to prevent intestinal infections . nowadays in eu , products such as mannan sc and gm are not authorized as zootechnical feed additives under the commission regulation ( ec ) 1831/2003 . in the future , both products could be authorized following concepts described in the opinion of efsa ( see ref . 23 ) concerning potential new categories of zootechnical feed additives . in this case it would be additives which improve product quality as microbial contamination controllers . however , today it is also possible to introduce them in the european feed legislation according to the regulation ( ec ) 767/2009 where there is a description of how ingredients can be used for particular purposes . in conclusion , we demonstrated that oligosaccharide structure is crucial for mannans to develop their prebiotic role to inhibit pathogenic adhesion and therefore to reduce proinflamatory immune response induced by the pathogen .

the supt16h gene known as factp140 is required for the transcription of other genes . for transcription , genes need to be complexed with accessory factors , including transcription factors and rna polymerase ii . one such factor , fact , interacts with histones h2a / h2b for nucleosome disassembly and transcription elongation . the supt16h gene has a transcript and many expressed sequence tags ( ests ) . we were especially interested in an malr - derived transcript ( est , bx333035 ) that included a new exon introduced by a transposable element , a mammalian apparent ltr retrotransposon ( malr ) . the malr was detected ranging from humans to galagos , indicating the malr in the supt16h gene is integrated into the primate ancestor genome . a new exon was created by alternative donor site provided by the malr . the original transcript and the malr - derived transcript were expressed in various human , rhesus monkey , and other primate tissues . additionally , we identified a new alternative transcript that included the malr , but there was no significant difference in the expression of the original transcript and the malr - derived transcript . interestingly , the new alternative transcript and the malr - derived transcript had the malr sequence in the new exon , but they had different structures by adopting different 3 splice sites . from this study , we verified transposable elements that contributed to transcriptome diversity .

transposable elements ( tes ) dispersed in the mammalian genome are on the rise , as completion of the whole human genome sequence . contrary to our expectations , tes , thought to be junk dna , were estimated to account for approximately 45% of the human genome ( international human genome sequencing consortium , 2001 ) . tes can be categorized by various features such as the capacity to mobilize themselves , dna / rna intermediation , and the existence of a long terminal repeat ( ltr ) . endogenous retroviruses ( ervs ) and long interspersed elements ( lines ) have gene - encoding enzymatic machinery that could mobilize to other genomic regions as autonomous elements . however , nonautonomous elements , especially alu elements , which are classified as short interspersed elements ( sine ) and sine - variable number tandem repeat - alu - like sequences ( sva ) , mobilize using the reverse transcriptase of the line . all of the tes described above are integrated into the host genome by their rnas , but the integration of dna transposons is mediated by dna . ervs and ltr retrotransposons have ltrs , but sines and lines are non - ltr elements [ 13 ] . one group of retrotransposon - like elements , mammalian apparent ltr retrotransposons ( malrs ) , accounts for approximately 3.65% and 4.82% , including solitary ltrs , of the human and mouse genome , respectively . . typical ervs consist of an internal region , gag , pol , and env with ltrs on both sides . ltrs are very important for integration and transcription because they function as a promoter element , transcription start site , and polyadenylation signal and site . compared to ervs , malrs have short internal sequences and ltrs at the 5 and 3 ends , but they do not have a transcription initiation site , reverse transcriptase , and primer binding site . recent studies have shown that tes contributed to primate genome evolution through gene structure variation and regulation by integrating into an intron , gene - flanking region , and an exon [ 7 , 8 ] . additionally , tes that integrate into introns could make alternative exons or cassette exons in a process termed exonization within human and mouse protein - coding genes ; in contrast , tes can make new introns in a process called intronization . these events could help with the understanding of transcriptome and proteome complexities [ 911 ] . the exonization of the primate - specific alu element is well established in the divergence of human and chimpanzee genomes as a major mechanism of exon creation [ 12 , 13 ] . ( 2003 ) reported that the alu consensus sequence could provide a splice site , leading to exonization . in the case of ltrs , 2007 ) reported that 256 of 1057 ltr retrotransposons related to human genes were observed in protein - coding regions , and 50 protein - coding exons of 45 genes were derived wholly from ltr retrotransposons . furthermore , they analyzed alternative exons of the interleukin 22 receptor , the alpha 2 gene ( il22ra2 ) , which was derived from malrs that provided splice sites , indicating the important roles of tes in human evolution ; this result coincided with those of previous studies [ 1 , 5 ] . importantly , the supt16h gene , also known as factp140 , is a component of the facilitates chromatin transcription ( fact ) complex . in eukaryotic cells , the transcription of genes requires rna polymerase ii and transcription factors with altered chromatin structure . in order to initiate transcription , dna needs to be in an open structure that is moderated by the alteration of chromatin with accessory factors , facilitating access to the dna . as described above , the supt16h gene has an original transcript consisting of 26 exons . however , the integration of an malr created a new exon that was expressed transcriptionally ( bx333035 ) . furthermore , we found a new alternative transcript , including the malr - derived exon . we assumed the time of integration of the malr into the genome by pcr amplification . all of the primates assayed had the malr in the supt16h gene , which was a natural result of its conservation in the mammalian genome . the expression patterns of the 2 transcripts in several tissues of humans , crab - eating monkey , marmoset , and squirrel monkey were ubiquitous with no significant tissue specificity or species specificity . structurally , the 2 transcripts included the exon - derived malr , but they adopted different splice sites , leading to exons of different sizes . taken together , we showed that the malr could regulate gene expression at the transcriptional level , that is , tes could provide transcriptome diversity , resulting in the proteome 's capacity for diversification , which potentially occupies an important role in the host genome . to identify the sequences related to the malr , we scanned the human reference genome sequence , human ests , and human refseq mrnas . using the malr consensus sequence as a query , we screened nonredundant databases through blast version 2.2.26 + to identify the novel hybrid transcripts of the malr and supt16h . then , we aligned the transcripts of the supt16h gene to identify the precise splicing patterns . tes included in the supt16h transcripts and their genomic loci were identified using the repeatmasker program ( http://www.repeatmasker.org/ ) , referencing a library containing the consensus sequences from repbase update . using the est and refseq mrnas , we accurately reconstructed the supt16h gene structure . to analyze the sequences of the various supt16h transcripts , we aligned them using the clustalw program . using the trizol reagent ( invitrogen ) , we extracted the total rna from the cerebrum , cerebellum , pituitary gland , heart , lung , spleen , liver , pancreas , kidney , and urinary bladder of the crab - eating monkey ; the heart , lung , brain , stomach , liver , kidney , pancreas , colon , spleen , and small intestine of the marmoset ; and the heart , esophagus , diencephalon , small intestine , lung , pancreas , cerebrum , colon , and stomach of the squirrel monkey . the total rna from human tissues , including the adrenal gland , cerebellum , whole brain , heart , kidney , liver , lung , testis , trachea , bone marrow , fetal brain , fetal liver , placenta , prostate , salivary gland , skeletal muscle , spinal cord , thymus , thyroid , and uterus , were purchased from clontech . we isolated the mrna from the total rna by using polyatract mrna isolation systems ( promega ) . we used the genomic dna of the following species : ( 1 ) hominoids : common chimpanzee ( pan troglodytes ) , gorilla ( gorilla gorilla ) , orangutan ( pongo pygmaeus ) , and gibbon ( hylobates agilis ) ; ( 2 ) old world monkeys : japanese monkey ( macaca fuscata ) , rhesus macaque ( m. mulatta ) , bonnet macaque ( m. radiata ) , mandrill ( mandrillus sphinx ) , mangabey ( cercocebus agilis ) , african green monkey ( chlorocebus aethiops ) , colobus ( colobus guereza ) , and langur ( trachypithecus cristatus ) ; ( 3 ) new world monkeys : night monkey ( aotus trivirgatus ) , squirrel monkey ( saimiri sciureus ) , and common marmoset ( callithrix jacchus ) ; and ( 4 ) prosimian : ring - tailed lemur ( lemur catta ) and a galago ( otolemur crassicaudatus ) . these genomic dna were isolated from the heparinized blood samples according to a standard protocol . after anticoagulant , and then , proteinase k and phenol equilibrated with tris - cl were treated for extraction . the viscous aqueous was transferred and ammonium acetate and ethanol were added to form a precipitate . lastly , the dna precipitate was washed , dried , and dissolved in te ( tris and edta ) . takenaka ( primate research institute , kyoto university ) kindly provided genomic dna of primates after conducting this procedure . using the genomic dna of humans and 17 primates , the malr in the supt16h gene m - mlv reverse transcriptase ( promega ) with an annealing temperature of 42c and an rnase inhibitor ( promega ) were used for the reverse transcriptions of the transcripts . the supt16h transcripts were amplified with a primer pair from genbank ( accession number nm001033560 ) consisting of s1 ( 5-aattggcggaaaggagaag-3 ) and as1 ( 5-gaggcagtcattccagctct-3 ) . the supt16h transcripts related to the malr were amplified with a primer pair from genbank ( bx_333035 ) consisting of s2 ( 5-tccagttctctgttctgcca-3 ) and as2 ( 5-tgtgtggagcttcaagcaag-3 ) . the pcr samples were subjected to an initial denaturation step of 4 min at 94c , followed by 30 cycles of pcr at 40 s of denaturation at 95c , 40 s of annealing at 55c , and 90 s of extension at 72c , followed by a final extension step of 7 min at 72c . as a standard control , gapdh was amplified with a primer pair consisting of gph - s ( 5-gagccccagccttctccatg-3 ) and gph - as ( 5-gaaatcccatcaccatcttccagg-3 ) from human gapdh ( genbank accession number nm002046 ) . the supt16h gene located on chromosome 14q11.2 encodes accessory factors that may facilitate access to dna by unpackaging the chromatin structure . it contains 26 exons with several transposable elements such as malrs ( ltr ) , alus ( sine ) , and l1s ( line ) distributed in the intronic regions . the supt16h transcript related to the malr ( malr - derived transcript ) has 3 exons , and its last exon was created by the integration of the malr ltr element in the opposite direction of the host gene ( figure 1 ) . malrs are considered to function in the mammalian genome by providing alternative splice sites , promoters , and other cis - regulatory elements , similar to other tes that have been conserved during primate evolution . for example , rnf19 has an alternative transcript resulting from the exonization of malr and alujo . specifically , the malr in the first exon has promoter activity in the sense orientation in the african green monkey fibroblast cell line , cos7 , and the human colorectal carcinoma cell line , hct116 . as described above , malrs are known to be present in genomes of rodents to primates and humans , indicating that they were distributed before the divergence of eutherian mammals . in order to compare the integration time of the malr in the supt16h gene with that from previous reports , we conducted pcr amplification using the genomic dna of human and other primates ( figure 2 ) . we identified an amplicon that was 481 bp long in all of the primates that we assessed , indicating that the malr in the supt16h gene integrated into the genome of a common ancestor before the divergence of the new world monkey and prosimians . in addition , from the sequencing alignment , we also identified 40 bp and 4 bp of specific sequences in japanese macaque ( figure 2(b ) ) . these sequences were not detected in any other primate , including the rhesus macaque , which belongs to the old world monkey family . tes distributed in the mammalian genome have played important roles in gene regulation and evolution . one of the typical characteristics of tes which is that they affect the transcription of cellular genes by exonization , which creates an alternatively spliced exon by integration into exonic or intronic regions , has been reported in many studies [ 24 , 25 ] . actually , these alternative transcripts are transient and flexible according to the cellular environment and stimuli . however , alternatively spliced variants can be stabilized and increased without selection , and exonization can lead to not only transcriptome diversity but also proteome diversity [ 2 , 10 ] . furthermore , these events occur differently or specifically according to the tissue , developmental stage , and disease ; thus , alternative transcripts are used to mine disease diagnostic markers [ 9 , 26 ] . to confirm whether the malr sequences are expressed and could regulate transcription , we conducted rt - pcr analysis of the refseq mrna ( original transcript ) and malr - derived transcript ( bx333035 ) from several human tissues . we analyzed its expression in tissues of other primates , including the crab - eating monkey , common marmoset , and squirrel monkey ( figure 3 ) . like the expression in humans , it was ubiquitously expressed in the tissues of these primates . because we identified japanese monkey - specific sequences , we conducted rt - pcr analysis in rhesus macaque samples to compare the expression in old world monkeys . however , we could not detect a difference in the supt16h expression between the japanese monkey and rhesus macaque ( data not shown ) . gene expression is a process that produces rna or proteins through transcription , splicing , and translation . the supt16h gene is a subunit of the fact complex that acts as a chromatin - specific transcription elongation factor and interacts with histone h2a / h2b to disassemble nucleosomes and promote transcription elongation . namely , the broad expression of supt16h in human and other primate tissues indicated its essential function for gene expression . as shown in figure 4 , a transcript related to the malr was also expressed broadly in human tissues . this product was also detected in tissues of crab - eating monkey , common marmoset , and squirrel monkey . there was hardly any difference in the expression of 2 transcripts which indicated malr did not affect the expression of the supt16h gene . its product was longer than that of the original transcript because alternative splicing retained an intronic region . the malr in the intron was not spliced and exonized ( exon 2 ) wholly between exons 2 and 3 ( figure 3 ) . comparing the new alternative transcript and the malr - derived transcript ( bx_333035 ) , we identified specific features of alternative splicing . entire sequences of the new exon of the alternative transcript identified in rt - pcr were derived from the malr . as shown in figure 5 , the malr provided both typical alternative splice sites , the acceptor site ( ag ) and a nucleotide ( g ) of the donor site ( gt ) . although these splicing sites were typical sequences recognized by the spliceosome , the splicing pattern could be changed . in case of the transcript related to the malr ( bx_333035 ) , the malr in the intronic region was also exonized to form a new alternative transcript . . only 1 site , the acceptor site , was recognized by the spliceosome , and the donor site was skipped . additionally , the elongated exon included malr - derived sequences and sequences of intron and exon 3 of the original and new alternative transcript . in spite of the different splicing pattern , the new alternative transcript was expressed broadly in tissues of humans and other primates , similar to the original and malr - related transcripts ( figures 3 and 4 ) . alternative splicing is a common mechanism by which most genes are processed [ 27 , 28 ] . in a previous report alternative splicing can be controlled by genetic mutation and epigenetic alteration [ 30 , 31 ] . however , the spliceosome can recognize splicing sites created by mutation and cryptic splicing sites , leading to alternative splicing . in addition to point mutations , the integration of transposable elements can induce alternative splicing as genetic mutations [ 2 , 25 ] . if the 2 transcripts showed different and tissue - specific expression patterns , we could assume that tissue - specific factors contributed to alternative splicing ; however , both transcripts were expressed broadly in tissues of humans and primates . various mechanisms can be considered to understand the differences between the 2 transcripts . compared to original transcript , however , the new exon includes intronic sequences by the typical splice site , ag - gt ( 35 ) , in the new alternative transcript , and ag ( 3 ) in the malr - derived transcript existing in the intron . if so , we could guess that the acceptor site and donor site of the new exon could be marked as a weak splice site by epigenetic regulation , such as histone modifications and dna methylationan , although it was the typical splice site . in a previous study , histone modification was reported as important factor for alternative splicing . among histone modifications such as h3k36me3 , h3k79me1 , h2bk5me1 , h3k27me1 , h3k27me2 , and h3k27me3 enriched in exon , the donor site of the new exon could be recognized weakly by the spliceosome with h3k36me3 which is found in weakly expressed exon [ 35 , 36 ] . additionally , the dna methylation and the cg content in the new exonic region could affect the expression of the 2 transcripts . zhou et al . ( 2012 ) reported that exclusive exons have lower levels of cg and methylated cg , whereas retained intron has higher levels . we suggest that the malr integrated into an intron of the supt16h gene before the divergence of new world monkeys and prosimians , and it played a biological role in the evolution of the supt16h gene during primate evolution by providing an alternative splice site . although further analyses are required to conclusively elucidate the functions of the transcriptional variants of the supt16h gene in primate genomes , the results of this study will help us understand the characteristics of the transposable element insertion driving the transcript diversification and its effect on the evolution of the host gene .

introduction : isolated pulmonary metastases from soft tissue sarcomas occur in 20 - 50% of these ( the issue is about metastases , not lung cancer ) patients , and 70% of these patients will present disease limited only to the lungs . surgical resection is well accepted as a standard approach to treat metastases from soft tissue sarcomas isolated in the lungs , and many studies investigating this technique have reported an overall 5-year survival ranging from 30 - 40% . the most consistent predictor of survival in these patients is complete resection . the aim of the present study was to determine the demographics and clinical treatment - related variables associated with long - term ( 90-month ) overall survival in patients with lung metastases undergoing pulmonary metastasectomy from soft tissue sarcomas.methods:we performed a retrospective review of patients admitted in the thoracic surgery department with lung metastases who underwent thoracotomy for resection following treatment of the primary tumor . data regarding primary tumor features , demographics , treatment , and outcome were collected.results:one hundred twenty - two thoracotomies and 273 nodules were resected from 77 patients with previously treated soft tissue sarcomas . the median follow - up time of all patients was 36.7 months ( range : 10 - 138 months ) . the postoperative complication rate was 9.1% , and the 30-day mortality rate was 0% . the 90-month overall survival rate for all patients was 34.7% . multivariate analysis identified the following independent prognostic factors for overall survival : the number of metastases resected , the disease - free interval , and the number of complete resections.conclusion:these results confirm that lung metastasectomy is a safe and potentially curative procedure for patients with treated primary tumors . a select group of patients can achieve long - term survival after lung resection .

sarcomas are a group of very uncommon malignant tumors that compromise 1% of all adult malignancies.1 approximately 11,590 new diagnoses and 3,560 deaths from soft tissue sarcomas ( sts ) and bone sarcomas occur annually in the united states.1 isolated pulmonary metastases from sts occur in 20 - 50% of these patients , and 70% of these patients will have disease limited only to the lungs.2 once systemic metastatic disease develops , the prognosis is typically poor.3 patients with extremity sarcomas are more likely to develop distant metastatic disease as the initial site of recurrence , whereas those with retroperitoneal and visceral sarcomas tend to present local recurrence.4 although considerable progress has been achieved in both chemotherapy and surgical treatment of sts , pulmonary metastases remain a major cause of mortality . given that a diagnosis of pulmonary metastases significantly affects outcome , treatment and prognosis , the detection of such pulmonary metastases is of vital importance.5 surgical resection is well accepted as a standard approach for the treatment of metastases from sts isolated to the lungs , and many studies investigating this technique have reported an overall 5-year survival ranging from 30 - 40% 6,7 . chemotherapy has not been shown to increase survival following the complete resection of pulmonary metastases.6 pulmonary metastasectomy is currently indicated for patients with the following criteria : primary tumor controlled , possibility of complete resection verified by computed tomography ( ct ) of the chest , pulmonary function and performance status compatible with the proposed lung resection , and lack of another available treatment that would be more effective than the surgical procedure.8 although the results obtained for pulmonary metastasectomy are encouraging , there is a need to better select and identify the patients who would benefit most from surgical removal of pulmonary metastases to avoid unnecessary surgical procedures . many authors have reviewed their experience in an attempt to clearly identify the variables associated with better survival . important predictors of survival include disease - free interval , histology of the tumor , the number of metastases and complete resection.5 - 7 among these , the most consistent predictor of survival is complete resection.6,7,9 the number of metastatic nodules and their impact on survival was initially considered to be an independent predictor of survival.10 however , if complete resection can be achieved , the number of pulmonary metastatic nodules does not seem to influence survival.6,7,11 since the early 1990s , our institution has employed a prospective protocol - oriented approach for the treatment of patients with resectable lung metastases to define factors related to outcome following lung metastasectomy . the aim of this study was to report the outcomes of patients with pulmonary metastases from sts who were submitted to surgical resection and to identify prognostic factors that affect overall survival in a group of patients from a single institution . the patients were referred to the thoracic surgery department of ac camargo hospital between 1990 and 2006 . the following characteristics of the primary tumors were recorded : primary tumor site ( extremity vs. others ) , completeness of surgical resection , histological type and grade , and neoadjuvant and adjuvant treatment . all patients were evaluated for the presence of lung nodules before treatment of the primary sts , followed by evaluations at routine follow - up appointments . the disease - free interval was defined as the time period between treatment of the primary tumor and the diagnosis of pulmonary metastases . patients with the following characteristics were considered eligible for pulmonary resection : 1 ) primary tumor controlled , 2 ) nodules confined to the lung parenchyma , 3 ) nodules that were amenable to complete surgical resection , 4 ) pulmonary function and clinical condition compatible with the scheduled operation , and 5 ) the unavailability of more effective treatment options for the patient at that time.in addition to the evaluations , we collected variables related to the pulmonary metastases , including the number of pulmonary nodules on the ct scan , the number of malignant pulmonary nodules resected , the size of the largest nodule resected and the laterality of the pulmonary nodules . all patients underwent a chest ct scan prior to surgery to evaluate the possibility of complete resection . radiological characteristics were analyzed in each patient as follows : the number of pulmonary nodules , the size of the largest nodule and the laterality of the nodules . the number of nodules reported in this study was the final number confirmed by the surgeon after complete resection . this procedure was typically initiated on the side where complete resection was deemed more difficult and included the number and size of the nodules identified by chest ct scan , the anatomical position of one or more nodules relative to the pulmonary hilum , and the potential need for a more complex and extensive lung resection . patients underwent surgery with general anesthesia and single lung ventilation . in all cases , we attempted a complete resection while preserving the lung parenchyma with a margin of 10 mm . , mediastinal lymph node dissection is not routinely performed in patients with lung metastases . in cases of a single pulmonary nodule at surgery , frozen sections were collected . if the histological evidence suggested the possibility of primary lung cancer , the patient was submitted to lobectomy with radical mediastinal lymph node dissection whenever possible . the date of surgery , type of resection ( complete or incomplete ) , number of malignant or benign nodules resected , size of the largest nodule , and type of lung resection ( wedge , segmentectomy , lobectomy or pneumonectomy ) were documented . some patients received chemotherapy based on the discretion of the medical oncologist , before or after surgical resection of the lung metastases . the type of radiological response ( partial , stable , complete , or progression ) as defined by recist12 criteria was documented . after surgery , the patients were discharged from the hospital following chest tube removal and in good clinical condition . after hospital discharge , the patients were followed - up with clinical and radiological examinations ( chest x - ray and ct scan ) every three months during the first two years and every six months subsequently until the fifth year . an annual radiological ( chest x - ray ) the observed postoperative complications were fever > 38c , bleeding , air leakage for more than seven days , intensive care unit stay for more than two days , cardiac complications ( arrhythmia , ischemia , and infarction ) , pulmonary atelectasis , embolism or respiratory failure , stroke and infection . whenever necessary , a histological confirmation of the new tumor was performed . if the recurrence was confined to the lungs and if the nodules were considered resectable by the thoracic surgeon , the patient was offered another attempt at complete disease resection . survival was calculated from the time of resection of the primary tumor and from the time of first metastasectomy to death or until the last follow - up . the log - rank and breslow tests were used to compare survival differences for each variable . multivariate analyses to determine the independent prognostic factors for overall survival were performed using the cox proportional hazard model14 as identified by the univariate analyses . survival time was determined whenever more than one pulmonary metastasectomy was performed , with time zero denoting the first thoracotomy . survival was calculated from the time of resection of the primary tumor and from the time of first metastasectomy to death or until the last follow - up . the log - rank and breslow tests were used to compare survival differences for each variable . multivariate analyses to determine the independent prognostic factors for overall survival were performed using the cox proportional hazard model14 as identified by the univariate analyses . survival time was determined whenever more than one pulmonary metastasectomy was performed , with time zero denoting the first thoracotomy . the patients included in this retrospective study ( n = 77 ) underwent a total of 122 thoracotomies ( mean of 1.6 thoracotomies / patient ) ; 273 nodules were resected , with a mean of 3.5 nodules per thoracotomy . five of the nodules were carcinomas and were treated as primary lung tumors , and more than four nodules were resected in each of 18 ( 18.1% ) patients . according to the primary sts , 66 ( 85.7% ) patients had extremity tumors , and 11 patients ( 14.3% ) had visceral or retroperitoneal tumors . in 64 patients ( 83.1% ) , the primary sts histology ( table 1 ) and grade ( table 2 ) were registered as well . in the 13 remaining patients , we could not find sufficient detailed information regarding the histological type and grade of the primary tumors in the medical records because the primary tumors of these patients had been resected at other institutions . based on the final confirmation of sarcomas from the pathology of the pulmonary metastases , these tumors were classified as undetermined and excluded from statistical analyses in which histology was considered . the median follow - up time was 36.7 months ( range : 10 - 138 months ) . twenty - nine patients were alive at the time of the final analyses , and 13 ( 16.9% ) showed no evidence of disease . thirty patients ( 39% ) died due to metastatic sts , and 18 ( 23.4% ) patients were considered lost to follow - up after missing two medical evaluations . more than one complication occurred in some patients , including fever , atelectasis , prolonged air leakage ( > 7 days ) , infection and a prolonged hospital stay ( > 30 days ) . the median overall survival for all patients was 36.7 months , with an estimated 5-year survival rate of 34.7% ( figure 1 ) . the median survival for patients with one , two , three or more pulmonary metastasis resections was 15.1 , 44.8 and 48.1 months , respectively . no significant difference in survival was detected between the groups with respect to the number of thoracotomies ( p = 0.07 ) . all other factors evaluated in the univariate analyses , including the response to adjuvant chemotherapy , type of resection , number of resected nodules , laterality of the nodules on the ct scan , number of thoracotomies and size of the largest nodule resected , were not statistically significant ( p>0.05 ) . multivariate analyses identified the number of pulmonary nodules resected ( p = 0.003 ) , the disease - free interval ( p<0.001 ) and complete resection ( p<0.001 ) as independent prognostic factors for overall survival ( table 4 ) . the resection of pulmonary sarcomatous metastasis remains the cornerstone for treatment of isolated pulmonary metastases in patients with sts.14 in the absence of other effective treatments and of contraindications , patients with lung metastases from sts benefit from surgical therapy.16,17 approximately 20 - 30% of patients undergoing pulmonary resection can experience long - term survival.5,6,18 improved survival following the resection of lung metastases has increased the surgical indications for such lesions , thus confirming that surgery itself affects the outcomes of patients who are candidates for a complete resection of nodules confined to the lungs . therefore , resectability should be evaluated preoperatively according to specific criteria . the recent introduction of positron emission tomography using 18-fluorodeoxyglicose ( fdg pet - ct ) has proven to be of clinical value for the proper staging of cancer patients to better select the best candidates for resection . according to pastorino et al . , this method can eliminate 21% of patients who are otherwise considered as surgery candidates.19 unfortunately , pet - ct was not available at our institution for most of the patients in this study . 5 - 7,17 the factors evaluated included the disease - free interval number of metastatic lesions resected , tumor doubling time , laterality , number of thoracotomies and resectability of the lesions.20 - 23 stss are an extremely heterogeneous group of tumors with a variable biological behavior . therefore , the histological type and grade of malignancy are the most important prognostic factors in the treatment of primary tumors.24 moreover , other studies have found histology to be predictive of pulmonary metastasis , resectability , and overall survival in extremity sts.6,25 the 77 patients included in this study underwent 122 thoracotomies for the treatment of lung metastases . the estimated 5-year overall survival of our patients was 34.7% at an average follow - up of 36.7 months . this outcome is similar to previous reports .5,7,9 in a review of the national cancer institute , the overall 5-year survival rate was 28% , with a 35% 3-year survival rate for patients who underwent complete resection . in that series , patients who experienced incomplete resection demonstrated a median survival of 9 months.26 with a median follow - up period of 27 months , the international registry of pulmonary metastases reported a 5- and 10-year survival rate in 936 patients with sts lung metastases of 30% and 22% , respectively .7 however , this result does not compare to the 14 - 21% overall survival reported by some studies with longer follow - up periods.6 several reports have demonstrated the prognostic importance of the number of nodules detected in the preoperative workup . the sensitivity of detecting pulmonary nodules varies depending on the technique used.23 in a prospective study including 182 patients with cancer and pulmonary metastases by younes et al . , ct scan detected the exact number of nodules resected in 47% of the patients . however , ct scan underestimated the actual number of lung nodules found during surgery in 28% of the cases and overestimated that number in 24%.27 in the present study , we discovered a poorer outcome in patients in which 4 nodules had been resected , as shown in the multivariate analysis . for some authors , the number of pulmonary metastases does not preclude resection because the ability to achieve complete resection is of greater prognostic value than the number of lesions removed.28 in a review of the european organization for research and treatment of cancer ( eortc ) database , the majority of patients undergoing resection ( 53% ) had only one lesion , although 15% of the patients had more than four lesions at the time of pulmonary metastasectomy.29 previous studies have examined the number of nodules resected and the presence of unilateral and bilateral metastatic disease as prognostic factors . casson et al . demonstrated that patients with three or fewer nodules survived significantly longer than did patients with four or more nodules resected9 . similar findings have been noted by putnam et al.5 who found that patients with four or fewer nodules survived longer than did patients with more than four nodules resected . the same group demonstrated that unilateral versus bilateral disease is not a significant indicator for prognosis . in most studies published to date , no differences have been observed in the survival of patients with metastases confined to one lung compared to bilateral lesions , despite the histology of the primary tumor 7 . the current study did not identify laterality as an independent indicator of prognosis . in the present study , the number of nodules resected had a significant effect on long - term survival in both univariate and multivariate analyses ( p = 0.003 ) . although the maximum number of pulmonary metastases often predicts survival,5,10 subgroup analyses have found that complete resection is a stronger predictor of survival , regardless of the number or size of the nodules.31 based on multivariate analysis , patients with synchronous metastases ( dfi<16 months ) presented with a significantly lower survival rate ( p<0.001 ) than did patients with metachronous disease , as reported in other studies.6,7,21,22,32 the correlation between dfi and survival is not consistent in the published data . depending on the study , dfi ranging from 8 months to as long as 5 years have been a significant prognostic factor ; however , the results are vague and inconclusive according to some authors.29,31 previous reports have been unable to consistently demonstrate a correlation between the primary site of sts and survival.29,33 in the current series , univariate analyses demonstrated better survival for patients with non - extremity primary tumors . in the multivariate analyses , however , the site of the resected primary tumor did not affect the long - term survival of patients submitted to pulmonary metastasectomy.6 similarly , no significant effect on outcome was observed between extremity tumors and those present in other primary sites . preoperative and postoperative systemic therapy had no significant effect on the long - term survival rate in this patient population , which is consistent with previously published data.34,35 although the patients ( n = 26 ) submitted to neoadjuvant treatment with chemotherapy prior to primary tumor resection demonstrated a numeric difference in the univariate analyses ( p = 0.018 ) , this variable did not have a significant effect in the multivariate analyses . this variable was a strong prognostic indicator in the multivariate analyses ( p<0.001 ) , which is consistent with previous reports.5 - 7,10,11,21,22 the median survival for patients who underwent complete resection was 44.8 months , as compared to 9.5 months in the group who underwent incomplete resection ( n = 15 ) . in the present study , 15 ( 19.5% ) patients were resubmitted to surgery for recurrent metastases , which ranged from one to four thoracotomies for complete resection . curiously , these patients demonstrated better survival ( median survival : 48.1 months ) than did those who underwent one thoracotomy for metastasectomy ( median survival : 15.1 months ) , which is consistent with previously published data.21,22,31,36 weiser et al . investigated a series of 86 patients who had undergone repeat resections . a poorer survival after repeat resection was associated with the following factors : more than three nodules , lesions larger than 2 cm , and high - grade histopathology . the presence of 0 - 1 adverse factors was associated with a disease - specific survival of 65 months . in comparison , the presence of 2 - 3 factors was associated with a disease - specific survival of 10 months.37 the type of surgical resection and the total number of thoracotomies failed to significantly influence patient outcome . the majority of our patient population ( 81.8% ) underwent a sublobar resection , and ten ( 15.6% ) patients required a lobectomy for complete resection . as reported previously , limited resection with lung parenchyma preservation is considered adequate to achieve clear margins and thus to provide local control of the disease . recently , cerfolio et al . showed that among 37% of patients who were candidates for video - assisted thoracic surgery ( vats ) , intraoperative palpation during a thoracotomy detected more nodules than had been identified using preoperative imaging by ct scan.38 there may be a role for minimally invasive surgery in the pulmonary metastasectomy setting ; however , additional prospective studies are required . although randomized controlled studies comparing observations to surgery are impossible , the current data suggest that the patients most likely to benefit from metastasectomy have a longer disease - free interval , single metastases , and complete resection of the metastases . the administration of neoadjuvant or adjuvant therapy may allow for further prolonged survival in some instances , despite the finding that surgery alone fails to treat lung metastases in the majority of patients . it should be emphasized that surgery does not change the biology of the tumor or the metastatic process , and a definitive cure for most patients represents the combination of host histology , tumor spread , response to systemic therapy , and surgical resection , which together render the patient free of disease . all patients with resectable nodules evaluated by ct scan and selected by the inclusion criteria should be considered as candidates for surgical resection because they might reveal an interesting overall survival , as shown by the present study .

obsessive compulsive disorder ( ocd ) is a common psychiatric disorder which is easily recognized . however , sometimes patients of ocd present in such an atypical or bizarre way that their problem comes to notice as being a psychiatric disorder after multiple consultations in different specialties . we are reporting a case of a man who had first sought opinion in the department of ear , nose and throat ( ent ) for hearing impairment . he was then referred to a neurologist and a general physician for evaluation of neurological cause of his symptom . as no pathology related to ent or neurology could be detected , he was referred to the department of psychiatry . the patient 's chief complaints were difficulty in hearing and inability to understand at once . he could be diagnosed as a case of ocd after meticulous evaluation and studying his response to treatment . there was significant improvement in all the presenting symptoms over a period of 6 weeks on 60 mg of fluoxetine .

obsessive compulsive disorder ( ocd ) is a common psychiatric disorder ( 2% - 3% of the general population ) which is diagnosed easily most of the time by the psychiatrist or even the physician . it is sometimes very difficult to properly diagnose a person for ocd based only on an interview . the presentation of ocd may be so atypical or unusual that the patients may be referred to different specialties before the actual diagnosis can be made . neuropsychological studies have consistently found cognitive impairment in the domains of memory and attention in patients of ocd . anxiety , lack of confidence , indecisiveness , associated clinical symptoms of ocd , along with impaired memory and attention , may further complicate or interfere in the delivery of information . a 36-year - old man used to request multiple repetitions of spoken words in order to understand what was said . he was then referred to a neurologist and a general physician for evaluation of neurological cause of his symptoms . even after multiple consultations with the ent , neurology and medicine specialists , no organic pathology to explain his symptoms could be detected . the patient lost considerable time and money because of delay in diagnosis caused by the atypical presentation to specialties other than psychiatry . at last , the patient was referred to the department of psychiatry for further evaluation . the patient 's chief complaints were difficulty in hearing , grasping or comprehending the components of conversations , interactions or clinical interviews at once . he had this problem for the last 6 months . during the interview , almost every question or the instruction had to be repeated slowly 2 to 3 times or more to make him understand . he was anxious and expressed regret repeatedly for the inconvenience caused to the doctor because of his disability . initially the patient was put on mirtazapine 15 mg / day and clonazepam 0.75 mg / day , keeping in mind the possibility of anxiety disorder nos ( not otherwise specified ) . mental state examination of the patient revealed impaired attention and concentration with depressive and anxiety symptoms . the possibility of ocd , predominantly obsessive type , was kept on the basis of answers of leading questions , and he was put on fluoxetine 60 mg / day ( titrated from 40 to 60 mg ) and clonazepam 0.5 mg / day . there was drastic improvement in all the presenting symptoms , including his ability to grasp and comprehend conversation , along with improvement in anxiety symptoms and clarity in thinking over a period of 6 weeks . the patient was then able to report his pre - treatment problem and the level of improvement comprehensively . he accepted that he was unable to understand or express his own problem before treatment . according to him , there were thoughts keeping his mind busy all the time , to the extent that he was unable to think or communicate his problem effectively . according to him , initially he used to make frequent unsuccessful attempts to get rid of these excessive and unnecessary thoughts . according to the wife of the patient , after treatment the patient was able to hear clearly . the diagnosis could be confirmed as ocd on the basis of patient 's spontaneous elaboration of symptoms and the level of improvement . in this case , the presentation of ocd is atypical , as the chief complaint was problem in hearing . he was able to hear on normal volume of sound , but the sentences needed to be repeated multiple times . moreover , no ent - related or neurological cause of hearing impairment was found in the patient . though hearing difficulty was not his chief subjective problem , he consulted the ent doctor only after repeated and forced advice by his wife and relatives . the hearing problem as perceived by the patient 's friends and relatives was basically a misinterpretation of the patient 's problem secondary to his psychiatric illness . also the patient was unable to express his actual problem at the time of presentation . the patient used to ask for repetition of the spoken sentences multiple times in order to grasp the components of interaction because of his significantly impaired attention . however , there could be the other possibility that this behavior could be a compulsive act of the patient to confirm his doubts . the patient could not express his problem elaborately , which was one of the reasons for not being able to make an early diagnosis . there were multiple factors that had complicated and hindered the narration of history by the patient . lack of clarity of thoughts and associated clinical symptoms of ocd ( especially predominantly obsessive type ) , indecisiveness , anxiety , lack of confidence , along with impaired memory and attention , were the possible factors in this case , as also reported in different studies . the patient could express details of his illness comprehensively once his above - mentioned signs and symptoms improved significantly . his wife and relatives also reported improvement in his hearing , which was actually the improvement in his attention and ocd symptoms . such an atypical presentation may lead to misdiagnosis , delay in diagnosis , loss of time and money as the patient may present to other specialties instead of psychiatry . high suspicion for diagnosis of ocd and response to treatment may help in managing such cases . obsessive compulsive disorder is usually easily recognized , but sometimes its presentation is so atypical or bizarre that the problem comes to notice as being a psychiatric disorder after multiple consultations in different specialties . but if proper evaluation is done , such cases can easily be recognized and treated effectively .

the new medicare prospective payment system has been challenged with regard to its fairness in reimbursing hospitals adequately , given the true resource needs in caring for patients . most of these criticisms are now labelled as issues about adjustments for severity of illness . critics point to the large amount of unexplained variation in charges and length of stay within the existing drg 's as indirect support for their contentions about inadequate adjustments.a paradigm is presented which argues that the key questions on the types of severity of illness measures to be utilized in future refinements of drg 's revolve around the extent and type of data which can feasibly be included in any workable reimbursement approach . a paradigm is presented on how these questions about information define a series of research options in the severity of illness arena .

the new drg - based prospective payment system ( pps ) for medicare is a revolutionary concept in health care finance . it introduces a new approach to reimbursement , which is based upon a defined clinical product . in contrast , under retrospective cost - based reimbursement , each component of the process of care ( each visit , x - ray , procedure , and day of care ) was paid for separately . that open - ended payment system has proved too inflationary ; federal outlays for medicare have increased from $ 1.1 billion in 1966 to $ 51 billion in 1982 , a rate of inflation which has outpaced the gross national product , the consumer price index , the median family income , and other benchmarks of the economy 's growth . under the prospective payment system the shifts in the medicare system should stimulate major efficiencies , as providers of care begin to consider which components should be introduced into the clinical production process , and which should be rejected . it is expected that there will be a major break in the upward - spiraling overutilization of services which produce no improvement in clinical care . it is hoped that the drg system will create incentives for physicians and hospitals to shorten hospital lengths of stay and incentives to decrease resource consumption per episode of illness . however , considerable debate has arisen over whether the current drg formulations in the prospective payment system represent the best approach for achieving these objectives . with tens of billions of dollars at stake each year , the health care financing administration ( hcfa ) will face innumerable challenges to the design and administration of the prospective payment system . these challenges are likely to encompass a broad spectrum : on one hand , there may arise overt challenges to the equity of the drg 's , particularly with respect to their ability to adjust fully for differences in severity of illness . on the other hand , there is the possibility of covert challenges , in particular , those which involve clinical opportunism . for example , under the pps , providers may seek to : 1 ) admit larger numbers of patients , especially patients with easily treated illnesses and short anticipated lengths of stay ; 2 ) split illnesses into two parts , in order to spread a patient 's care over two hospital admissions ; 3 ) unbundle diagnostic procedures , shifting some to the ambulatory setting ( outside the pps ) ; 4 ) upgrade primary and secondary diagnostic codes , in order to obtain a higher - paying drg assignment ; 5 ) perform more complex surgical procedures to inflate the drg ( procedure inflation ) ; and 6 ) prolong the hospital stay of patients with lingering illnesses , as the outlier trim point is approached . when a patient has more than one clinical problem , there arises the possibility of gaming the data about the correct principal diagnosis ; there also arises the possibility of outright fraudulent representations . hcfa can respond to these challenges by improvements in design of the basic drg formulation and administrative rules and procedures . it is likely that changes in drg structure and design can best deal with the equity and overt challenges to the system ; administrative controls will likely have their greatest strength in dealing with the covert challenges . a cornerstone of the new prospective payment system for medicare is the use of a diagnosis - related group case classification system . the drg system employs existing discharge abstract data on the patient 's principal diagnosis , secondary diagnoses , surgical procedures , age , sex , and discharge status , in order to classify patients into different groups that are clinically coherent and homogenous with respect to resource use the current drg 's have been criticized for failing to account for severity of illness . controversies about severity are based upon the assumption that , within each drg , there are sicker patients who will cost more . hospital administrators and physicians have expressed concern that they will be unfairly penalized if their patients are sicker and consume greater resources . medicare administrators have concerns of their own : to the extent that providers can measure or anticipate patient severity , they may reject patients with severe illnesses in order to maximize their profit . among policy analysts and economists , severity has become the buzzword to describe the sources of otherwise unexplained variations in resource use within the drg 's . it is the authors ' opinion that the objectives of a system for defining severity of illness under drg 's ( with respect to medicare reimbursement ) should be threefold : 1 ) to place patients into categories which are homogeneous with respect to resource need ; 2 ) to categorize patients in such a way that manipulations of data are minimized ; and 3 ) to avoid producing distortions of provider behavior that could adversely affect the outcome of patient care . ( for example , with respect to the third objective , hcfa has refrained from using death as a classification factor in most drg 's . unless this is a common outcome , in heart attacks hcfa does not want to reward hospitals for letting patients die . ) one of the major assumptions by many critics of the drg system , particularly those who point to large variations in resource use within drg 's , is that the major source of this variation is the absence of equitable severity of illness measures . . these may be responsible for a substantial portion ( or even the majority ) of the variation in costs and length - of - stays observed to date in drg 's and may be even more important as covert challenges to restraining medicare costs . for example : 1 ) situations exist where there are no clear rules for determining an individual 's correct principal diagnosis or the validity of the presence of a complication ; 2 ) existing practice patterns frequently involve substantial proportions of medically inappropriate use , and 3 ) clinical practice patterns exist where definition of an appropriate episode of medical care can be split to produce two or more reimbursable episodes . stated another way , variation in resource use within drg 's may be due not only to inadequacies of measurements and adjustments , but also to the above types of problems . in this article , these three basic sources of confounding will be reviewed , and the measurement of severity of illness will be discussed , focusing on an applied research paradigm for testing new methods of incorporating such measurement approaches into the medicare prospective payment system . the major factor which determines the drg assignment , and thus determines the payment of the provider , is the physician 's assessment of the patient 's diagnosis . the accuracy and completeness of the diagnostic and procedure data are fundamental to the drg system . regulations require that physicians record all diagnostic and procedures data on the face sheet in an honest and responsible manner . peer review organizations are charged with monitoring the validity of diagnostic information provided by the hospital for the purposes of payment . however , hcfa has accumulated evidence suggesting serious errors in the description and coding of the principal diagnoses and principal procedures . according to studies by the institute of medicine and the department of management and research of the university hospitals of cleveland , errors and discrepancies in the listing of the principal diagnoses range from 20 - 40 percent ( demlo et al . , 1978 , much of this error was probably due to sloppi - ness and to use of unqualified record abstractors . these errors represent a threat to the validity of all analytic studies employing historical hospital diagnostic data , and almost certainly contribute to the variance found in some previous drg analyses . the extent of the sloppiness problem should , however , rapidly diminish under pressure from concerned administrators and from hcfa oversight through professional review organizations ( pro 's ) and fiscal intermediaries . creation of each drg category starts with consideration of the patient 's primary diagnosis and the performance or nonperformance of an operating room surgical procedure . a substantial number of the drg categories are then further divided , depending upon the presence or absence of significant complications or comorbid conditions . a complication is a condition other than the principal diagnosis which occurs after the onset of the principal diagnosis ( and usually after the patient is admitted to the hospital ) . many ( if not most ) medicare patients admitted to the hospital have one or more comorbid or complicating conditions in addition to their primary diagnosis . in the planning of the drg system , it was reasoned that the length of stay and the consumption of resources ( and , of course , reasonable reimbursement ) are likely to be higher if a substantial complication or comorbidity is present . therefore , specific lists of such conditions have been developed , based upon the judgment of the physician teams who assisted the drg design team at yale . a substantial comorbidity or complication is defined as any secondary diagnosis likely to prolong the hospital stay of at least 75 percent of patients by one day or more ( yale university , 1982 ) . one example of a substantial comorbid - complicating condition is international classification of diseases , ninth revision , clinical modification ( icd-9-cm ) code 202.98 ( hodgkin 's disease ) . if one or more of these is present and is listed on the discharge data abstract sheet as a secondary diagnosis , the patient 's care needs are considered more resource - intensive . therefore , he or she is usually placed in a drg category for which the hospital is awarded a higher reimbursement for the episode of illness . the concept of a complicating condition or a comorbid condition requiring additional resources has validity and should contribute to a reduction in variance among drg categories . at the same time , operationalization of the concept in a prospective payment system raises some serious medical and definitional problems . one obvious problem is that the source of the information used to classify each patient into a drg is the face sheet of the medical record . for example , the presence or absence of a substantial complication or comorbid condition can only be determined from the list of all secondary diagnoses on the face sheet . however , as noted earlier , there is considerable error in hospital discharge data . another serious problem is whether rewarding hospitals for more complications is a fundamentally sound approach . if a comorbid condition is present upon admission , its inclusion as a factor discriminating between types of patients may be valid . for complications which arise after admission , however , their inclusion may reward less careful practitioners , while penalizing those who make investments to prevent such untoward events . might a hospital which reduces its infection control procedures benefit if its infection rates subsequently rose ( thus sending additional patients into higher reimbursement drg categories ) ? the current drg classification system , however , does not distinguish a comorbid problem present at the time of admission from a complicating condition which occurs subsequently . this dichotomy , although conceptually attractive , would be extremely difficult to operationalize ; identification of many comorbid conditions at admission might require a series of investigational procedures , which are not normally done and which might involve additional cost and risk for patients . an even more difficult problem is how to define and validate the actual presence of a substantial comorbidity or complication . for example , diabetes mellitus with renal manifestations is one of the drg substantial comorbid / complicating conditions . does the presence of an abnormal glucose tolerance test and the finding of trace proteinuria constitute icd-9-cm 250.4 for the purposes of reimbursement ? similarly , icd-9-cm 780.0 , the diagnosis of coma ( including stupor , drowsiness and somnolence ) is a substantial comorbid / complicating condition . how is drowsiness validated by the pro ? how is respiratory failure ( icd-9-cm 799.1 ) defined ? would the presence of a positive culture from the wound site for any organism be sufficient ? would it depend upon the colony count of infecting organisms or the types of organisms found ? would it require that the infection be significant to the point where fever occurs or antibiotic therapy is instituted ? if the lowest level of this hierarchy of definitions is chosen , every patient will have their wound site cultured , and many with clinically insignificant bacterial organisms ( many of which are normally present on the skin ) will suddenly start to become included in the complicated condition category . if sufficient evidence is antiobiotic treatment , we are likely to see many elderly patients ( who are already at higher risk of adverse drug reactions ) having such agents administered more frequently , because of the bias introduced by the drg reimbursement system . as long as the definitions of comorbid and complicating conditions remain vague , clinicians and data abstracters have an incentive to include larger numbers of such conditions on the data abstract sheet , whether or not such conditions are clinically relevant . indeed , most of the current drg scheme makes no distinction between complicating / comorbid conditions related and not related to principal diagnosis . related comorbidities or complications may contribute more ( or less ) to resource consumption than unrelated conditions ( louis and heineccius , 1983 ) . it should be noted that these definitional issues plague not only the assessment of complicating or comorbid conditions , but also affect the determination of the principal diagnosis . for example , when does a patient with peptic ulcer disease fit into drg 175 ( gastrointestinal hemorrhage , age less than seventy without c / c ) , rather than into one of the other peptic ulcer categories ( drg 176 or drg 178 ) ? is hemorrhage evidenced by a positive stool guaiac test for blood ? is it , instead , any aspiration of hematest - positive material through a nasogastric tube ( and if so , does it count any fresh bleeding which may have been induced by insertion of the nasogastric tube ) ? or is adjacent drg categories are frequently separated only by such terms as hemorrhage or complication . different parties are likely to define such terms differently , depending upon economic and other pressures . obviously , many of the adjacent drg 's require more precise separation and definition , for they rank among the high cost diagnoses for medicare patients . the boundaries separating many such drg 's are indistinct and , potentially , malleable . in maryland , for example , the mean charge for drg 140 ( angina ) was $ 2,409 in 1981 versus a mean charge for drg 143 ( chest pain ) of $ 1,925 . there are no well - accepted rules delineating the correct assignment of a patient to one or the other of these categories . thus , the incentives to label a case as angina , with a 25 percent greater reimbursement , will be substantial . there will still remain , however , a more serious basic issue how to relate a patient 's clinical reasons for hospitalization to the icd-9-cm coding system used in drg groupings . the international classification of diseases , now in its ninth revision , is still based on an anatomic approach to disease developed in the late nineteenth century by the great french forensic pathologist , dr . jacques bertillon . this system , despite its decennial updating and an american clinically modified version , was never designed to deal with clinical severity of illness . it is still not well - suited to this task . nor was the icd-9-cm coding structure even intended to accommodate a reimbursement system or withstand the pressures for precision that this requires . icd-9-cm 's central flaw is that there are no clear rules for determining when a patient has a particular problem which fits an icd-9-cm code . while a pro policing effort may prevent massive definitional abuse , the pressing need is for a research and development program to develop at least consensus guidelines and criteria for use of icd-9-cm terms . where there are objectively verifiable criteria , these should be disseminated by hcfa throughout the hospital and pro community nationwide . where there are no such criteria , definitional guidelines can be generated through consensus conferences ( such as the national institutes of health utilizes ) , opinion surveys of specialists and generalists , and other such means . certainly , the medical community will continue to be plagued with serious definitionally - induced variance unless such steps are initiated . in the current health system patients are often admitted to the hospital for problems such as low back pain , which can be treated at home , and cancer chemotherapy , which may be safely administered on an outpatient basis . if these current patterns of unnecessary admissions those not severe enough to warrant hospital admission are allowed to continue , medicare will continue to spend billions of dollars unnecessarily . many of the most common drg 's , particularly those not associated with surgery , involve conditions with a wide spectrum of clinical manifestations and needs for acute care . the decision whether or not patients in these categories require hospitalization instead of outpatient treatment involves a set of physiologic and clinical assessments which are often not captured by the diagnostic , procedure , and demographic factors used to designate drg 's . for example , the category heart failure ( drg 127 ) might include a patient with such excess fluid load that he or she was having severe difficulty breathing and was in need of aggressive therapy in a hospital environment . however , this drg category would also apply to a patient with long - standing heart disease who had gained some extra fluid weight as a result of dietary indiscretion and was noticing a mild increase in shortness of breath when walking a short distance . the latter case would not require hospitalizaion in most circumstances , but with the drg system there might be an incentive to admit such a patient for several days of therapy for the patient 's convenience and for the financial benefit of the provider . this situation involves no manipulation of the diagnosis as suggested by the term drg creep , but rather involves playing on a central weak point of the drg system a lack of severity of illness standards which distinguish inappropriate hospital admissions . other high frequency categories such as drg 182 ( gastroenteritis and miscellaneous digestive diseases ) and drg 132 ( artherosclerosis in patients over 70 ) involve the same potential problem of classification of disease by diagnosis , by procedure , and by age which could run the gamut from life - threatening acute situations to chronic , stable manifestations which can be effectively treated on an ambulatory basis . while it may be fraudulent to deliberately change the diagnosis of a patient so as to gain higher drg reimbursements , there are no accepted legal or procedural barriers to physicians lowering their severity standards for admitting patients within these medical drg categories . a study of over 8,000 medicare and non - medicare patients hospitalized in 41 massachusetts hospitals in 1973 and 1978 found that almost two out of every five days for medical patients with a length of stay ( los ) under ten days were inappropriate a rate higher than that of medical patients staying longer than ten days ( gertman et al . , 1982 ) . studies done by the boston university school of medicine indicated that in 1980 , among selected professional standards review organizations , levels of inappropriate hospital admissions among medicare beneficiaries ranged from a low of 12 percent to over 31 percent ( restuccia et al . , 1984 ) . both of these studies employed the appropriateness evaluation protocol methodology developed for hcfa ( gertman and restuccia , 1981 ) . part of the mysterious variation in days of care per 1,000 medicare beneficiaries across the country may represent variation in inappropriate admissions . assessments by systemetrics incorporated , using their standardized medreview instrument ( smi ) found that a smaller but still substantial amount of medicare admissions were inappropriate and that levels varied geographically ( systemetrics , inc . , january 1983 ) . given that neither of these instruments challenged the necessity of elective surgery ( i.e. , was the hysterectomy indicated ) , these must be conservative estimates . the joint massachusetts blue cross - massachusetts hospital association 's ancillary services review program has documented average levels of inappropriate laboratory tests , electrocardiograms and respiratory therapy in the 20 percent to 30 percent range ( hughes et al . , 1984 ) . after adjustments for certain severity of illness factors and for pricing differences , the dollar amount of inappropriate use of an ancillary service for a specific diagnosis varied over tenfold from the highest to the lowest hospital . these data suggest that a major source of variation among institutions for any given diagnostic grouping may not be due to inadequacies in the grouping method , but rather , largely due to inappropriate admissions , inappropriate days of care and inappropriate ancillary utilization . this possibility can be addressed by a major program of research on the correlation of unexplained drg variation with measures of inappropriate use . if this , along with the other basic factors , is the principal source of variation , then the current drg system may , in the near term , need only modest refinement rather than a dramatic overhaul . the drg case - mix classification system defines the relevant unit of service for reimbursement as the illness episode , which lasts from the time of admission to the hospital until discharge . the greater the number of admissions a hospital has for a fixed number of bed days and available staff , the greater its revenue will be . this could create incentives for shortening hospital lengths of stay and decreasing consumption of resource per episode . for some conditions a medical episode of illness has a clear beginning and end point which correspond fairly closely with initial admission to the hospital and subsequent discharge . for many conditions , however , the potential exists for deliberate abuse of the admission payment concept , by artificially dividing an episode of illness into two hospitalizations . in other words , patients may be admitted for several days or weeks , then discharged , and later readmitted for a procedure or for continued medical treatment . the medicare regulations recognize the potential for such deliberate abuses , and the admission pattern monitoring program is specifically charged with looking for such attempts . unfortunately , for some conditions , the proper end points of hospital care are less clearly defined . the single admission - to - discharge concept remains most problematic in the following two areas . there are a substantial number of common conditions where medical opinion may differ and individual physician judgment is used to determine whether a patient 's course of treatment should be completed during one or more hospitalizations . some physicians believe that patients should first be admitted for medical treatment , which includes fluids , antibiotics and nasogastric suction . other physicians and surgeons believe it is safe and proper to operate on the patient who is improving during a single hospitalization . at the present time , no medical review panel could fairly penalize competent physicians for choosing one course over the other . similarly , a patient hospitalized with symptoms of coronary insufficiency may undergo cardiac catheterization during his hospitalization and be discharged for a period of recovery and risk - factor modification ( e.g. , stopping smoking ) prior to coronary bypass surgery ; alternatively , these procedures might occur in the same admission . another example is the procedure - linked diagnosis of benign prostatic hypertrophy ; patients may be admitted with symptoms of urinary obstruction and then scheduled for prostatectomy either during the same hospitalization or at a subsequent admission in the near future . to illustrate this issue , the authors reviewed the top 100 drg 's and identified 32 examples of medically acceptable situations where , in their opinion , multiple procedure - linked admissions might occur ; these are listed in table 1 . in each case , for some physicians , it might seem preferable to complete the medical treatment , the diagnostic evaluation and the operative therapy in a single hospitalization . however , depending upon the habits and style of practice of the physicians in charge and depending upon the economic incentives which are allowed to come to the fore , a single process of diagnosis and treatment might be split into two care episodes . there are usually no well established guidelines to determine whether split admissions are appropriate for a given clinical problem . the older the patient and the more complicated his medical care , the more often physicians may decide to partition care into two or more hospitalizations . the second problematic area related to illness episode involves chronic diseases requiring episodic hospital care . in managing many illnesses , such episodes of illness are very difficult to monitor or regulate under the drg system . particular areas of difficulty are psychiatric illnesses such as alcoholism . if , after seven days , an alcoholic patient signs out of the hospital against medical advice , how hard should the hospital or physician fight to convince him to stay ? similarly , if a psychiatric patient is almost well , a potentially inappropriate incentive exists to allow him to return home too early . in such cases , there is a high probability the patient will return in the days or weeks to come . indeed , there are no clearly defined lengths of stay for inpatient treatment of alcoholism and psychiatric illnesses . such patients are usually treated to the point where they are able to function outside of the hospital environment . the more vigorously the acute episode of heart failure or chronic lung disease is treated , the less likely that the patient will be readmitted in the near future . a study conducted at university hospital and boston city hospital demonstrated that readmissions for heart failure were inversely related to the vigor with which excess fluid was removed during the hospital stay ( gertman and stanton , 1975 ) . patients who were discharged early with persisting but modest signs of fluid accumulation had a higher rate of readmission in the ensuing year . yet , examining only an individual case after the fact , a medical panel could not have successfully proven undertreatment of any single patient . relatively little research has been done , even at a descriptive level , on patterns of care for older individuals over time ; this deficiency is particularly great for those with multiple active chronic health problems . prior use , as discussed later , may in part explain subsequent hospital admission resource use . how patterns of care affect resource use and outcomes is a topic which requires extensive research . at the least , such research is necessary to minimize confounding efforts to properly adjust for severity of illness ; beyond this , any study of drg impacts on quality of care must develop such information . there is considerable concern among providers about whether the current drg system optimally achieves the stated objectives of the medicare prospective payment system . specifically , many fear that the pps may not equitably deal with systematic differences in severity of patient illnesses across all hospitals . as much as one might wish that 467 drg 's controlled for interhospital differences in patient severity , the large amount of unexplained variance in some drg 's is disturbing . of course , much of it may be simply random variation , with zero net effect across all admissions . on the other hand , there may be systematic variations by one or more hospital characteristics ( e.g. , teaching status , ownership , location , etc . ) , resulting in windfall gains or unfair losses . inevitably , however , an inaccurately defined output leads to inequities and , what is worse , gaming of the system . while all parties to this debate over the equity of drg 's generally understand the term severity and would agree that in any group of patients there are those with more severe and less severe illnesses , there is no agreement on how to : quantify differences in severity on a continuous scale of measurement ; translate such severity measures into uniform resource need measures ; and fairly monetize the resource measures in medicare reimbursement procedures . in fact , the authors would argue that there is no fully valid way to accomplish any one of these three measurement tasks . the best one can do is make approximations of the concept which reduce the extent of disagreement . recognizing that , even if one could remove all confounding factors , no perfect severity measure exists , hcfa and health care providers must move forward to make the best approximations possible subject to a host of practical constraints . the old drg system devised by the yale university group was subjected to extensive methodological criticism for not adequately dealing with major differences in severity among patients . several research organizations , including blue cross of western pennsylvania , systemetrics , inc . , susan horn 's team at the johns hopkins school of public health and others have demonstrated that they can achieve superior reductions in length of stay variance by alternative methods compared to the drg 's used in the old system ( young , 1979 ; garg et al . , 1978 ; horn , 1983 ) . horn has compared four existing classifications systems ( icd-9-cm drg 's , the old new jersey drg 's , disease staging , and generalized patient management paths ) with her severity of illness index for their respective variance reduction characteristics on a disease - specific basis across different hospitals . generally , horn 's chart abstract method achieves far greater subgroup homogeneity ( total charges and length of stay ) than do any of the other computerized , discharge abstract - based classification systems . horn and her co - workers have also tested whether their approach is superior to the new medicare drg system which has attempted to address some of the prior criticisms on severity adjustment . in a study of ten new drg 's at four hospitals , they have reportedly been able to achieve better than 40 percent additional reduction in variance by their severity of illness index ( horn et al . , 1983 ) . the issue for hcfa is not whether to simply stand pat with the drg system as currently defined versus attempting to identify possible improvements . rather , the issue is : what is the most cost effective way to proceed in enhancing the drg 's . while basic research must proceed on the sources of confounding , such as definitional imprecision , inappropriate use and variance in practice patterns , efforts to explore alternative severity of illness adjustments in the medicare pps must move forward as well . to assist in illustrating how a drg / severity of illness policy research program could be organized , figure 1 shows a decision tree model for questions which might be explored in the pps severity of illness revisions . the authors believe that the central issue structuring a practical severity of illness research paradigm is the amount and type of information available . the most important practical question to address is whether to limit enhancements of the case - mix method to those measures available only in the uniform hospital discharge data set ( uhdds ) . while some might argue that this is only a procedural constraint and should not have such an important place in the research agenda paradigm , the availability of information and the logistics of acquiring that information is in fact the most critical decision that medicare must address now if it wants to have an operationally revised system in the near future . within the current information set available to medicare , the next question which must be addressed is whether the focus of revisions should be to fine - tune the existing drg system or to alter it ? the former course leads to research option area a ; here the principal research work would be to evaluate better trim points , test use of dollar ( cost ) dependent variables more extensively than was possible in the initial drg design , structure definitional requirements more tightly to prevent abuse , develop a system of potential correction factors for inappropriate admissions , etc . alternatively , under research option area b , hcfa could decide to modify the drg 's by creating additional drg subcategories . applying the primary staged conditions and staging levels to selectively create subcategories where there is a clear need for reduction in currently large variances in costs per admission ( systemetrics , inc . , revisions might also be designed that anticipate issues of major quantitative importance in the near future . a pioneering institution in the field , brigham and women 's hospital in boston , has now started to extensively perform multiple major joint replacements in a single hospital admission episode rather than having two or more admissions . in their own internal , drg - oriented management information system , they have now subdivided drg 209 into two groups based on whether there is a single major joint replacement or multiple major joint replacements . this is an important , common surgical procedure where cost - effective and quality - of - care advances in surgical practice , involving multiple joint replacements per admission , are likely to disseminate fairly rapidly over , the next several years . research option area c , using the current uhdds dataset , would set aside the current yale drg framework and evaluate completely different ways of defining severity of illness differentials between institutions for purposes of reimbursement . these research options might involve use of multivariate quantitative models as opposed to a step - wise classification methodology to determine severity - adjusted reimbursement formulas . carol fernow at health care systems international , for example , has taken commission on professional and hospital activity ( cpha ) data and developed log - linear models which apply specific quantitative coefficients to factors such as specific secondary diagnoses ( e.g. , diabetes as a secondary diagnosis in cholelithiasis ) , types of secondary operation procedures , etc . , to compute case - mix intensity and expected lengths of stay ( fernow , 1983 ) . the potential flexibility of a multivariate model based on medicare uhdds data sets is shown in tables 2 , 3 , and 4 which were part of a teaching example developed at the boston university school of medicine in the spring of 1983 . in this hypothetical case situation , 27 patients who were discharged alive were admitted for heart attacks to three different hospitals in the same community . the current drg algorithm categorizes such live discharges sequentially by whether or not they have had a permanent pacemaker implant , and whether or not they have had cardiovascular complications . under the existing drg framework for classifying heart attacks , the only surgical data used is whether or not there is a permanent pacemaker implant and the complications factors are related solely to the cardiovascular system . as shown in table 2 , after adjusting for the highly significant class variable ( p = .0004 ) of the current drg category , no differences could be demonstrated among the hospitals ( p = .27 ) . in contrast , a simple multivariate model incorporated an age factor , a factor for whether or not the patient had any nonoperating room procedures ( such as insertion of a swan - ganz catheter , an endoscopic procedure , etc . ) and whether or not the patient had a prior history of a heart attack ( which could be determined fairly readily in a longitudinal data file ) . in this model , while the pacemaker variable is still the most important single factor explaining variation in charges , all of the other factors contributed and produce an r value 50 percent greater than a drg hospital comparison . additionally , the conclusion of the analysis using the multivariate model is that there are significant differences in severity among the three different hospitals ; in fact , it reverses the unadjusted results . what appears to be the most expensive hospital in terms of the unadjusted charges is actually the least expensive hospital after adjustment through the multivariate model . while this is obviously a simplified example , it does illustrate the potential to use more clinical diagnostic and procedure data than is employed in the current drg framework and to potentially obtain superior reductions in variance through these techniques . revisions of severity adjusters potentially unfettered by the serious clinical constraints of current drg branched groupings might provide substantial additional explanatory power and could , if useful , be quickly implemented because the data is readily at hand . another example of a factor which could be incorporated is whether there is a history of any socio - medical problem such as alcohol abuse , even though that may not be an active clinical issue during the admission . data collected by the health data institute , inc . on auto workers indicated that for major nonsubstance abuse admissions , individuals with a history of alcoholism have more expense per admission than persons admitted without such a prior history , controlling for all other relevant case - severity factors ( mcguire and fair bank , 1984 ) . if the research on revision and enhancement of drg 's is not constrained to working with just the current uhdds data items , a series of other research options become available . again , data issues are key . the next practical question is whether any expansion of the items needs to be limited to a small number of additional fields ( e.g. , five to ten characters ) or whether the process can be more open - ended in the types of data elements that are used . a further subdivision is whether common elements are to be used as adjusters for all diagnoses or procedures ; if so , then the focus would be on generic , nondiagnostic clinical measures such as vital signs ( research option area d ) . factors like the admission temperature , the highest peak temperature , the lowest systolic blood pressure during the hospitalization , etc . , would frequently indicate the severity of a condition , the presence of comorbid conditions or the development of complications . table 5 shows how one of these clinical factors , peak temperature , can considerably enhance the explanation of variance within a single drg , uncomplicated heart attacks . in a project for alcoa , cpha data sets were obtained for six southern community hospitals which were the sole source of health care in the community , as part of an effort to understand why the alcoa corporation 's length of stay experience at its main aluminum plant in marysville , tennessee was so high ( lind et al . , 1979 ) . as part of that effort , an analysis was done for a set of common conditions to pinpoint differences in resource utilization which employed standard diagnostic and procedure information . the analysis also attempted to adjust for severity of illness by use of the medical audit component of the cpha hospital discharge abstract form . peak temperature was by far the most significant factor in explaining variations in length of stay among hospitals for this drg and also turned out to be the most significant factor in explaining variations in charges among physicians at alcoa 's principal community hospital . table 4 shows that it is possible to obtain additional reductions in variance by use of other treatment and clinical factors within a defined drg category . clinical variables not currently in the uhdds data set may be conceptually more attractive as severity adjusters because : 1 ) they are more directly tied to a patient 's actual clinical status than is a diagnosis , and 2 ) they can be selected to more likely be exogenous rather than endogenous variables . thus , an admission blood pressure level can not readily be directed by the provider , while performing surgery can be done with considerable discretion . if variable severity elements are allowed for different conditions ( research option area e ) , type and extent - of - disease spread variables could be incorporated for malignancies , medically derived cardiovascular classes could be identified ( e.g. , american heart association functional rating for general cardiac patients , killip classifications for heart attacks , plum - pozner codes for strokes ) , trauma severity index for injuries and other limited data elements shown in clinical trials to be important prognostic severity of illness measures . if variable special elements could be incorporated , the availability of four , six or ten additional characters of data might dramatically improve the severity of illness adjusters and thus the clinical homogeneity of the groups created for reimbursement purposes . in fact , hcfa need not be limited to adding data fields to the uhdds data set , but rather might replace fields that are currently reserved for other purposes . for example , by dropping the five digits reserved for the fifth diagnosis an item with potentially extremely small marginal utility one could encode in two character fields the peak temperature ( e.g. , 99.6 , 103.5 , etc . ) and the lowest systolic blood pressure ( which would occupy only three character fields ) . if extensive additional data elements could feasibly be added to the uhdds dataset for medicare , then much more clinically - oriented physiologic parameters could be used in the severity adjustment methodology ( research option area f ) . one example of this is the acute physiology and chronic health evaluation ( apache ) system used at george washington university . in apache , more than thirty additional clinical measurements , including blood gas determinations , are incorporated into the classification weighting scheme for intensive care patients ( knaus et al . , 1981 ) . finally , if one is not limited to the uhdds dataset , severity of illness classification based on judgmental criteria such as the severity of illness index of susan horn and her associates and the relative intensity measures ( rims ) nursing care weighted system , etc . , could be tested ( research option area g ) . a critical policy decision on the approach to incorporating severity measures into the drg revision process is whether an attempt should be made to develop a comprehensive set of revisions which would be introduced en masse , or whether revisions should be promulgated through a piecemeal or phased - in approach . ( by the latter , we do not mean a phase - in of a comprehensive set of revisions because , if evidence was present of a superior method of classification , it would probably be politically unacceptable to the losers under the old approach for hcfa to not introduce all of the revisions at once . ) the arguments for and against either of these approaches are basically political rather than technical . yet , the decision on which is the preferable course to the government and the health care industry is important to provide guidance for an efficient research strategy and would prioritize among the areas . some approaches , such as the severity of illness index technique , are essentially comprehensive approaches and do not lend themselves to partial introduction for the drg list of categories . on the other hand , approaches which would further subdivide or minimally reorganize terminal drg 's within a major diagnostic category , such as the systemetrics approach , using adjacent diagnosis - related groups or additional age splits , are more reasonable on a selective introduction basis . there is a final issue which will plague the drg reimbursement system even if all data confounding and severity problems could be resolved perfectly the issue of effectiveness of treatment . there has been an assumption in many quarters that if any group of patients had exactly the same clinical needs , their physicians or any outside physicians would agree on the product to be delivered . the actual situation today in american medicine is that there are sometimes vast , well - reasoned and sincere differences among outstanding physicians about the best course of diagnosis and treatment . if these differences are associated with substantial and systematic differences in resource use within a drg or other adjustment schemes , then american medicine will continue to resist the system , labelling it as inequitable and as potentially containing adverse quality of care consequences .

backgroundcardiac arrhythmias and sudden cardiac death are more frequent in patients with obstructive sleep apnea ( osa ) . osa is associated with qt prolongation , and qt prolongation is an independent risk factor for sudden cardiac death . because qt prolongation can be mediated by potassium channel loss of function , we tested whether osa or continuous positive airway pressure therapy altered mrna expression of circulating white blood cell potassium channels.methods and resultsin total , 28 patients with osa newly diagnosed by polysomnogram and 6 participants without osa were enrolled . potassium channel levels in white blood cells at baseline and at a 4week followup visit were compared . there was a significant inverse correlation between the severity of the osa stratified by apnea hypopnea index and mrna expression of the main potassium channels assessed : kcnq1 ( r=0.486 , p=0.007 ) , kcnh2 ( r=0.437 , p=0.016 ) , kcne1 ( r=0.567 , p=0.001 ) , kcnj2 ( r=0.442 , p=0.015 ) , and kcna5 ( r=0.468 , p=0.009 ) . in addition , kcnq1 , kcnh2 , and kcne1 inversely correlated with the oxygen desaturation index 4 . after 4 weeks of continuous positive airway pressure therapy , circulating kcnq1 and kcnj2 were increased 1.40.4fold ( p=0.040 ) and 2.11.4fold ( p=0.046 ) in the moderate osa group . compared with patients with mild or moderate osa , patients with severe osa had a persistently higher apnea hypopnea index ( mild 2.01.8 , moderate 1.00.9 , severe 5.85.6 ; p=0.015 ) , perhaps explaining why the potassium channel changes were not seen in the severe osa group.conclusionsthe mrna expression of most potassium channels inversely correlates with the severity of osa and hypoxemia . continuous positive airway pressure therapy improves circulating kcnq1 and kcnj2 in patients with moderate osa .

obstructive sleep apnea ( osa ) is a highly prevalent and underdiagnosed disease.1 a recent large clinical trial established osa as a novel risk factor for sudden cardiac death ( scd).2 after 20 years of followup , the busselton health study cohort showed that moderate to severe sleep apnea was independently associated with an increased risk of allcause mortality.3 in a longitudinal study of 10 701 adults , osa was associated with scd , and the magnitude of risk varied with osa severity.2 qt prolongation is an independent risk factor for scd,4 , 5 , 6 and osa is associated with longer qt intervals.7 , 8 this electrocardiographic effect is thought to be caused by systemic hypoxia , hypercarbia , or acidosis . the most effective treatment for osa is continuous positive airway pressure ( cpap ) therapy , which improves oxygenation and decreases sleep fragmentation . cpap improves prolonged cardiac repolarization9 and decreases the rate of death.10 cpap withdrawal is associated with prolongation of the corrected qt interval.11 the genetic link of scd and qt interval to potassium channels has been well established in inherited long qt syndromes.12 lossoffunction gene mutations of cardiac potassium channels contribute to different types of long qt syndromes , such as kcnq1 ( long qt syndrome type 1 [ lqt1 ] ) , kcnh2 ( lqt2 ) , kcne1 ( lqt5 ) , kcne2 ( lqt6 ) , and kcnj2 ( lqt7).13 , 14 other potassium channels expressed in the heart include kcnd3 , encoding the potassium channel that underlies the transient outward current ito15 ; kcna5 , encoding the potassium channel that underlies ikur 16 ; and kcnj11 , encoding kir6.2 , which underlies the inward rectifying channel.17 in summary , osa is associated with qt prolongation and scd . cpap improves qt prolongation.9 , 11 qt interval is influenced by potassium channels . in our previously published study , the levels of circulating cardiac sodium channel fulllength mrna and splicing variants in white blood cells were representative of levels in the myocardium.18 based on these observations , we reasoned that circulating white blood cells may experience conditions similar to those of cardiomyocytes and may reveal insights into the mechanism of qt changes with osa and cpap . we hypothesized that prolonged qt in osa represented altered potassium channel regulation and that this regulation would be reflected in circulating white blood cells . sodium channel splicing in obstructive sleep apnea ( socsosa ) and was conducted at the lifespan health system ( rhode island hospital and the miriam hospital ) in providence , rhode island , and at the university of illinois at chicago ( uic ) in chicago , illinois . participants were adults ( aged 18 years ) with osa newly diagnosed by polysomnogram who agreed to cpap treatment ( osa group ) or adults without osa ( control group ) . all trial participants were screened with the stopbang questionnaire19 before proceeding to the sleep study . based on the severity of apnea hypopnea index ( ahi ) , osa patients were assigned to 3 groups : mild ( ahi 515 ) , moderate ( ahi 1530 ) , or severe ( ahi > 30).20 the ahi was defined as the number of apneas and hypopneas per hour and calculated by adding the total number of apnea and hypopnea events and dividing by the total number of minutes of actual sleep time multiplied by 60 . the overnight sleep study consisted of continuous polygraphic recordings from 10 pm to 7 am , and the baseline parameters were measured . arterial oxygen saturation was recorded by digital pulse oximeter , and the nadir arterial oxygen saturation levels ( nadir o2 ) were obtained . the oxygen desaturation index is the number of times per hour of sleep that the blood 's oxygen level drops by 3% or 4% ( ie , odi4 ) from baseline . to quantify overall nocturnal desaturation , we used the cumulative percentages of sleep time spent at saturations < 90% or < 85% . respiratory disturbance index was defined as the average number of episodes of apnea , hypopnea , and respiratory event related arousals per hour of sleep documented in the polysomnography . after 4 weeks of cpap therapy , an ahi score was measured and documented as residual ahi , which was used to assess the efficacy of cpap therapy . the osa patients were evaluated with the epworth sleepiness scale ( ess)21 and the functional outcome of sleep questionnaire 10 ( fosq10)22 at the time of initial diagnosis . improvement of the symptoms were reevaluated by the ess and fosq10 after 1 month of cpap treatment . the exclusion criteria included chronic use of hypnotics ; current drug or alcohol addiction ; a rhythm other than sinus at enrollment ; mandatory and biventricular pacing ; a history of heart transplant or left ventricular assist device ; active use of intravenous vasodilators , vasopressors , or inotropes ; hemodialysis or peritoneal dialysis ; active infection ; acute coronary syndrome ; major trauma or surgery ; malignant neoplastic disease on active treatment including chemotherapy and radiation therapy ; life expectancy < 1 year ; collagen vascular disease on active treatment including steroids and other immunomodulating drugs ; systemic steroid use ; or concomitant use of an investigational drug . blood samples were collected in paxgene blood rna tubes ( qiagen ) and were stored in a freezer at 80c before processing . total rna was isolated with the use of the paxgene blood rna isolation kit and was then converted to cdna with the use of the superscript iii cdna reverse transcription kit ( life technologies ) . only samples with an optical density of 260/280>2.0 and 260/230>1.7 quantitative reverse transcriptase polymerase chain reaction was performed to detect the abundance of potassium channels using iqsybr green supermix ( biorad laboratories ) and the 7500 fast realtime pcr system ( life technologies ) . the primer sequences used were hkcnq1f ( 5ttgggaagccctcactgttc3 ) , hkcnq1r ( 5ctggtg aagcatgtcggtga3 ) , hkcnh2f ( 5tcaccgccctgtacttcatct3 ) , hkcnh2r ( 5caggccttgcataca ggttca3 ) , hkcne1f ( 5gaaccccaccactggctaaa3 ) , hkcne1r ( 5taatcca cccctcacccctt3 ) , hkcne2f ( 5atttcatcctgcccacacact3 ) , hkcne2r ( 5 ccagcgtctgtgtgaaattgg3 ) , hkcna5f ( 5agaggagtccagcggaaggt3 ) , hkcna5r ( 5actgtctgcattctccagggt3 ) , hkcnj11f ( 5tccagggtgttacaa ggcac3 ) , hkcnj11r ( 5gaattgggttgggaggagca3 ) , hkcnj2f ( 5tgtcacgg atgaatgcccaa3 ) , hkcnj2r ( 5ctgcgccaatgatgaaagca3 ) , hkcnd3f ( 5t gtacgaacctccaccatcaa3 ) , and hkcnd3r ( 5atgggtagttctgcattgaact ct3 ) . quantitative reverse transcriptase polymerase chain reaction thermal cycling conditions were initial incubation at 50c for 60 minutes and then termination of the reactions at 85c for 5 minutes . polymerase chain reaction was performed with an initial denaturation step at 95c for 20 seconds , followed by 40 cycles of denaturation at 95c for 3 seconds and annealing and nucleotide prolongation at 60c for 30 seconds . each sample was measured for the target genes kcnq1 , kcnh2 , kcne1 , kcne2 , kcna5 , kcnj11 , kcnj2 , kcnd3 , and actin . continuous variables were described in terms of descriptive statistics ( mean and standard deviation ) . independent samples t tests and pearson chisquare tests were used for bivariate analysis comparisons of continuous and categorical variables , respectively . the pearson correlation coefficient was used to assess the correlation between the parameters measured in the sleep study , the severity of osa , and the mrna levels of potassium channels . differences between gene expression at baseline and at the followup visit were examined by means of paired wilcoxon tests . sodium channel splicing in obstructive sleep apnea ( socsosa ) and was conducted at the lifespan health system ( rhode island hospital and the miriam hospital ) in providence , rhode island , and at the university of illinois at chicago ( uic ) in chicago , illinois . participants were adults ( aged 18 years ) with osa newly diagnosed by polysomnogram who agreed to cpap treatment ( osa group ) or adults without osa ( control group ) . all trial participants were screened with the stopbang questionnaire19 before proceeding to the sleep study . based on the severity of apnea hypopnea index ( ahi ) , osa patients were assigned to 3 groups : mild ( ahi 515 ) , moderate ( ahi 1530 ) , or severe ( ahi > 30).20 the ahi was defined as the number of apneas and hypopneas per hour and calculated by adding the total number of apnea and hypopnea events and dividing by the total number of minutes of actual sleep time multiplied by 60 . the overnight sleep study consisted of continuous polygraphic recordings from 10 pm to 7 am , and the baseline parameters were measured . arterial oxygen saturation was recorded by digital pulse oximeter , and the nadir arterial oxygen saturation levels ( nadir o2 ) were obtained . the oxygen desaturation index is the number of times per hour of sleep that the blood 's oxygen level drops by 3% or 4% ( ie , odi4 ) from baseline . to quantify overall nocturnal desaturation , we used the cumulative percentages of sleep time spent at saturations < 90% or < 85% . respiratory disturbance index was defined as the average number of episodes of apnea , hypopnea , and respiratory event related arousals per hour of sleep documented in the polysomnography . after 4 weeks of cpap therapy , an ahi score was measured and documented as residual ahi , which was used to assess the efficacy of cpap therapy . the osa patients were evaluated with the epworth sleepiness scale ( ess)21 and the functional outcome of sleep questionnaire 10 ( fosq10)22 at the time of initial diagnosis . improvement of the symptoms were reevaluated by the ess and fosq10 after 1 month of cpap treatment . the exclusion criteria included chronic use of hypnotics ; current drug or alcohol addiction ; a rhythm other than sinus at enrollment ; mandatory and biventricular pacing ; a history of heart transplant or left ventricular assist device ; active use of intravenous vasodilators , vasopressors , or inotropes ; hemodialysis or peritoneal dialysis ; active infection ; acute coronary syndrome ; major trauma or surgery ; malignant neoplastic disease on active treatment including chemotherapy and radiation therapy ; life expectancy < 1 year ; collagen vascular disease on active treatment including steroids and other immunomodulating drugs ; systemic steroid use ; or concomitant use of an investigational drug . blood samples were collected in paxgene blood rna tubes ( qiagen ) and were stored in a freezer at 80c before processing . total rna was isolated with the use of the paxgene blood rna isolation kit and was then converted to cdna with the use of the superscript iii cdna reverse transcription kit ( life technologies ) . only samples with an optical density of 260/280>2.0 and 260/230>1.7 were used . quantitative reverse transcriptase polymerase chain reaction was performed to detect the abundance of potassium channels using iqsybr green supermix ( biorad laboratories ) and the 7500 fast realtime pcr system ( life technologies ) . the primer sequences used were hkcnq1f ( 5ttgggaagccctcactgttc3 ) , hkcnq1r ( 5ctggtg aagcatgtcggtga3 ) , hkcnh2f ( 5tcaccgccctgtacttcatct3 ) , hkcnh2r ( 5caggccttgcataca ggttca3 ) , hkcne1f ( 5gaaccccaccactggctaaa3 ) , hkcne1r ( 5taatcca cccctcacccctt3 ) , hkcne2f ( 5atttcatcctgcccacacact3 ) , hkcne2r ( 5 ccagcgtctgtgtgaaattgg3 ) , hkcna5f ( 5agaggagtccagcggaaggt3 ) , hkcna5r ( 5actgtctgcattctccagggt3 ) , hkcnj11f ( 5tccagggtgttacaa ggcac3 ) , hkcnj11r ( 5gaattgggttgggaggagca3 ) , hkcnj2f ( 5tgtcacgg atgaatgcccaa3 ) , hkcnj2r ( 5ctgcgccaatgatgaaagca3 ) , hkcnd3f ( 5t gtacgaacctccaccatcaa3 ) , and hkcnd3r ( 5atgggtagttctgcattgaact ct3 ) . quantitative reverse transcriptase polymerase chain reaction thermal cycling conditions were initial incubation at 50c for 60 minutes and then termination of the reactions at 85c for 5 minutes . polymerase chain reaction was performed with an initial denaturation step at 95c for 20 seconds , followed by 40 cycles of denaturation at 95c for 3 seconds and annealing and nucleotide prolongation at 60c for 30 seconds . each sample was measured for the target genes kcnq1 , kcnh2 , kcne1 , kcne2 , kcna5 , kcnj11 , kcnj2 , kcnd3 , and actin . continuous variables were described in terms of descriptive statistics ( mean and standard deviation ) . independent samples t tests and pearson chisquare tests were used for bivariate analysis comparisons of continuous and categorical variables , respectively . the pearson correlation coefficient was used to assess the correlation between the parameters measured in the sleep study , the severity of osa , and the mrna levels of potassium channels . differences between gene expression at baseline and at the followup visit were examined by means of paired wilcoxon tests . overall , 28 osa patients and 6 participants without osa met eligibility criteria and completed the baseline and followup visits . the mean age ( sd ) was 50.710.1 years in osa patients and 43.616.3 years in participants without osa . the baseline body mass index was 36.48.1 in osa patients and 39.510.7 in participants without osa . at baseline , there was no significant difference in demographic characteristics between groups ( table 1 ) . clinical characteristics of the socsosa study ahi indicates apnea hypopnea index ; bmi , body mass index ; cpap , continuous positive airway pressure ; ct85 , the cumulative percentages of sleep time spent at saturations < 85% ; ct90 , the cumulative percentages of sleep time spent at saturations < 90% ; odi3 , the number of times per hour of sleep that the blood 's oxygen level drops by 3% from baseline ; odi4 , the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline ; osa , obstructive sleep apnea ; rdi , respiratory disturbance index ; residual ahi , the apnea hypopnea index after 4 weeks of continuous positive airway pressure therapy . p value from t tests for continuous variables and chisquare tests for categorical variables . the sleep characteristics and cpap treatment among participants with osa are shown in table 1 . as expected , cpap therapy was associated with significant reductions in ahi . nevertheless , compared with patients with mild or moderate osa , patients with severe osa had significantly higher residual ahi ( mild 2.01.8 , moderate 1.00.9 , severe 5.85.6 ; p=0.015 ) . the stopbang questionnaire was used to screen for osa . compared with patients with mild osa , patients with moderate osa had markedly higher scores ( mild 4.11.4 , moderate 6.01.3 , severe 5.41.1 ; p=0.022 ) . there was no statistically significant difference between moderate and severe osa , and that might be explained by use of the stopbang questionnaire , which is highly sensitive at low scores and highly specific at high scores for osa.23 the osa patients were evaluated with the ess questionnaire to measure daytime sleepiness before and after cpap therapy . the ess score significantly decreased after 4 weeks of cpap in mild and moderate osa patients ( mild 12.65.2 versus 9.26.1 , p=0.028 ; moderate 11.74.5 versus 7.35.7 , p=0.034 ; severe 12.36.6 versus 10.75.3 , p=0.497 ) . we observed a significant inverse correlation between the severity of osa stratified by ahi and the mrna expression of the main potassium channels assessed : kcnq1 ( r=0.486 , p=0.007 ) , kcnh2 ( r=0.437 , p=0.016 ) , kcne1 ( r=0.567 , p=0.001 ) , kcnj2 ( r=0.442 , p=0.015 ) , and kcna5 ( r=0.468 , p=0.009 ) ( figure 1 and table 2 ) . in addition , kcnq1 ( r=0.404 , p=0.027 ) , kcnh2 ( r=0.416 , p=0.022 ) , and kcne1 ( r=0.465 , p=0.010 ) inversely correlated with odi4 , indicating the oxygen desaturation status ( figure 2 and table 2 ) . a , correlation between mrna level of kcnq1 ( ordinate ) and ahi ( abscissa ) . b , correlation between mrna level of kcnj2 ( ordinate ) and ahi ( abscissa ) . correlation of the expression of baseline potassium channels with hypoxia ahi indicates apnea hypopnea index ; nadir o2 , nadir arterial oxygen saturation levels ; odi4 , the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline . a , correlation between mrna level of kcnq1 ( ordinate ) and odi4 ( abscissa ) . b , correlation between mrna level of kcnj2 ( ordinate ) and odi4 ( abscissa ) . odi4 indicates the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline . for patients with newly diagnosed osa initiating cpap for the first time , circulating kcnq1 and kcnj2 were increased 1.40.4fold ( p=0.040 ) and 2.11.4fold ( p=0.046 ) , respectively , after 4 weeks of cpap therapy in the moderate group ( figure 3 ) . although they did not reach statistical significance , cpap improved most potassium channel gene expression in moderate osa : kcne1 1.5fold , kcne2 2.9fold , kcna5 1.9fold , kcnd3 1.3fold , and kcnj11 1.6fold . p<0.05 comparing the postcpap fold change of kcnq1 or kcnj2 to the baseline in the moderate osa group . overall , 28 osa patients and 6 participants without osa met eligibility criteria and completed the baseline and followup visits . the mean age ( sd ) was 50.710.1 years in osa patients and 43.616.3 years in participants without osa . the baseline body mass index was 36.48.1 in osa patients and 39.510.7 in participants without osa . at baseline , there was no significant difference in demographic characteristics between groups ( table 1 ) . clinical characteristics of the socsosa study ahi indicates apnea hypopnea index ; bmi , body mass index ; cpap , continuous positive airway pressure ; ct85 , the cumulative percentages of sleep time spent at saturations < 85% ; ct90 , the cumulative percentages of sleep time spent at saturations < 90% ; odi3 , the number of times per hour of sleep that the blood 's oxygen level drops by 3% from baseline ; odi4 , the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline ; osa , obstructive sleep apnea ; rdi , respiratory disturbance index ; residual ahi , the apnea hypopnea index after 4 weeks of continuous positive airway pressure therapy . p value from t tests for continuous variables and chisquare tests for categorical variables . the sleep characteristics and cpap treatment among participants with osa are shown in table 1 . as expected , cpap therapy was associated with significant reductions in ahi . nevertheless , compared with patients with mild or moderate osa , patients with severe osa had significantly higher residual ahi ( mild 2.01.8 , moderate 1.00.9 , severe 5.85.6 ; p=0.015 ) . the stopbang questionnaire was used to screen for osa . compared with patients with mild osa , patients with moderate osa had markedly higher scores ( mild 4.11.4 , moderate 6.01.3 , severe 5.41.1 ; p=0.022 ) . there was no statistically significant difference between moderate and severe osa , and that might be explained by use of the stopbang questionnaire , which is highly sensitive at low scores and highly specific at high scores for osa.23 the osa patients were evaluated with the ess questionnaire to measure daytime sleepiness before and after cpap therapy . the ess score significantly decreased after 4 weeks of cpap in mild and moderate osa patients ( mild 12.65.2 versus 9.26.1 , p=0.028 ; moderate 11.74.5 versus 7.35.7 , p=0.034 ; severe 12.36.6 versus 10.75.3 , p=0.497 ) . we observed a significant inverse correlation between the severity of osa stratified by ahi and the mrna expression of the main potassium channels assessed : kcnq1 ( r=0.486 , p=0.007 ) , kcnh2 ( r=0.437 , p=0.016 ) , kcne1 ( r=0.567 , p=0.001 ) , kcnj2 ( r=0.442 , p=0.015 ) , and kcna5 ( r=0.468 , p=0.009 ) ( figure 1 and table 2 ) . in addition , kcnq1 ( r=0.404 , p=0.027 ) , kcnh2 ( r=0.416 , p=0.022 ) , and kcne1 ( r=0.465 , p=0.010 ) inversely correlated with odi4 , indicating the oxygen desaturation status ( figure 2 and table 2 ) . a , correlation between mrna level of kcnq1 ( ordinate ) and ahi ( abscissa ) . b , correlation between mrna level of kcnj2 ( ordinate ) and ahi ( abscissa ) . correlation of the expression of baseline potassium channels with hypoxia ahi indicates apnea hypopnea index ; nadir o2 , nadir arterial oxygen saturation levels ; odi4 , the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline . a , correlation between mrna level of kcnq1 ( ordinate ) and odi4 ( abscissa ) . b , correlation between mrna level of kcnj2 ( ordinate ) and odi4 ( abscissa ) . odi4 indicates the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline . for patients with newly diagnosed osa initiating cpap for the first time , circulating kcnq1 and kcnj2 were increased 1.40.4fold ( p=0.040 ) and 2.11.4fold ( p=0.046 ) , respectively , after 4 weeks of cpap therapy in the moderate group ( figure 3 ) . although they did not reach statistical significance , cpap improved most potassium channel gene expression in moderate osa : kcne1 1.5fold , kcne2 2.9fold , kcna5 1.9fold , kcnd3 1.3fold , and kcnj11 1.6fold . p<0.05 comparing the postcpap fold change of kcnq1 or kcnj2 to the baseline in the moderate osa group . the main findings of this prospective study are that the mrna expression of at least 5 of 8 cardiac potassium channels correlated with the severity of osa and that cpap treatment improved circulating kcnq1 and kcnj2 in patients with moderate osa . there were trends toward increases in 7 of 8 potassium channels in patients with moderate osa . qt prolongation is an independent risk factor for scd . in the oregon sudden unexpected death study , idiopathic abnormal prolongation of the corrected qt interval was associated with 5fold increased odds of scd.5 recent genetic studies have established a clear inverse relationship between qt interval and expression and function of potassium channels . gene defects resulting in loss of function of voltagegated potassium channels , for example , are associated with long qt syndromes : lqt1 ( kcnq1 ) and lqt2 ( kcnh2 ) , encoding subunits of the potassium channels iks and ikr ; lqt5 ( kcne1 ) and lqt6 ( kcne2 ) , encoding subunits of the potassium channels iks and ikr ; and lqt7 ( kcnj2 ) , encoding the inward rectifier potassium channel kir2.1.12 , 13 , 14 , 15 , 16 , 17 osa is associated with scd and long qt . osa predicted incident scd in a longitudinal study of 10 701 adults.3 in osa patients who had no evidence of underlying cardiac , pulmonary , or central nervous system disease , the qt interval was prolonged at the onset of apnea.7 because osa is associated with long qt , and long qt can be caused by potassium channel loss of function , we tested whether osa was associated with circulating potassium channel changes . a total of 8 potassium channel genes were studied , and we found that the mrna levels of at least 5 of 8 potassium channel genes assessed correlated inversely with hypoxemia and osa severity . of these channel genes , 2 were statistically significantly increased and 5 more showed trends toward an increase with cpap therapy . this could be explained if correction of hypoxia were the major driver for improvements in channel levels . consistent with this idea , in a population of 10 701 adults , the magnitude of scd risk was predicted by ahi and nocturnal oxygen desaturation.3 in our study , even with cpap , severe osa patients had higher residual ahi with statistical difference , and there was no change in potassium channel levels with cpap . this supports the idea that the degree of hypoxia , rather than some other aspect of cpap , was mediating the changes in mrna abundance . in our patients with mild osa , hypoxia was not severe and changed little with cpap . the transcription factor hypoxiainducible factor 1 ( hif1 ) plays a key role in cellular response to systemic oxygen levels in humans . hif1 is a subunit of a heterodimeric hif1.24 , 25 there are 3 putative hif1 transcription factor binding sites located in the upstream sequence of the human kcnq1 gene and 4 hif1 binding sites located in the upstream sequence of the human kcnj2 gene ( data not shown ) . the systemic oxygen level affects hif1 through multiple mechanisms , such as hif1 conformational changes and stability , subcellular localization , and transcriptional activity . hif1 regulates the expression of > 60 genes involved in angiogenesis , cell proliferation , cell survival , and glucose and iron metabolism.26 the presence of hif1 binding sites in the promoters of potassium channels may provide the mechanism by which systemic oxygen level affects the gene expression of potassium channels . although the paired design controlled for interparticipant variability , the small sample size precluded comprehensive multivariable adjustment . in addition , because of the short study duration , it is not clear if the changes in potassium channel expression with cpap will be durable or if they correlate with improved outcomes . furthermore , we did not correlate potassium channel mrna levels in white blood cells with levels or currents in the myocardium . the mrna expression of most potassium channels inversely correlated with the severity of osa and hypoxemia . if downregulations in circulating potassium channel genes with osa are mirrored in the heart , as may happen if systemic hypoxemia is a causal factor , then downregulation of potassium channel genes in the heart may explain the long qt and increased risk of scd in osa . moreover , it suggests that potassium channel gene levels may represent a new circulating marker for arrhythmic risk in osa . the present work was supported in part by national institutes of health grants r01 hl104025 and hl106592 .

generation of reactive oxygen species during dopamine ( da ) oxidation could be one of the factors leading to the selective loss of nigral dopaminergic neurons in parkinson s disease ( pd ) . vesicular monoamine transporter type 2 ( vmat2 ) proteins in nerve terminals uptake dopamine into synaptic vesicles , preventing its cytoplasmic accumulation and toxic damage to nigral neurons . polymorphisms in vmat2 gene and in its regulatory regions might therefore serve as genetic risk factors for pd . in the present study , we have analyzed 8 single - nucleotide polymorphisms ( snps ) located within / around the vmat2 gene for association with pd in an italian cohort composed of 704 pd patients and 678 healthy controls . among the 8 snps studied , only the 2 located within the promoter region ( rs363371 and rs363324 ) were significantly associated with pd . in the dominant model , odds ratios were 0.72 ( 95% confidence interval [ ci ] : 0.60.9 , p < 0.005 ) for rs363371 and 0.76 ( 95% ci : 0.60.9 , p = 0.01 ) for rs363324 ; in the additive model , odds ratios were 0.78 ( 95% ci : 0.650.94 , p = 0.008 ) for rs363371 and 0.85 ( 95% ci : 0.720.92 , p = 0.04 ) for rs363324 . there were no significant relationships between the remaining snps ( rs363333 , rs363399 , rs363387 , rs363343 , rs4752045 , and rs363236 ) and the risk of sporadic pd in any genetic model . this study adds to the previous evidence suggesting that variability in vmat2 promoter region may confer a reduced risk of developing pd , presumably via mechanisms of gene overexpression .

parkinson 's disease ( pd ) is a debilitating neurologic disorder characterized clinically by bradykinesia , resting tremor , rigidity , and postural instability with a therapeutic response to levodopa . pathophysiologically , pd is characterized by the loss of dopamine neurons in the substantia nigra ( sn ) pars compacta and by the presence of intracellular inclusions known as lewy bodies , which are composed primarily of alpha - synuclein protein aggregates . although the disease etiology remains largely unclear , generation of reactive oxygen species during oxidation of dopamine ( da ) could be one of the factors leading to the selective loss of nigral dopaminergic neurons in pd ( spina and cohen , 1989 ) . the vesicular monoamine transporter type 2 ( vmat2 , slc18a2 ) uptakes cytosolic monoamines , including dopamine , into intracellular secretory vesicles , preventing their toxicity in the cytosol and discharging them into the extracellular space by exocytosis . altered function of vmat2 proteins may therefore cause cytoplasmic accumulation of free da , leading to dopaminergic neuron death , therefore being a risk factor for pd . vmat2 is expressed in central , peripheral , and enteric neurons as well as in platelets ( peter et al . , 1995 ) . vmat2-deficient mouse exhibits increased oxidative stress , progressive loss of da terminals and cell bodies in the sn pars compacta , alpha - synuclein accumulation ( caudle et al . , 2007 ) , and increased sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) toxicity ( mooslehner et al . , 2001 ) ; interestingly , this model displays also motor and nonmotor symptoms of pd ( taylor et al . , 2011 ) . in humans , the involvement of vmat2 in pd pathogenesis is supported by positron emission tomography studies showing significantly lower vmat2 densities in the putamen , caudate , and sn of pd patients ( bohnen et al . , 2006 ; , 2008 ; okamura et al . , 2010 ) ; interestingly , reduced vmat2 messenger rna ( mrna ) levels were observed in platelets from patients ( sala et al . , 2010 ) . polymorphisms in the vmat2 gene or in its regulatory regions that affect its quantitative expression or protein function might therefore represent genetic risk factors for pd . some common gain - of - function haplotypes in vmat2 promoter have been reported to confer a protective effect against pd in women ( glatt et al . , 2006 ) , whereas no studies have so far addressed specifically the association of intragenic vmat2 variability with pd in caucasian populations . in light of these premises , we carried out an association study to evaluate whether genetic variants in vmat2 gene and in its regulatory regions might influence either susceptibility to pd or age of onset of the disease in a series of italian sporadic pd patients and unrelated healthy controls . the study included 704 pd patients recruited consecutively from the outpatient clinic for movement disorders at the department of neurology of san gerardo hospital in monza and at the parkinson institute in milan . pd patients were diagnosed according to the standard criteria ( gelb et al . , 1999 ) . among patients , 45 ( 7.5% ) reported a family history of pd in at least a first - degree or second - degree relative . pd cases with an age at onset ( aao ) of < 40 years were screened to exclude that they were carriers of recognized pd - causing mutations ( parkin , pink1 , and lrrk2 genes ) ; the remaining familial cases were screened for the highly penetrant g0219s mutation in the lrrk2 gene that was absent in all the examined subjects . all the 45 cases with a family history of pd were included in the analysis . control population consisted of 678 individuals , comprising healthy spouses and former blood donors . both patients and controls were caucasians , and the controls none of the controls had clinical evidence of neurologic disease ( as assessed by neurologic examination ) or familial history of neurodegenerative diseases . none of the patients and of the controls had a current or former history of alcohol use disorders . the present analysis comprised a total of 8 single - nucleotide polymorphisms ( snps ) . snps were selected according to the intermarker distance , their minor allele frequency ( maf > 5% ) , the illumina design scores ( > 0.7 ) , and the previous publications showing associations with other dopamine - related traits . in detail , the 2 snps in the promoter region ( rs363371 and rs363324 ) previously identified by resequencing and included in haplotypes have been shown to be associated with alcoholism ( lin et al . , 2005 ) , rs363333 and rs363387 have been previously shown to be associated with substance dependence ( schwab et al . , 2005 ) , and rs363399 and rs4752045 with depression ( christiansen et al . , 2007 ) . lastly , considering a possible role in vmat2 mrna stability , an additional candidate snp in the 3-untranslated region ( rs363236 ) was also selected . table 1 highlights the genomic positions and the type of the genotyped markers . genomic dna was extracted from peripheral venous blood using a qiagen blood kit ( qiagen , milan , italy ) . snps were typed using the veracode goldengate genotyping assay on illumina beadxpress reader platform according to the manufacturer 's protocol ( illumina , san diego , ca , usa ) . goodness of fit of the genotype frequencies to hardy weinberg ( hw ) expected proportions in control subjects was examined . chi - square tests were used to compare sex distribution and genotype frequencies between patients and controls and to test for deviation from hw equilibrium . we used logistic regression analysis to test for the association between the vmat2 gene selected variants and pd risk . the estimated odds ratios ( ors ) and relative 95% confidence intervals ( cis ) were adjusted for sex , age at enrollment , and smoking ( ever / never ) . we performed analyses assuming autosomal dominant , log - additive , and recessive models of inheritance . in the test , all p values for individual snps , or , and 95% ci are presented as noncorrected for multiple testing . analyses were performed for subjects overall and stratified by family history of pd , age at study ( divided in quartiles ) , and sex . the association of each genetic variant with aao of pd was assessed using cox proportional hazard models , adjusted for sex . for each genetic variant , we calculated a hazard ratio , a 95% ci , and a 2-tailed p value . power calculations were carried out using the quanto software version 1.2 , under a dominant model of inheritance . the pairwise linkage disequilibrium ( ld ) between markers the ensembl genome browser ( http://www.ensembl.org/index.html ) was used to identify and to annotate nearby snps in ld ( proxies ) based on hapmap ceu . the overall difference in haplotype frequencies between cases and controls was assessed using a global score test . haplotype - specific tests were performed only including haplotypes with frequencies > 1% , using the additive model with the most frequent haplotype as the referent group in the regression analysis . correction for multiple testing was performed by applying the simulate = true parameter in haplo.score that gives simulated p values based on 10,000 permutations . the demographic data for the 704 pd patients were as follows : mean age at study was 65.14 9.3 years ( range 3290 ) and mean aao of symptoms was 57.28 10 years ( range 2787 ) . the 678 control subjects consisted of 375 men and 303 women ; their mean age at examination was 64.2 7.7 years ( range 4490 ) . there was no statistical difference in the age at study and sex between patients and controls ( p > 0.05 ) . the control genotype distributions were in agreement with the hw equilibrium for all markers ( hw p value > 0.05 ) . table 3 lists the estimated ors and associated 95% cis from the association analysis between each snp and pd , assuming autosomal dominant and trend ( additive ) models of inheritance . two out of the 8 investigated snps , both located in the putative promoter region at the 5 untranslated region , were inversely associated to pd when assuming dominant ( rs363371 : or 0.72 , ci 0.620.97 , p = 0.004 ; rs363324 : or 0.76 , ci 0.610.95 , p = 0.01 ) and trend model of inheritance ( rs363371 : or 0.78 , ci 0.650.94 , p = 0.008 ; rs363324 : or 0.85 , ci 0.720.92 , p = 0.04 ) . after bonferroni correction for multiple comparisons , only rs363371 remained significantly associated with pd ( corrected p value = 0.03 , dominant model ) . the remaining intragenic snps were not significantly associated with pd overall or in strata defined by age at study , sex , and family history ( data not shown ) . furthermore , we tested the possible association among all the different combinations of alleles at the 8 polymorphic loci with pd . four common haplotypes ( gatttagt , frequency in cases / controls = 0.30/0.29 ; gatttact , frequency = 0.23/0.21 ; gatttacc , frequency = 0.07/0.07 ; and agtctagt , frequency = 0.05/0.06 ) and several rare ones ( with frequency < 5% ) were inferred . none of the common haplotypes was associated with the overall risk for pd ( global haplotype association p value = 0.56 ) . given the observed ld pattern between the studied snps ( with poor linkage between the 5 half and the 3 half markers ) , we then partitioned the whole haplotype into smaller segments ( composed of 24 adjacent markers ) and tested them for association with pd . consistent with the individual snp analysis , a significant association with pd was observed for the haplotype including the 2 promoter variants , rs363371 and rs363324 ( global p value = 0.04 ) . in particular , the all - minor allele haplotype ag ( the second most frequent haplotype in both case and control populations ) was significantly underrepresented in cases ( frequency 19% ) as compared with controls ( frequency 23% ) , hence being associated with a reduced pd risk as compared with the most frequent haplotype ga ( or 0.78 , 95% ci 0.650.94 , permutation - based p value = 0.03 , additive model ) . finally , in analyses restricted to pd cases , none of the studied snps displayed a significant association with age at disease onset under additive or dominant models , either in the overall sample or after stratification by sex ( data not shown ) . in our study , we found that 2 polymorphisms located in the putative promoter region of the vmat2 gene were inversely associated to pd , whereas the other snps within the gene were not related to the risk of developing pd or to disease aao . the human vmat2 gene consists of 16 exons and 15 introns and has been localized to chromosome 10q25 . the recently launched website pdgene ( http://www.pdgene.org/ ) , which systematically reviews genetic association studies of pd including genome - wide association studies ( gwas ) , confirms that information regarding the association of vmat2 gene with pd is still deficient . a single case control study comprising 190 japanese patients and 190 controls failed to identify any significant association among 3 intronic snps in the gene ( rs2072362 , rs3523 , and rs363334 ) and pd ( mizuta et al . , 2006 ) . although none of the snps analyzed in the present study was specifically tested in the publicly accessible gwas ( as assessed using the national center for biotechnology information database of genotypes and phenotypes , http://view.ncbi.nlm.nih.gov/dbgap ) , the national institute of neurological disorders and stroke ( ninds ) genome - wide genotyping in parkinson 's disease ( dbgap study accession number : phs000089.v3.p2 ) and the center of inherited disease research ( cidr ) : gwas in familial parkinson disease ( phs000126.v1.p1 ) contained respectively 2 ( rs363327 and rs1860404 ) and 1 ( rs1860404 ) snps in high ld with our variants rs363343 and rs363333 , which similarly were not associated with pd . hence the present study , extending the analysis to more snps inside the gene ( both in coding and noncoding regions ) , adds to previous evidence showing no association between intragenic vmat2 variability and pd . a few more studies focused on the association of variants in the vmat2 promoter region with pd . in stark contrast to the very low degree of variability of the coding sequence , a high degree of genetic variability in the vmat2 promoter region has been identified ( glatt et al . , 2001 , 2006 ) . ( 2005 ) sequenced the 17.4-kb vmat2 promoter region in 23 caucasian individuals and identified 47 polymorphisms conferring 13 haplotypes associated to different promoter activities , as suggested by in vitro luciferase - transport assays . of note , both the snps in the promoter region ( rs363371 and rs363324 ) genotyped in our study were among the 4 haplotype - defining snps identified by lin et al . the g allele ( wild type ) of rs363371 is embedded in a consensus sequence for transcriptional repressor gc - binding factor ( gcf ) that is expressed in many tissues , whereas the minor a allele is not ( kageyama and pastan , 1989 ) . on the other hand , rs363324 is in considerable ld with rs363371 ( d = 1 , r = 0.61 ) and with another variant at the 5 untranslated region , 2504t > c ( rs2619096 ) , which is probably a transcription factor binding site , because the switching from t ( wild - type allele ) to c ( variant allele ) could double the promoter activity in luciferase assays in shsy cells ( lin et al . , 2005 ) . interestingly , regulatory haplotypes including these variants have been associated also with alcohol dependence , a different dopamine - related phenotype , supporting the hypothesis that vmat2 gene expression has in vivo physiological relevance to dopamine homeostasis ( lin et al . , 2005 ) . another study screened the vmat2 gene promoter region for common haplotypes and tested their functional effects in reporter gene assays , showing that only the gain - of - function haplotypes ( which displayed significantly increased transcriptional activity in vitro ) were associated to a reduced risk of developing pd ( glatt et al . , 2006 ) in women . in a previous work , we analyzed the relationship between the 2 variants in the promoter region ( rs363371 and rs363324 ) and vmat2 mrna levels in platelets from a subgroup of the subjects genotyped for the present study , but we failed to detect any association ( sala et al . , 2010 ) . nevertheless , because this was not the primary aim of that study , we were probably underpowered to detect subtle differences in mrna platelet levels according to distinct genotypes / haplotypes ; furthermore , the in vitro data may not necessarily reflect the snp activity in vivo because of other unknown activities and known mechanisms such as dna methylation . although the quantitative effect of the variants in vmat2 promoter region on transcriptional activity is expected to be small , and it is unclear how our findings might relate to in vivo rna and protein levels , the reported inverse association might still be biologically meaningful : even slight variations in vmat2 expression might affect pathogenic mechanisms involved in nigrostriatal degeneration ( chen et al . , 2008 ) or in the defense against pd - related environmental toxins ( lee et al . , 2000 ) . in contrast to glatt 's and our findings , 2 polymorphic sequences upstream the vmat2 promoter region were not associated to pd in the japanese population ( kariya et al . , 2005 ) ; however , the different type ( polymorphic microsatellites ) and location of the studied polymorphisms as well as the relevant ethnic differences previously observed in the genotype and allele frequencies of the polymorphisms in / around this gene ( crowley et al . , 2008 ; glatt et al . , the aforementioned gwas ninds and cidr examined only 2 variants ( rs10886051 and rs2532798 ) in the vmat2 putative promoter region , which were in high ld with rs363371 and rs363324 , respectively . whereas in the ninds study both variants were not significantly associated with pd ( association p values = 0.41 and 0.47 , respectively ) , in the cidr study only the snp in ld with rs363371 showed a tendency toward inverse association with pd ( or = 0.8 , p = 0.08 ) , which is in favor of a possible involvement of rs363371 rather than rs363324 in disease risk . although our reported associations of the promoter variants with pd may be spurious , it is also recognized that efforts to minimize the false - positive rates in gwas may lead to missed associations . replication of our findings in independent populations as well as marker saturation of this still understudied vmat2 regulatory region will be required to determine if and how variability in vmat2 promoter affects susceptibility to pd . we adjusted our analyses for possible confounders ( sex , age at study , and smoking ) . the sample size employed and the maf for the selected snps provided sufficient statistical power for the main effect analyses . assuming the population susceptibility allele frequency to be the values observed in controls and a population prevalence of 0.02 , our study had 80% power ( alpha = 0.05 , uncorrected for multiple testing ) to detect ors as small as 1.43 ( or 0.71 or smaller ) for the snp with the highest maf and as small as 1.5 ( or 0.63 or smaller ) for the snp with the lowest maf under a dominant model of inheritance . we also considered multiple genetic models and stratified our sample for multiple variables to explore possible effect modifications , in particular by sex . although we planned our study to include most haplotype - defining snps based on the previous data from the literature , our coverage of vmat2 variability is inevitably incomplete , given the low ld across and around the gene , as inferred from hapmap ceu data ; furthermore , nonsynonymous variants in coding regions of the gene ( most likely to alter its function ) , as well as rare polymorphisms ( maf < 5% ) and copy number variations , have been excluded from our study . hence , the possibility remains that intragenic vmat2 variability contributes to pd susceptibility or interacts epistatically with other genes . another limitation of our study might be that , because we did not conduct correction for multiple testing , false - positive findings remain possible ; hence , our results must be considered as preliminary until replication in additional series is obtained . with use of the bonferroni criteria to adjust for multiple comparisons , the association with pd of only 1 of the 2 snps in the promoter ( rs363371 ) remained significant ( corrected p value = 0.03 , dominant model ) . however , the bonferroni correction for multiple testing takes no account of ld between adjacent variants , thus leading to overcorrection and possible rejection of true - positive findings ( attia et al . , 2009 ) . in conclusion , the current study suggests that variability within the vmat2 promoter region may confer a reduced risk of developing pd . if confirmed and supported by further functional studies , genetic association of these variants with pd susceptibility might also support molecular targeting of the vmat2 gene or gene products as a therapeutic strategy for the disease .

background and objective : pelvic abscesses are a well - known complication of intestinal diseases or abdominal surgery . we report our case series concerning transrectal drainage by endoscopic ultrasound ( eus).methods : between january 2010 and august 2014 , seven patients received transrectal drainage by endoscopic ultrasound ( eus ) were selected and analyzed.results:two pigtails was positioned under fluoroscopic and eus control . the success rate was 100% and complication rate was 0% . the median time of hospitalization was 10 days [ range 4 - 25].conclusions : the technique appears to be safe and feasible in all etiologies . in our experience , we can considerate transrectal drainage by eus like a first - line technique in experienced hands .

we did a single - center retrospective analysis of seven patients with pelvic abscess treated by eus transrectal drainage between january 2010 and august 2014 . there were four men and three women , with a median age of 50 years ( range 22 - 68 ) . a concomitant intravenous antibiotic treatment was always administered be for eus and for a minimum of 7 days after drainage . the method of drainage was done with a linear interventional echoendoscope ( pentax ) ( eg 3870utk pentax - hitachi , hambourg , germany ) . eus - doppler evaluation was first performed to exclude the presence of intercalated blood vessels before puncture with a 19-gauge access needle ( cook ) ( 19 g , echotip access needle , cook ireland ltd . , limerick , ireland ) . the puncture tract was then enlarged with a 10 french diathermic cystostomy over a tetrafluoroethylene ( tfe)-coated 0.035-inch guidewire ( cook endoscopy , winston - salem , nc , usa ) . a second guidewire was then put into the cavity , and two 7 french double pigtail plastic stents were positioned ( 4 or 7 cm length ) . eus drainages were done for a majority of abscesses post surgery ( n = 5/7 ) . the treatment was feasible and effective in 100% of cases [ figures 17 ] , without supplementary radiological or surgical intervention [ table 1 ] . the median time of hospitalization was 10 days ( range 4 - 25 days ) . the main difficulty related to this technique is the positioning of the double pigtail plastic stents , induced by pus outflow that can reduce the endoscopic visibility . two of our patients were embarrassed by the length of the double pigtail plastic stents which protruded in the anal canal . pelvic abscess before drainages pelvic abscess after drainage sagittal view of pigtail stent in pelvic abscess fluoroscopic view of first pigtail insertion endoscopic ultrasound ( eus ) view of douglas abscess eus view of abscess puncture endoscopic view with first guidewire insertion etiology and outcomes since 2003 , eus pelvic abscesses drainage is performed ; given the proximity between the rectal lumen and the abscess , with a mini - invasive technique compare to radiological or surgical drainage . moreover , percutaneous drainage may be difficult to realize and uncomfortable , especially if the collection is in deep location which needs a posterior drainage . our technique is safe , without adverse events observed ; and is actually well standardized , in analogy with 10 years of practice for eus drainage of pancreatic pseudocysts . the use of the fluoroscopy and cystostomy facilitates the procedure and can be easily used by experienced operators . the length of double pigtail plastic stents should be as short as possible , especially if drainage is done in the lower rectum , because it may cause discomfort if it touches the anal canal . we can also conceive that the length of hospital stay could be further reduced if the antibiotic treatment is continued orally in selected patients . eus transrectal drainage is a safe and an efficient method for therapeutical treatment of pelvic abscess . in our experience , we recommend eus transrectal drainage for pelvic abscess as the first - line technique for experienced hands .

backgroundthe reovirus 2 protein catalyzes mrna capping , that is , addition of a guanosine to the 5 ' end of each transcript in a 5'-to-5 ' orientation , as well as transfer of a methyl group from s - adenosyl - l - methionine ( adomet ) to the n7 atom of the added guanosyl moiety and subsequently to the ribose 2'-o atom of the first template - encoded nucleotide . the structure of the human reovirus core has been solved at 3.6 resolution , revealing a series of domains that include a putative guanylyltransferase domain and two putative methyltransferase ( mtase ) domains . it has been suggested that the order of domains in the 2 protein corresponds to the order of reactions in the pathway and that the m7 g ( cap 0 ) and the 2'-o - ribose ( cap 1 ) mtase activities may be exerted by the mtase 1 and the mtase 2 domains , respectively.resultswe show that the reovirus mtase 1 domain shares a putative active site with the structurally characterized 2'-o - ribose mtases , including vaccinia virus cap 1 mtase , whereas the mtase 2 domain is structurally similar to glycine n-mtase.conclusionson the basis of our analysis of the structural details we propose that the previously suggested functional assignments of the mtase 1 and mtase 2 domains should be swapped .

the reovirus 2 protein catalyzes mrna capping , that is , addition of a guanosine to the 5 ' end of each transcript in a 5'-to-5 ' orientation , as well as transfer of a methyl group from s - adenosyl - l - methionine ( adomet ) to the n7 atom of the added guanosyl moiety and subsequently to the ribose 2'-o atom of the first template - encoded nucleotide . the structure of the human reovirus core has been solved at 3.6 resolution , revealing a series of domains that include a putative guanylyltransferase domain and two putative methyltransferase ( mtase ) domains . it has been suggested that the order of domains in the 2 protein corresponds to the order of reactions in the pathway and that the mg ( cap 0 ) and the 2'-o - ribose ( cap 1 ) mtase activities may be exerted by the mtase 1 and the mtase 2 domains , respectively . we show that the reovirus mtase 1 domain shares a putative active site with the structurally characterized 2'-o - ribose mtases , including vaccinia virus cap 1 mtase , whereas the mtase 2 domain is structurally similar to glycine n - mtase . on the basis of our analysis of the structural details we propose that the previously suggested functional assignments of the mtase 1 and mtase 2 domains should be swapped . methylated 5'-terminal cap structures have been described in most eukaryotic and many viral mrnas . in all cap structures , including the ' minimal ' cap 0 ( mg(5')ppp(5')n ) , an n7-methylguanosine ( mg ) is attached through a 5'-5 ' triphosphate bridge to the penultimate nucleoside . cap 0 is usually synthesized in the nucleus by the sequential action of three enzymatic activities : mrna triphosphatase , guanylyltransferase ( gtase ) , and mg - methyltransferase ( mtase ) ; this pathway was defined originally during studies of vaccinia and reovirus mrna synthesis . in some molecules , additional 2'-o - ribose methylations are found at the penultimate and the antepenultimate nucleosides , forming the cap 1 ( mg(5')ppp(5')nm2'o ) and cap 2 ( mg(5')ppp(5')nm2'opnm2'o ) structures , respectively ( reviewed in ) . the capping apparatus differs significantly in fungi , metazoans , protozoa and viruses in respect of the evolutionary origin and structure of individual domains , and the arrangement of domains within subunits . hence , the capping enzymes encoded by viral , fungal and protozoal pathogens are attractive targets for specific inhibitors that would exert a limited effect on the host enzyme . reoviruses are non - enveloped icosahedrally symmetric viruses with two concentric protein capsids surrounding and protecting the multisegmented , double - stranded ( ds ) rna genome ; they replicate in the cytoplasm of the eukaryotic host cell ( reviewed in ) the reovirus core particle can produce mgpppgm2'opc(pn)n - oh plus - strand rna from each genomic double - stranded rna segment in vitro , indicating that it contains all of the enzymes necessary for de novo synthesis of capped mrna . on the basis of sequence analysis and the results of uv - crosslinking and site - directed mutagenesis it has been suggested that the 144-kda 2 protein possesses an s - adenosyl - l - methionine ( adomet)-dependent methyltransferase ( mtase ) domain ( or domains ) , which mediates both the cap 0 and cap 1 methylation activities in forming a 5 ' cap structure on reovirus mrna . nevertheless , the failure of isolated 2 to exhibit methylation activity limited studies to dissect its putative methyltransferase activities and domain structure at a molecular level . the structure of the reovirus core has been recently solved at 3.6 resolution by reinisch et al . ( entry 1ej6 in protein data bank ) , revealing a series of seven domains in the 2 monomer , of which one domain ( amino acids 1385 ) was identified as a guanylyltransferase and two domains ( 434691 and 8041,022 ) were found to share the three - dimensional fold with typical adomet - dependent mtases ( reviewed in ) . in the turret formed by 2 pentamers , the gtase site from one monomer is closer to the mtase 1 in the clockwise neighbor than to other mtase domains . hypothesized that a correlation may exist between the spatial arrangement of domains in 2 , and the order of reactions in a pathway ( guanylyl transfer n7 methylation 2'o methylation ) . thus , it has been proposed that mtase 1 is the cap 0 mtase and mtase 2 is the cap 1 mtase . however , no groove to guide the mrna from one active site to another could be identified and the problem of specificities of reactions carried out by the individual mtase domains was left open . in the course of a large - scale analysis of mtase sequences and structures we observed low similarity between various ribose 2'-o - mtases from eubacteria and negative - strand rna viruses and a fragment of the grass carp reovirus protein corresponding to the carboxy - terminal part of mtase 1 of human reovirus ( j.m.b . and l.r . disappointingly , sequence database searches carried out using psi - blast initiated with the 2 sequence and its fragments revealed significant similarities only between the human reovirus and grass carp reovirus proteins . nonetheless , vast searches of the protein data bank revealed high similarity of both putative mtase domains to numerous known mtase structures . detailed inspection of the coordinates superimposed using swiss - pdb viewer allowed us to predict which amino - acid residues might be involved in cap 0 and cap 1 methylation of reovirus mrna . according to vast , mtase 1 showed highest similarity to the rrmj ( 1ejo ; p - value = 10 ) and vp39 ( 1av6 ; p - value = 10 ) proteins . the mutual similarity of mtase 1 and mtase 2 , which can be regarded as a reference , was evaluated as high , albeit significantly lower ( p - value = 10 ) . rrmj and vp39 are both 2'-o - ribose mtases ; rrmj targets u2552 in eubacterial rrna , whereas vp39 is a bonafide cap 1 mtase of vaccinia virus mrna . remarkably , simultaneous superposition of the three structures revealed perfect conservation of the k - d - k - e tetrad of putative catalytic residues ( figures 1,2,3 ) , which has been observed by us in other families of genuine and putative 2'-o - mtases ( and j.m.b . and l.r . , unpublished data ) . these results strongly argue that mtase 1 is more likely to be a cap 1 mtase than a cap 0 mtase . in the mtase 1 structure we could identify no aromatic residues that would superimpose well with y22 and f180 from vp39 , which form enhanced stacking interactions with the n7-methylated cap . moreover , a region corresponding to the guanine - binding pocket in vp39 is blocked off by a differently positioned loop in the reovirus 2 protein structure . nevertheless , we identified a cluster of aromatic side chains ( y460/464 , f461/465 and f618/f622 ) that are invariant between the human and grass carp reovirus sequences and map in the vicinity of the methylated cap , if the mrna coordinates are copied from the superimposed vp39 structure ( figure 2 ) . it is tempting to speculate that a conformational change in this region occurring on mrna binding would , for instance , reallocate y460 and f618 residues of human reovirus to positions equivalent to y22 and f180 from vp39 . the sequence alignment ( figure 3 ) has been manually adjusted to illustrate this hypothesis . the mtase 2 structure showed highest similarity to the glycine n - mtase ( gnmt ; 1xva ; p - value = 10 ) . it is noteworthy that the mtase 2 domain showed lower similarity ( p - value = 10 ) to vp39 than to gnmt and the mtase 1 domain showed lower similarity ( p - value = 10 ) to gnmt than to the 2'-o - ribose mtases . figure 4 shows that in addition to the common catalytic domain , gnmt and mtase 2 share a topologically equivalent ' lid ' domain made of three antiparallel strands . in gnmt , the -lid domain forms a wall of a large ' molecular basket ' structure , which may accommodate a variety of small molecules , including adomet , tetrahydrofolate and polycyclic aromatic hydrocarbon molecules such as benzopyrene ( reviewed in ) . in mtase 2 , the strands forming the -lid domain are much shorter and the pocket is also smaller . however , a guanine moiety can be docked into that pocket in a way that the n7 nitrogen is presented for the methyl group donor ( data not shown ) . most of the amino acids that line up the putative guanine - binding pocket of mtase 2 ( for instance q925 , n927 , f951 , r956 , and e958 ) are conserved between the human and grass carp reovirus sequences ( figure 5 ) . to our knowledge , all structurally characterized mtases that modify bases in nucleic acids and do not employ covalent bond formation with the target use aromatic or aliphatic side chains to bind the base to be methylated and stabilize it in the active site . examples of such mtases , in which the structure of the active site was determined experimentally or predicted from sequence analysis , include enzymes generating n - methyladenine in dna and rna ( reviewed in ) , n - methylcytosine in dna , and n - guanine in rna . we hypothesize that the invariant f951 and l890 ( substituted by f897 in grass carp reovirus ) may be involved in van der waals interactions with an aromatic ring of guanine . but because of the low resolution of the original structure and lack of a precise docking model , the detailed contacts between the target and the enzyme could not be predicted . nevertheless , we believe that our model will be a useful guide for site - directed mutagenesis experiments that would allow to elucidate the role of individual side chains of the putative cap 0 mtase . the data presented suggest that mtase domains 1 and 2 of reovirus 2 protein function as cap 0 and cap 1 mtases , respectively ; that is , that the previous assignments should be swapped . on the basis of detailed comparison of the available mtase structures , we predicted the substrate - binding mode for both mtase domains and proposed which residues are likely to participate in catalysis we suggest that substitution of the predicted cap - binding or catalytic residues in the mtase 1 domain should result in accumulation of cap 0-methylated viral mrnas that nevertheless lack cap 1 modification . we also report for the first time a remarkable structural similarity between the predicted mg mtase domain and gnmt , suggesting a common evolutionary origin for the two n - mtase families . it is noteworthy that threading analysis and subsequent homology modeling suggest a similar structure for a family of cellular and poxviral cap 0 mtases that show no significant sequence similarity to predicted cap 0 mtases from the sindbis - like supergroup of positive - strand rna viruses nor to the predicted cap 0 mtase from reoviruses analyzed herein ( j.m.b . and l.r . on the other hand , structure prediction for the family of aminoglycoside - resistance 16s rrna : mg mtases suggests that it shares only a common catalytic domain , but not the -lid domain or predicted target - binding residues , with other mg mtases . it will be interesting to determine how many times the mechanism of guanine - n methy - lation appeared in the evolution of adomet - dependent mtases and what is the phylogenetic origin of the individual viral cap 0 mtase families . this work was supported by kbn ( grant 8t11f01019 to j.m.b . ) and bioinfobank . we would like to thank karen reinisch for comments and suggestions that helped to improve the manuscript . stereoview ribbon diagrams of superimposed structures of the mtase 1 domain ( green ) , vp39 ( blue ) and rrmj ( magenta ) . mtase 1 shows elaborations of the common fold similar to those of vp39 , which are altogether absent from the rrmj structure and which are predicted to take part in cap recognition and binding . adomet ( copied from the rrmj structure ) and the k - d - k - e tetrad of putative catalytic residues are shown in wireframe representation . sequence alignment of the predicted human reovirus cap 1 mtase with its counterpart from grass carp reovirus ( gcrv ) and vaccinia virus ( vp39 ) . residues that are invariant between the reoviruses and those predicted to participate in binding of the adomet , the cap moiety , and in catalysis in all cap 1 mtases , are highlighted in black ; other conserved residues are highlighted in gray . stereoview of superimposed structures of the mtase 1 domain ( ribbon in green , side chains and labels in red ) and vp39 ( ribbon and labels in blue , side chains in cyan ) , delineating the proposed binding sites for the guanine moiety and the target ribose 2'-oh . capped mrna is shown in orange and the methylated guanine ( black label ) is sandwiched between y22 and f180 of vp39 . a conformational change of both the protein and the rna is required for the cluster of conserved aromatic residues of the reovirus mtase to bind the cap structure in a similar manner . stereoview of superimposed structures of the mtase 2 domain ( green ) and gnmt ( blue ) showing extensive similarities of both domains .

objective : helicobacter pylori ( h. pylori ) is the most common chronic bacterial infection in humans . chronic colonization increases the risk of duodenal ulcer and gastric cancer . the risk factors for acquiring the infection have been extensively studied . however , there are conflicting results on the role of breastfeeding in the prevention of h. pylori infection . we conducted a study to evaluate the effects of breastfeeding on the h. pylori infection in kurdish children in sanandaj , ir iran.methods:a historical cohort study was carried out from january 2011 through december 2012 . totally 221 children who were going to attain 2 years old during the study period were randomly enrolled . they were divided into two groups , i.e. breastfed and non - breastfed . we used h. pylori stool antigen test to detect infection in the selected group of children after age of 2 years and cessation of breastfeeding . each group was subdivided into two subgroups , infected and non - infected . the associations of breastfeeding with h. pylori infection was assessed using statistical software.findings:we found no difference in the odds of infection between breastfed and non - breastfed groups ( or=0.809 , 95% ci [ 0.4531.444 ] ) . an association between age and the prevalence of infection was found ( p=0.008 ) . there was an increase in the odds of infection as the family size grew ( or=1.93 , 95% ci [ 1.043.6 ] ) as well as increasing housing density ( or=2.12 , 95% ci [ 1.104.10]).conclusion : the data suggests that breastfeeding in infancy does not protect against h. pylori infection for long duration among studied children in iran . the protective effects of breastfeeding , if any , are at most transient .

helicobacter pylori is the most common chronic bacterial infection in humans , affecting 50% of the world population . chronic colonization increases the risk of duodenal ulcer and gastric cancer , including adenocarcinoma and mucosa associated lymphoid tissue ( malt ) lymphoma . the relative risk of gastric carcinoma in infected persons is 2.3 to 8.3 times greater than that in normal population . according to the world health organization ( who ) classification , h. pylori has been classified as group i carcinogens . although family members are the most likely source of infection , the factors that make some people more susceptible to infection have not been determined . it has been suggested that infection with this organism starts in infancy , and most cases occur before the age of three . the mechanism of transmission of this organism is unknown , but the most likely route of transmission is fecal - oral or oral - oral . the prevalence of h. pylori infection is much higher in developing countries . in children , the risk factors for h. pylori infection include socioeconomic status , number of siblings , race / ethnicity , rural residence , institutional residence , lack of maternal education and infection status of family members . the protective effect of breast milk and its beneficial effects in preventing respiratory infections and diarrhea have long been known . several studies have suggested that breastfeeding can also prevent bacterial colonization of h. pylori during childhood . however , other studies had conflicting results and the importance of breastfeeding in the prevention of h. pylori is still under question . in fact , there are studies that have reported that breastfeeding may even increase the risk of h. pylori infection . breast milk may be infected by h. pylori and horizontal infection through breastfeeding may occur . we conducted the present study to evaluate the effects of breastfeeding on h. pylori infection in kurdish children in sanandaj , ir iran , by using the h. pylori stool antigen test ( hpsa ) . sanandaj is the center of kurdistan province , in west iran , with a population of about 450000 . a previous study has detected a high prevalence of h. pylori in children in this city . the study had a historical cohort design that was carried out from january 2011 through december 2012 in the pediatric department of besat tertiary hospital affiliated to kurdistan university of medical sciences . participants were selected from a list of healthy children in 12 primary healthcare centers across the city . we selected them from children who were going to attain 2 years old during the study period . the rationale for selection of these age groups was that the breastfeeding practice usually continues 2 years or even longer in our community . simple random sampling was used in breastfed children . however , in non - breastfed children , we had to enroll all of them due to paucity of cases . a computer based program generated random numbers in order to draw a selected group of children from the lists . the sample size for both breastfed and non - breastfed groups was calculated as at least 90 patients for each group . the sample size calculation was based on at least 20% prevalence in the community , 40% prevalence in studied children , 20% power , an odds ratio ( or ) of 2.5 , and 95% confidence interval ( ci ) , computed from previous epidemiological studies . all procedures and tests were carefully explained to the parents of children and informed consent was obtained . through interviews with the parents , the data regarding age , sex , parents education , duration of breastfeeding , family size , previous antibiotic usage , height and weight , were recorded for each child . breastfed child was defined as a child that has had at least 1 month duration of continuous breastfeeding . status of breastfeeding until the date of stool sampling ( at least age of 2 years or older ) was questioned and on the base of the breastfeeding status the children were divided into two groups , i.e. breastfed and non - breastfed . these two groups were compared in terms of the absolute breastfeeding ( no formula exclusively ) , those with mixed feeding with breast milk and formula for various durations of time and , those with no breastfeeding ( no breast milk exclusively ) . infants that have received antibiotics or proton pump inhibitors more than a week in the last 2 months were excluded from the study . a stool sample was collected from the enrolled children who attained at least 2 years old by parents with varying time after breastfeeding cessation . we used premier platinum hpsa plus ( meridian diagnostics , cincinnati , ohio , usa ) kits to detect infection in children . the stool assay kit was purchased from a local representative of meridian company , netherlands . it is cleared by the fda and ce - marked with high sensitivity and specificity in comparison to other stool antigen tests . the maastricht iv / florence consensus states that the diagnostic accuracy of the stool antigen test ( sat ) is equivalent to the urea breath test ( ubt ) if a validated laboratory - based monoclonal test is used . the sensitivity and specificity for the premier platinum hpsa plus test were reported as 92.2% and 94.4% , respectively , in a recent study . the test procedures are described in detail and available online ( http://www.meridianbioscience.com/diagnostic-products/h-pylori/premier/premier-platinum-hpsa-plus.aspx ) . according to the manufacturer s instructions , results are divided into two groups for h. pylori infection : negative ( the optical density at 450/630 nm < 0.100 ) and positive ( the optical density at 450/630 nm 0.100 ) . chicago , il , usa ) . by using descriptive statistics , frequency distribution tables and graphs were drawn and crude odds ors and 95% confidence interval ( ci ) were estimated . we calculated z scores for the indicators of the attained growth standards ( weight - for - age , height - for - age , weight - for - height ) based on who standards , using the respective spss macro ( syntax ) files . we used t test to compare z scores in infected and non - infected groups . a total of 221 children were selected and tested for h. pylori antigen in stool ( table 1 ) . a number of 130 children had a history of variable duration of breastfeeding time ( table 2 ) . age , gender and growth characteristics of studied children numbers in each gender group ; sd : standard deviation prevalence of h. pylori infection based on duration of breast milk feeding and gender a stool sample was tested for helicobacter infection after varying times of breast milk cessation . the age range of enrolled children at the time of stool was 2 to 4 years . we found no difference in the odds of infection between breastfed and non - breastfed groups ( or=0.809 , 95% ci [ 0.4531.444 ] ) . the studied children were divided into five groups as not - breastfed , 16 , 712 , 1318 , and 1824 months of breastfeeding . there was a marginal decrease in the odds of infection as the duration of breastfeeding increased ( p=0.07 ) . we compared the chance of acquiring infection for the two groups of children that lied at the two ends of the spectrum , the first group fed the longest time with breast milk ( 1824 months ) and the second group with no breastfeeding . there were no differences in the odds of infection of infection ( or=1.043 , 95% ci [ 0.5711.904 ] ) . there was no correlation between the prevalence of infection and either the type of feeding in the female gender group ( or=0.44 , 95% ci [ 0.181.056 ] ) nor the duration of breastfeeding among girls . similarly , there was no correlation between prevalence of infection and either the type of feeding in male gender group ( or=1.28 , 95%ci [ 0.5692.864 ] ) , nor the duration of breastfeeding among boys ( =8.16 , p=0.08 ) . when the frequency of h. pylori infection was calculated in various age groups ( table 3 ) , there was a statistically significant association ( =11.92 , p=0.008 ) . association of h. pylori infection with age , family size , home size , body weight , father education and mother education chi square test was used 7 ( 3% ) values for weight were missed 4 ( 2% ) values for weight were missed fisher s exact test was used p values assessing the association of infection with categorized z scores or : odds ratio ; ci : confidence interval ; hpsa : h. pylori stool antigen test the prevalence of infection marginally increased as the family size grew ( = 5.08 , p=0.07 ) . however , when we compared the family size in three member families with four and more member families , we found significant differences in odds of infection ( or=1.93 , 95%ci [ 1.043.6 ] ) ( table 3 ) . we corrected these numbers by dividing family size by home size , i.e. calculated housing density , and observed a significant increase in the odds of infection , as the density increased ( or=2.12 , 95% ci [ 1.104.10 ] ) . we calculated the z scores for weight - for - age , height - for - age , and weight - for - height for both non - breastfed and breastfed groups based on who standards and the prevalence of infection in respective groups ( table 3 ) . the mean z score for weight - for - age in infected and non - infected children was calculated as 0.071.45 and 0.541.16 , respectively , which was statistically a significant difference ( p=0.02 ) . the mean z score for height - for - age in infected and non - infected children was calculated as 0.141.73 and 0.42 1.85 , respectively , which was not statistically significant ( p=0.3 ) . the mean z score for weight - for - height in infected and non - infected children was calculated as 0.051.61 and 0.221.79 , respectively which similarly was not statistically significant ( p=0.5 ) . we were unable to confirm the protective association between breast milk feeding in prevention of h. pylori infection . we found higher odds of infection in the selected ages of the two groups , breastfed and non - breastfed . the prevalence of infection was higher in these age groups in comparison to previous studies . there are multiple research articles that have surveyed the effect of breast milk on infection by h. pylori . our study found no protective effect of breast milk against h. pylori infection . a systematic review by chak et al suggested that breastfeeding is protective against h. pylori infection . however , they reported that the studies conducted in developed countries did not achieve statistically significant enough or to conclude that breastfeeding has a protective effect against h. pylori infection . most of the studies with significant or were in the low and middle income countries . the prevalence of h. pylori infection in each community depends on two factors , the rate of acquisition and the rate of persistence of infection . in developing countries , most of the acquisition of this infection occurs in early childhood and continues as chronic infection to adult life . unhygienic behavior , especially during early childhood , frequent contacts with the sources of infection and frequency of other risk factors lead to persistence of infection . based on data from united nations secretariat , iran consequently , lack of protection by breast milk feeding against h. pylori in this country , may be more consistent with the results that have been obtained in developed countries . however , the high prevalence of h. pylori in many epidemiologic studies in iran is more compatible with the epidemiology of h. pylori in low income countries . the components of breast milk , such as antibodies , lactoferrin , lysosomes , and kappa - casein possess antibacterial and antiviral characteristics that protect infants against many infectious agents causing diarrhea and pneumonia . however , these types of immunity are mostly passive and are not long - lasting enough to protect the infant after cessation of breastfeeding . once breastfeeding has ended , it is likely that it is the better nutritional status and the well development fostered by breastfeeding that protect the child against infections , and not the direct effect of breastfeeding ( i.e. the immunity acquired passively by breastmilk ) . one assumption regarding the different results found in our study is that , in the low and middle income countries , malnutrition is very common . there is no large variation between nutritional status of infants fed with breast milk and formula fed infants as seen in the low income african countries such as gambia or egypt . unlike low income countries , many types of substitute feedings including various formulas for infants are readily available to even low income people in our country . as there are no significant differences in nutritional status of breastfed and non - breastfed children in our county , we may be unable to detect differences between the prevalence of helicobacter infection in these two groups . one study in gambian children reported a high prevalence of h. pylori infection in children with malnutrition or chronic diarrheal disease . in our study , the mean z scores for body weight in non - breastfed children in non - infected and infected groups was calculated as 0.00 and 0.29 , respectively ( p=0.4 ) . in other words , there was no association between the infection odds and lower z scores for body weight in non - breastfed children . however , in breastfed - children , the mean z scores for body weight in non - infected and infected groups were calculated as 0.12 and 0.69 , respectively ( p=0.02 ) . there was a significant correlation between odds of infection and lower z scores in breast milk fed children . in the absence of correlation of the other malnutrition indicators ( height - for - age and weight - for - height ) with the rate of infection , it seems that , in iran , the infection is more associated with a mild type of acute malnutrition . with other words , in the context of h. pylori infection , maternal infection with h. pylori plays a major role in the acquisition of this infection in the early years of life . long term and close contact with mother , despite presumptive passive protection by breast milk feeding , involve frequent exposure to infectious sources of h. pylori . non - breastfed infants have less contact with their mothers . in iran , on the other hand , many mothers avoiding breast milk feeding because of their employment are from high socioeconomic graduated class . these factors may explain the mechanism by which the infection rates increase in breast milk fed infants . moreover , breast milk feeding causes a decrease of other infectious diseases and less need for antibacterial drugs . less exposure to antibacterial drugs results in the persistence of h. pylori in the stomach , which is usually primarily eradicated by accidental administration of antibacterial drugs for respiratory or diarrheal diseases . high infection odds due to high carriage rate may be a reason for the lack of protective effect of breast milk feeding in our study . there may be a relative protective effect for breastfeeding that is overcasted by the high burden of h. pylori infection . in most of the studies that reported a protective effect from breast milk infection , the prevalence of infection was notably below 4050% of the study population in lower age groups . there is a general agreement that the protective role of breastfeeding against h. pylori infection is passive , and therefore is not long lasting . reinfection is possible after waning of the protective effect of maternal antibodies occurring with the cessation of breast milk feeding . in our study , the mean age of the studied children was 39 months and the mean interval between sampling time and the cessation of breastfeeding was calculated as 13 months . considering the high carriage rate in our community , this interval may be long enough for waning immunity , reinfection and surpassing the suspicious protective effect of breastfeeding . the prevalence of exclusive breastfeeding and the mean duration of breastfeeding in iran are above 75% and 23 months duration , respectively . the prevalence of continuing breastfeeding for a minimum of 1215 months is of 84% in iran . on the other hand , the prevalence of h. pylori infection in iran has consistently been reported as well above 50% in many studies conducted in this country . consideration of these facts challenges largely against the protective effects of breastfeeding in our community . lengthening breastfeeding duration to multiple months in the early years of age had no effect on the acquisition of infection . however , this is a contradiction in articles advocating for the protective effects of breastfeeding against h. pylori infection . if one assumes any protective effect for breast milk against h. pylori infection , it should be a dose - response relationship . moreover , in many of these articles , we found no case definition for breastfeeding based on duration . in one study , a prolonged time of breastfeeding was associated with an increased chance of acquiring infection . the majority of the studies regarding the protective effects of breast milk against h. pylori infection have used laboratory tests , other than stool antigen test , as diagnostic tools . this test has low sensitivity and specificity in comparison to the stool antigen test . these variables may cause confusing results in the study of the protective effect of breast milk against infection . it is a worthwhile way with high sensitivity and specificity , comparable to hpsa test . however due to the high cost , limited availability in developing countries and the complexity of the test , it is less applicable on large scale in healthy children . a recent meta - analysis discussed that the use of 13c labeled isotope ubt ( 13c - ubt ) tests are less accurate for the diagnosis of h. pylori infection in young children . our search for the epidemiologic studies that used hpsa as a diagnostic tool to evaluate effects of breast milk feeding have revealed only limited results . we were not able to identify the time of acquisition of infection and its relationship to breastfeeding to unveil any short term protection . another prospective cohort study may help to solve this problem and clarify the effects of breast milk feeding on h. pylori infection . the presented data suggest that breastfeeding in infancy does not protect against h. pylori infection for long duration among studied children in iran . the protective effect of breastfeeding , if any , is at most a transient one . j. soltani , b. nikkhoo , j. khormehr , p. ataee , f. gharibi : substantial contributions to conception and design , acquisition of data , analysis and interpretation of data . all authors participated in drafting the article and they critically reviewed the manuscript and approved the final manuscript as submitted .

our research was conducted to determine the algorithm changes during the treatment of submandibular sialolithiasis . two time periods were compared between 20042008 and 20092012 . the turning point was december 2008 , when sialendoscopy procedure was introduced . in the first period , 48 patients were treated : 31 outpatient duct incisions with stone evacuation and 17 surgical excision of submandibular gland . in the second period , 207 sialendoscopy procedures were performed on 197 patients . out of this particular group , 158 patients were diagnosed with pathological obstruction of salivary glands and 64 of them were confirmed to have sialolithiasis of submandibular gland . deposits of calcifications in 40 individuals ( 62.5 % ) affected by sialolithiasis were removed endoscopically ; however , in 21 patients , due to the increased circumference of the stone , the intimate association of deposits within the wall of the duct along with its presence inside the deep portions of the gland , double approach ( incision of the floor of the mouth in hilar area and sialendoscopy ) was performed . three individuals had their salivary glands totally removed due to the presence of calcified deposits within the glandular parenchyma . our results allow us to affirm that sialendoscopy is the current treatment of choice for submandibular glands affected by sialoliths . indication for a complete removal of the gland is becoming uncommon as a first line treatment although still indispensable in chosen cases .

chronic sialadenitis is one of the major disorders that can cause salivary hypofunction and correct diagnosis and management is essential for its recovery . the classification of this pathological condition has changed in the past decade and nowadays was revised and modified , for new diagnostic ( high - resolution ultrasonography , ct and mr sialography and sonoelastography ) and therapeutic methods ( sialendoscopy ) were introduced . sialolithiasis is the most common cause of inflammatory diseases of large salivary glands and occurs in about 1.2 % of the population [ 9 , 21 ] mostly in the submandibular gland87 % . salivary gland stones are single or multiple , located in the efferent duct distally or proximally , rarely occur intraparenchymally , representing various shapes and sizes . the annual increase in size of salivary stones is estimated at 1 mm , and thus the duration of complaints history is crucial for treatment planning . contemporary achievement in endoscopy caused strong common belief that stones of up to 45 mm in diameter can be successfully removed through sialendoscopy ( se ) . this applies especially to stones which lie freely in the lumen of the duct and are mobile . in these cases , the stones can be extracted under endoscopic control in more than 80 % of cases [ 8 , 9 , 14 ] . larger sialoliths may , however , be fragmented in the lumen of the duct , either mechanically or using a laser beam . lithotripsy ( eswl ) is another possibility for the fragmentation of large sialoliths of any size and location ; although up to three sessions of lithotripsy may be required . thus , the introduction of sialendoscopy has significantly reduced the number of submandibular glands removal in the course of sialolithiasis [ 5 , 6 , 8 , 9 , 14 , 17 ] . according to literature data , the use of lithotripsy is effective in 75 % of cases , and in turn , allows for the complete retrieval of stones in half of the cases [ 2 , 3 , 6 , 7 , 22 ] . the number of successes in the use of lithotripsy clearly decreases with increase in the stone diameter . despite notable technological progress , 510 % of patients with sialolithiasis can not be successfully treated using minimally invasive techniques . the main cause appears to be the large size of the stones and long - standing history of recurrent inflammations , which lead to the impaction of the sialolith to the wall of the efferent duct . in these cases , the aim of the study was to analyze the trends in the treatment methods in submandibular sialolithiasis in the past decade , the effectiveness of particular methods and to present treatment schedule proposed by the authors . 112 patients with submandibular gland sialadenitis were treated in tertiary university centre ( otolaryngology , head and neck surgery department pozna medical university ) in the years 20042012 . data were analyzed retrospectively on the basis of medical documentation ( outpatient charts , operating protocols ) . the epidemiological data ( gender , age ) , duration of complaints , the treatment method and its effectiveness were analyzed and compared in both groups . in preoperative diagnosis , additionally , in the recent 3 years , ct was performed in 11 cases . during first period , patients were treated in outpatient department by incision of mucosa of floor of the mouth in local anesthesia . following the introduction of sialendoscopy , patients were admitted to the hospital for 1 day . during interventional sialendoscopy , 1.3 and 1.6 mm diameter endoscope ( karl storz tutlingen , germany ) was used . stones were removed with the help of the basket and forceps , introduced through the working canal . the se procedure was carried out under local anesthesia after premedication with ( midazolam , 7.5 mg ) . once the size of the stone localized in submandibular hilum was determined to be larger than 67 mm and endoscopic removal was deemed impossible , the decision of combined approach was made . the combined approach ( incision of the floor of the mouth at the level of submandibular hilum and sialendoscopy ) was performed also under local anesthesia . in case of failure of this treatment and when stone was primarily localized in gland parenchyma , the decision of eswl or total removal of the gland was undertaken . the open surgery was performed under general anesthesia with the help of facial nerve monitoring , with leads from area of marginal branch around the mouth . although the paper had a predominantly descriptive character , some statistical analysis was performed using spearman and kruskal wallis tests . there were 48 patients with submandibular sialolithiasis : 31 outpatient duct incisions with stone evacuation and 17 surgical excision of submandibular gland in the first 5 year period of time . there were 21 men and 27 women , aged from 20 to 73 , mean 43 . the length of complaints ranged from 3 months to 20 years , mean being 3.4 years . the number of treated patient ( in whom the removal of submandibular gland was undertaken ) in those years was as follows : 20041 patient , 20052 , 20063 , 20074 , 20082 , 20094 , 20100 , 20111 . in the second , 3.5-year period wharton duct stenosis was diagnosed in 33 patients ; 64 out of 97 patients with submandibular sialadenitis suffered from lithiasis . there were 38 men and 26 women , aged from 14 to 68 , mean 45 . the length of complaints ranged from 2 months to 17 years , median 2.9 years . endoscopic removal of stones was possible in 40 cases ( 62.5 % ) . in 37 patients , double approach ( incision of the floor of the mouth in hilar area and sialendoscopy ) in patients with refracted , big stones and long history of lithiasis was performed successfully in 21 out of 64 ( 32.8 % ) patients . surgical excision of the submandibular gland was performed in 3 out of 64 ( 4.7 % ) patients between 2009 and 12 . follow - up in this group of patients ranged from 48 to 6 months , mean being 19.6 months . comorbidities and patients age had no correlation with sialendosoppy stone removal rates ; these were , however , found to be statistically dependent from the duration of complaints and a history of more than 5 years doubled the risk of failure ( p < 0.005 ) . various methods of treatment through two distinct time intervals are depicted in table 1.table 1various methods of treatment throughout two time intervalssubmandibular gland calculiyears 20042008years 20082012removal of calculi through incision of mucous membrane of floor of the mouth in hilar area310sialoendoscopy064endoscopic removal of calculi040 ( 62.5 % ) double approach021 ( 32.8 % ) surgical removal of submandibular gland173total4864 various methods of treatment throughout two time intervals according to bigler , harrison , yoel , work , wang et al . , there are two particular subdivisions : chronic obstructive ( sialolithiasis , stenosis of the duct , inflammation of the glandular tissue with recurrent stenosis or enlargement of the duct ) and non - obstructive group of inflammations this agonizing experience may even occur without any component of mechanical obstruction , although presence of lithiasis is the main cause in 50 % of affected individuals . high - resolution ultrasonography could be utilized as an optional diagnostic method for visualization of calcified deposits or exclusion of tumor presence . the main feature of sialography is excision , constriction or enlargement of the excretory ducts . ultrasonographic confirmation of either ductal stricture or presence of inraparenchymal stones allows postponing the sialoendoscopy procedure until the acute state has subsided [ 6 , 8 , 13 ] . high - resolution computer tomography is still considered to be the most sensitive method for the determination of stones , whereas the ultrasonographic technique allows to view a sialolith > 2 mm . increased accumulation of inorganic calcified deposits within the center of the gland gives off a strong echo signal ; therefore , it is not uncommon to overlook miniscule remnants of the stones due to insufficient signal saturation . however , it is important to emphasize that false positive results could be obtained in case of excessive hyperemia caused by inflammation of the duct . in these particular situations , classic sialography , sialography performed by utilization of computer tomography or by magnetic resonance imaging , is an instrumental addition to the diagnostic evaluation ; however , many authors prefer high - resolution ultrasonography . the use of endoscopic and minimally invasive techniques allows for the greater preservation of the major salivary glands in cases of sialolithiasis . according to literature data , 8090 % of patients with parotid gland sialolithiasis can be treated using minimally invasive techniques such as sialendoscopy and eswl [ 5 , 6 , 8 , 9 , 14 , 15 , 17 , 19 , 27 ] . it should be remembered that stones larger than 6 mm in diameter and impacted in the wall of the duct limit the possibility of using sialendoscopy [ 8 , 9 , 14 , 15 , 19 ] . after performing eswl , larger stones ( larger than 810 mm in diameter ) our initial , 5-year emergency department data indicate that 48 patients who were admitted with the symptoms of sialolithiasis . in 31 patients , stones were removed through an incision in the mucous membrane . due to complications and persistent symptoms , the remaining 17 patients had their submandibular glands removed . following the introduction of sialendoscopy procedure to our department , 3.5 years worth of clinical data show 64 patients admitted with symptoms indicating submandibular sialolithiasis . double approach ( incision of the floor of the mouth in hilar area and sialendoscopy ) with the successful removal of stones was carried out in 21 out of 64 ( 32.8 % ) patients . three patients , however , had their submandibular glands totally removed due to several complications . it is valuable to include a diagnostic and therapeutic procedure developed by koch ( fig . 1 ) . sialendoscopy is considered as a significant diagnostic and therapeutic method of primary treatment . according to our data , 48 patients treated throughout the 5 year time interval without utilization of sialendoscopy , when compared with those 64 patients who were treated with sialoendoscopy procedure , showed mediocre outcomes . it is , therefore , imperative to point out that the introduction of sialendoscopy and superior results obtained throughout its treatment of chronic inflammation of glands affected by sialolithiasis allowed us to gradually adapt to the published literature [ 6 , 11 , 13 , 17 , 19 , 20 ] . in situation in which there is a limited access to eswl , a double approach could be used as an alternative method of treatment in both parotid and submandibular glands [ 10 , 13 , 16 , 18 , 23].fig . 1algorithm of salivary gland obstructive pathology treatment , according to koch et al algorithm of salivary gland obstructive pathology treatment , according to koch et al the most conservative method of treatment , as well as the one that provides us with the least amount of unfavorable outcomes , is the main objective for those affected by a chronic inflammatory process . according to the literature and our current data , in the vast majority of cases , surgical intervention could be replaced with a minimally invasive procedure by utilization of diagnostic and therapeutic sialendoscopy procedures [ 6 , 11 , 13 , 19 ] . according to data obtained at our center , the development of sialendoscopy , however , allowed us to treat our patients with a minimally invasive procedures . similarly , in the year 2004 through 2008 , 17 surgical removals of submandibular glands were performed due to lithiasis or advanced inflammatory states ; however , in the year 20092012 , only three were performed . with the advent of new diagnostic and therapeutic methods , it is imperative to verify current classification of chronic inflammation of large salivary glands . critical analysis of literature reviews and our own data indicate that continuous improvement of current methods and introduction of new ones , such as utilization of sialendoscopy are crucial in treatment of pathological obstructions of salivary glands . indication for a complete removal of the gland is becoming uncommon as a first line treatment although still indispensable in chosen cases .

backgrounddiabetes mellitus ( dm ) , particularly type-2 is a major public health concern worldwide . not much information is available with regard to the incidence of dm in united arab emirates ( uae ) . this study aimed at determining the incidence rate of diabetes mellitus among emirati population in ajman , uae . this is a retrospective cohort study.methodsthis study was conducted in all primary health care centers and shaikh khalifa and gmc hospitals , ajman , uae where the emirati population primarily go for diagnosis and treatment . the incident cases of diabetes mellitus were collected during the period 2010 january to december.resultsa total of 158 cases newly detected in 2010 , 54 were among non - emiratis and one was type-1 diabetes . eliminating these , the remaining 101 were included in the analysis . the age of the patients ranged from 23 years to 78 years ; 35 ( 34.7% ) males and 66 ( 65.3% ) were females . the overall incidence observed was 4.8/1,000 person - years ( py ) with a female predominance of 6.3/1,000 py against incidence among males of 3.3/1,000 py . with regard to age specific incidence rate among males , it increases with age till 60 years and then showed a decreasing trend . among females also the same trend was observed but not as similar to males.conclusionthe highest incidence rate was observed in the 55 - 59 age group among males , 23.4/1,000 py and females , 32.4/1,000 py . among males the incidence rate was much less compared to females in - the age groups older than 59 years .

diabetes mellitus ( dm ) is one of the most common chronic metabolic diseases , which is on the rise globally.123 ) the dm prevalence for all age groups worldwide was estimated to be 2.8% in 2000 and projected to be 4.4% by 2030 . during this period , the total number of people with dm is expected to rise from 171 million to 366 million.4 ) several explanations have been provided for the increase in dm - prevalence , such as population growth , aging , urbanization , increasing obesity prevalence , and physical inactivity.5 ) diabetes poses a challenge not only to those living with the disease,6 ) but also to their families , communities , and the health care system.789 ) monitoring the dm prevalence and incidence trend is important for assessing the national burden of the disease to describe the impact of risk factors , develop interventions , and project needs for future health services.410 ) the states in the co - operation council for the arab states of the gulf ( gcc ) have some of the highest rates of type 2 dm in the world . five of the international diabetes federation 's top ten countries for dm prevalence in 2011 and projected prevalence in 2030 are in this region.11 ) the recent and rapid socioeconomic development of the gcc countries has been associated with this rising prevalence.12 ) according to the international diabetes federation , the number of people with dm in its middle east - north africa region will increase to 83% by 2030.11 ) in the united arab emirates ( uae ) , the dm prevalence has reached 24% and 17.4% among citizens and expatriates,13 ) respectively . the dm prevalence in the uae was ranked to be the 2nd highest in the world ( 19.5% ) in 2007 , and the expected prevalence for 2025 is 21.9%.12 ) the uae has developed a national diabetes care continuum program with guidelines to combat this disease.13 ) based on a population study conducted in al ain , in the uae , the age - standardized prevalence rates for dm ( diagnosed and undiagnosed ) and pre - diabetes among people aged 30 - 64 years were 29.0% and 24.2% , respectively.14 ) no published data are available about the age - standardized incidence rates of dm in the uae . such data are important , because information on incidence will help plan specific preventive and control strategies , and will enhance our knowledge about the profile of this disease in the uae . therefore , this study was conducted to determine the age and gender specific incidence rate of dm among nationals in ajman medical district in 2010 . the total population of ajman , uae as per 2005 census was 206,997 ( men , 131,684 ; women , 75,313 ) . among this , 39,231 were emiratis ( men , 20,155 ; women , 19,076 ) and the remaining were other nationals ( 11,529 males and 56,237 females ) . the age and gender distribution of the ajman population ( estimated for 2010 using the geometric growth method ) is shown in table 1 . health care services for the target population were mainly provided through 5 primary healthcare centers and 2 general hospitals ( sheikh khalifa hospital and gmc hospital , ajman ) in ajman during the study period , and most of the diabetic patients were presumed to be managed at these sites . the data were collected by using a proforma that was prepared by the team of investigators . every copy of the research proforma was allocated a code number to identify the primary health care center . the pro forma consisted of the domains , such as socio - demographic characteristics , clinical data , biochemical parameters , and mode of management . the emirates or health insurance identification number of the nationals and expatriates were recorded to eliminate duplication . approval from the ethics and research committees of gulf medical university and ministry of health was obtained before commencing this study , and a prior approval was sought from the director of ajman medical district to conduct the study at primary health care centers ( phc ) . participants were not identified by their names , and any information that could reveal the identity of the subjects was not recorded on the proforma . furthermore , confidentiality of the research data was maintained and anonymity was preserved . confidentiality was maintained by maintaining all research documents at the statistical support facility for 3 years , and only the researchers , ethics and research committee members , and statistical support facility members had access to the data with appropriate regulatory sight . any information that can be used to reveal the identity of a participant will not be included in any publications or presentations . cases of dm were documented retrospectively by studying the medical records from january 1 , 2010 to december 31 , 2010 . the cases diagnosed for the first time during the study period were included as incident cases . the following diagnostic criteria were used : ( 1 ) diabetes was defined as a fasting plasma glucose ( fpg ) concentration of 126 mg / dl or previous first diagnosis of dm within the same time period . impaired fasting glucose and normoglycemia were defined as a fpg concentration of 110 - 126 mg / dl and < 110 mg / dl , respectively;13 ) ( 2 ) when criterion ( 1 ) was not met , we relied on the notes written by doctors elsewhere given that the first diagnosis by the other physician was within the same time period . 2010 spreadsheet were transformed to ibm spss ver . 20.0 ( ibm co. , armonk , ny , usa ) for statistical analysis . the incidence rate was presented for different age groups ( i.e. , 5-year intervals ) , and confidence intervals for the proportions were estimated . the crude incidence rates were calculated by considering the population of ajman in 2010 and newly diagnosed dm cases . the dm cases and total population were arranged into different age groups with 5-year intervals to separately calculate age specific dm incidence rates for men and women . a total of 158 new dm cases were diagnosed during 2010 ; 102 of these cases were emiratis residents and the remaining cases were other nationalities . among the 102 emiratis cases , there was only 1 type 1 dm case , and the remaining 101 type 2 dm cases were included in the estimation of incidence rate . the overall incidence rate of dm cases observed among those aged 20 years was 4.8/1,000 person - years ( py ) . in the present study , 35 patients ( 34.7% ) were men and 66 patients ( 65.3% ) were women , indicating female predominance . the gender specific incidence rate was 3.3/1,000 py for men and 6.3/1,000 py for women . the incidence rates in men and women were almost similar until the age of 39 years . however , women who were 40 years showed a higher incidence rate than men . the highest incidence rate for both genders ( men , 23.4/1,000 py ; women , 32.4/1,000 py ) was observed in the 55 - 59 years age group , and then a downward trend was observed . in this study , we evaluated the incidence of dm among emirati residents because they constitute a more stable population than expatriates . future studies involving this population will reflect changes in the epidemiology of this problem that can be linked to lifestyle , socioeconomic , or health service factors . according to the present study , a similar type 2 dm incidence were observed both among emirati men and women in ajman , uae in the age group of 20 - 39 years , and a female preponderance in the incidence rate of dm in the later age groups . the incidence of dm was 4.4/1,000 py in 2005 according to a study from the united kingdom ( uk).15 ) the crude incidence rate of type 2 dm was 515/100,000 ( or 5.15/1,000 py ) in 2010 based on more recent data from the uk.16 ) according to this study , the incidence of dm increased with age , and was similar among men and women up to 40 years of age . after 40 years of age , men had a higher incidence rate of dm than women . the gender specific incidence rate for men and women was 4.86/1,000 py and 4.31/1,000 py , respectively.15 ) an overall dm incidence rate of 10/1,000 py with a male predominance was observed in a study from the united states of america ; using data from the new york city community health survey , the incidence rates were 11/1,000 py and 9/1,000 py in 2008 for men and women , respectively.17 ) the overall incidence of dm was 6.3/1,000 py in 2011 based on the report , " diabetes in canada , " and a male predominance was observed in the gender - specific rates ( 6.8/1,000 py in men compared to 5.7/1,000 py in women in the year 2009).18 ) a cohort study conducted over a period of 2000 - 2009 reported an overall incidence rate of dm in china was 9.5/1,000 py with a male predominance ( 11% men vs. 6.2% women).19 ) according to a previous study in the year 2000 - 2009 , the overall ( 20 - 79 years of age ) incidence rate of dm in taiwan was 11.6/1,000 py.20 ) in a report from sweden,21 ) the incidence rate ( 20 - 100 years of age ) in men and women were nearly similar ( 3.92/1,000 py and 3.64/1,000 py in men and women , respectively ) . the comparison of crude incidence rates in different countries is provided in figure 2 . an age - wise increasing trend in the age specific rates of dm however , only the 10-year age specific incidence rate was available , and the highest rate was observed in the 70 - 79 years age group ( 9.94/1,000 py and 7.77/1,000 py for men and women , respectively . the age specific incidence rates of dm were 0.20 , 0.54 , 2.37 , 4.68 , and 7.46/1,000 py for men and 0.29 , 0.70 , 1.71 , 3.57 , and 6.51/1,000 py for women in the 20 - 29 , 30 - 39 , 40 - 49 , 50 - 59 , and 60 - 69 years age groups , respectively.21 ) the age specific incidence rate of dm in canada was comparable with the present findings ( figures 3 , 4 ) . the rate of dm was almost similar at the age of 40 years in the present study . the peak incidence rate of dm was observed in the 55 - 59 years age group and then a downward trend in the incidence rate of dm was observed . in canada , a steady increase in the incidence rate of dm was observed with increasing age , and the incidence rate in women was much higher in ajman than in canada . a variation in the dm epidemiological pattern was observed in the available data in developing compared to developed countries . according to the present study , dm appears to affect mostly younger participants ( range , 44 - 64 years ) , while in developing countries , the majority of diabetic patients were > 64 years of age.4 ) the higher incidence rate of dm observed among women compared to men after the age of 40 years in this study was incompatible with data from the uk . a further study is needed to investigate the possible related factors ( i.e. , hormonal , dietary , lifestyle , and social factors ) that can restrict the physical activity of older women . both men and women have a nearly similar global prevalence of dm with slightly higher rates in men < 60 years of age and women at older age , greater than 60 years.4 ) it is worth noting that , according to the international diabetes federation,7 ) the estimated percentage of undiagnosed diabetic patients in middle - eastern high - income countries ( i.e. , uae ) was 40.6% in the year 2011 . therefore , the actual incidence of dm is about 40% higher than what was observed in this study . an important finding in this study was that the incidence of dm started to increase after the age of 30 years , which implies that earlier screening , rather than the currently recommended screening at 45 years of age for people without risk factors by the american diabetic association,22 ) is necessary for this disease in the uae . in this study , we have provided evidence to support recommendations by the uae national diabetes committee,13 ) which is included in the periodic health check up program23 ) to start screening for dm in all individuals in the uae at the age of 30 years . other researchers24 ) from the uae explored the need for such a modification to the current american diabetes association guidelines based on their finding of a low prevalence rate of undiagnosed dm ( 1.5% ) among low risk individuals ( age , < 45 years ; body mass index , < 25 kg / m ) . certainly , the final decision about the age to initiate screening for dm is affected by cost - effectiveness and health system capacity factors in addition to the incidence / prevalence of the disease.25 ) we recommend future studies to be conducted in these areas . the incident cases of dm were collected from 5 phcs at shaikh khalifa hospital and gulf medical college hospital in ajman , uae . it is possible that the incidence rate of dm was underestimated in this study because emirati residents at other hospitals were not considered . even with this limitation , we can conclude that the incidence rate of dm was higher than in the uk and sweden . the highest incidence rate of dm was observed in the 55 - 59 year age group ( men , 23.4/1,000 py ; women , 32.4/1,000 py ) . the incidence rate among men was much lower than in women in the > 59 years age groups . the incident cases of dm were collected from 5 phcs at shaikh khalifa hospital and gulf medical college hospital in ajman , uae . it is possible that the incidence rate of dm was underestimated in this study because emirati residents at other hospitals were not considered . even with this limitation , we can conclude that the incidence rate of dm was higher than in the uk and sweden . the highest incidence rate of dm was observed in the 55 - 59 year age group ( men , 23.4/1,000 py ; women , 32.4/1,000 py ) . the incidence rate among men was much lower than in women in the > 59 years age groups .

this pilot study tested the effect of cognitive behavioral therapy ( cbt ) on parent - reported autism symptoms . nineteen children with autism spectrum disorders and an anxiety disorder ( 711 years old ) were randomly assigned to 16 sessions of cbt or a waitlist condition . the cbt program emphasized in vivo exposure supported by parent training and school consultation to promote social communication and emotion regulation skills . parents completed a standardized autism symptom checklist at baseline and posttreatment / postwaitlist and 3-month follow - up assessments . cbt outperformed the waitlist condition at posttreatment / postwaitlist on total parent - reported autism symptoms ( cohen s d effect size = .77 ) . treatment gains were maintained at 3-month follow - up . further investigation of this intervention modality with larger samples and broader outcome measures appears to be indicated .

core autism symptoms such as social communication deficits have proven difficult to change through social skills training ( rao et al . 2008 ) . although several large - scale interventions for young children with autism such as pivotal response treatment meet american psychological association criteria for probable efficacy , therapeutic treatment programs for school - aged children with an autism spectrum disorder ( asd ) have not achieved this level of empirical support . however , there are no known methodologically rigorous studies of cognitive behavioral therapy ( cbt ) for core autism symptoms in spite of the fact that cbt may well suited for this indication . this paper presents preliminary results on the impact of cbt on parental reports of core autism symptoms in 711-year - old children . a common therapeutic approach that has been used to address core autism symptoms in school - aged children with asd up to this point has been social skills training ( rao et al . 2008 ) . this approach often involves practicing specific social skills ( e.g. , greetings ) in regularly - meeting groups of children with asd using hypothetical or dramatized scenarios as the principle learning mechanism . studies of this approach have generally ( a ) relied on diagnostically nonspecific measures of social skills rather than measures of core autism symptoms that are typically used in the diagnosis of asd and ( b ) either have found little evidence of generalization of treatment effects or have used uncontrolled designs that preclude conclusions about efficacy ( rao et al . 2008 ) . in comparison to social skills training , cbt relies on a cognitive science - informed model of psychological change that aims to promote retrievable memories of adaptive responses that can successfully compete with and suppress memories of previously learned maladaptive responses evoked under real world conditions outside the therapy office ( e.g. , brewin 2006 ) . in developing cbt treatment methods for children with asd , target goals ( e.g. , social skill development and generalization ) need to be matched with procedures for enhancing memory retrieval . we have outlined cbt procedures for addressing numerous core autism symptoms elsewhere ( sze and wood 2007 , 2008 ; wood et al . given space limitations , two examples of such procedures are given : ( 1 ) to promote reciprocal conversation skills , the encoding specificity principle from basic memory research suggests that skill learning should occur in the actual settings where social deficits are exhibited , rather than in simulated social situations such as therapy settings , as is often done in traditional social skills training . teachers and parents can be trained on simple social coaching procedures that promote the child s use of reciprocal conversation skills ( e.g. , posing questions ) in real - world settings immediately preceding actual social interactions ( i.e. , moments before , rather than hours or days before , the interactions ) to address this clinical need . ( 2 ) research on levels of processing in human memory has demonstrated that deep semantic processing rather than rote memorization increases the chance of the retrieval of a target memory ( e.g. , for a social skill ) . to promote deep semantic processing of new concepts , socratic questions ( questions that incorporate hints of the correct answer ) can be posed by the therapist to encourage children to put accurate answers in their own words ( sze and wood 2008 ) . the combination of repeated in vivo rehearsal of social skills in real - world settings coupled with socratic discussions about the positive effects of such skills may promote deep semantic processing and increase the memory retrieval of the targeted skills in naturalistic contexts while helping to suppress memories of habitual maladaptive responses such as social avoidance . cbt has achieved large , clinically significant outcomes in other childhood disorders ( e.g. , walkup et al . 2008 ) and high - functioning children with asd are considered promising candidates for this treatment approach . studies of information processing and cognition in high - functioning children with asd show that contrary to common speculation , their capacity for abstract reasoning is varied rather than absent ( e.g. , minshew et al . it is therefore plausible that the cognitive aspects of cbt would be understandable for many high - functioning school - aged children with asd , as long as the nature of asd is taken into account in the presentation of skills and concepts ( anderson and morris 2006 ) . in a recent randomized , controlled trial , a cbt program was found to have a positive impact on parent- and diagnostician - reports of anxiety in children with asd ( wood et al . anxiety disorders are common among children with asd and are associated with greater impairments in social responsiveness and more repetitive behaviors ( sukhodolsky et al . ( 2007 ) have proposed that anxiety in asd may be an indicator of a meaningful subclass of children with a greater likelihood of benefitting from the intervention techniques used in cbt . in the present study , it was hypothesized that cbt would outperform a waitlist condition on these outcomes and that a reduction of anxiety symptoms during the trial would be associated with a reduction of autism symptoms . the sample included 19 children , ranging in age from 711 years ( m = 9.37 , sd = 1.42 ) , and their primary parent(s ) ( defined as parent(s ) who were responsible for overseeing the child s daily activities ) living in a major metropolitan area of the western united states ( see wood et al . the 19 children in this sample include the final 11 participants from the wood et al . ( 2009 ) trial as well as eight additional children subsequently randomized into the same treatment protocol : ( a ) beginning with the 29th child enrolled in the wood et al . ( 2009 ) trial , the social responsiveness scale ( srs ; constantino and gruber 2005 ) was added as an outcome measure . once the 40th child completed treatment , anxiety disorder outcomes were evaluated as planned and the paper ( wood et al . ( 2009 ) paper was submitted , we randomized eight additional children into the same study protocol and collected srs data from their parents . ( c ) hence , the final 19 of the 48 total participants who had been randomized to condition had srs data . the cbt intervention was identical for all 48 participants and there were no extra treatment components administered to children whose parents completed the srs . children were referred by a medical center - based autism clinic , regional centers , parent support groups , and school personnel such as inclusion specialists . children met research criteria for asd ( see below ) and at least one anxiety disorder . psychiatric medication , if used , was at a stable dose prior to intake and throughout the duration of the trial . children with verbal iqs less than 70 or who were in concurrent psychotherapy were excluded . most children were boys ( n = 16 ; 84% ) and most primary parents had a college degree ( n = 15 ; 79% ) . children s race / ethnicity varied : caucasian ( n = 10 ; 53% ) ; asian ( n = 4 ; 21% ) ; native american / caucasian ( n = 2 ; 11% ) ; latino ( n = 1 ; 5% ) ; latino / asian ( n = 1 ; 5% ) ; african american / caucasian ( n = 1 ; 5% ) . therapists included four doctoral students in clinical or educational psychology and one postdoctoral fellow in psychology . therapists received at least 8 h of initial training on the intervention , read the treatment manual and attended weekly hour - long supervision with the treatment developers . therapists worked with families for 16 weekly sessions , each lasting 90 min ( about 30 min with the child and 60 min with the parents / family ) , implementing a version of the building confidence cbt program ( wood and mcleod 2008 ) modified by the study authors for use with children with asd . as with other cbt programs for child anxiety disorders , the manual includes coping skills training followed by in vivo exposure . a hierarchy is created in which feared situations are ordered from least to most distressing . children work their way up the hierarchy and are rewarded as they attempt increasingly fearful activities . in asd - specific treatment modules , children and parents are taught friendship skills ( e.g. , giving compliments , acting like a good sport , hosting peer get - togethers successfully , etc . ) and children are given social coaching by the therapist , parents , and available school providers on appropriate ways to enter interactions and ( later in treatment ) maintain conversations with peers . unlike traditional social skills training , social coaching is provided on - site immediately before attempting to join a social activity at school or home or in public and discussed in terms of others thoughts and feelings . these skills are practiced in session , at school , and during play dates and are reinforced with a comprehensive reward system that relies on both daily privileges and longer - term incentives . to address the social isolation that many children with asd experience at school , peer buddy and mentoring programs are set up during the school meeting . two meetings are scheduled at the child s school to teach the social intervention techniques to relevant school providers ( e.g. , aides , teachers ) . all skill development and practice efforts are supported by guided conversations in which the therapist uses socratic questioning to promote conceptual development and particularly , perspective taking ( e.g. , immediately before entering a playground interaction : if you offered her a turn , can you think of a nice thought she would have about you ? children s circumscribed interests and stereotypies are incorporated into the intervention in two ways . to enhance rapport , therapeutic concepts ( e.g. , emotion recognition , cognitive restructuring ) are taught using children s special interests as examples ( e.g. , for a child primarily interested in a particular cartoon character , the character s feelings and thoughts in socially awkward situations could serve as the basis of discussion ) and as rewards ( e.g. , granting access to the preferred stimulus ) . later in treatment , after rapport has been established ( as evidenced by the therapist s and supervisor s observation of at least three consecutive sessions of high child involvement during therapy ) , a suppression approach is introduced , in which increasing amounts of time per day are devoted to consciously refraining from discussing or engaging in activities related to the circumscribed interest or engaging in stereotypies such as flapping . to help children understand the rationale for suppression , information about social expectations and acceptance is provided during these modules ( e.g. , that these behaviors are fine in private but tend to confuse peers and get in the way of friendship ) . asd diagnoses were assigned with an algorithm using the autism diagnosis interview - revised ( adi - r ; le couteur et al . 2002)module 3 , a checklist regarding circumscribed interests , and a review of previous assessment records ( also see wood et al . the adi - r and ados were administered by doctoral students and doctoral - level psychologists who received standardized training and assessment certification . the srs ( constantino and gruber 2005 ) is a standardized , 65-item , 4-point likert style parent - report form assessing children s autism - specific characteristics such as social communication deficits and repetitive behaviors . the srs provides a total score and five subscales , and has demonstrated robust reliability and validity . the anxiety disorders interview schedule : child and parent versions ( adis - c / p ) ( silverman and albano 1996 ) is a semi - structured diagnostic interview with favorable psychometric properties that generates diagnoses as well as clinician s rating scale ( crs ) scores ranging from 0 to 8 ( with higher scores representing more severe anxiety ) . parents gave written informed consent and children gave written assent to participate in the study . children who met all inclusion / exclusion criteria were block randomized by a research assistant to either immediate treatment ( it ) or a 3-month waitlist ( wl ) using a computer randomization program ( the randomization sequence was concealed from investigators until interventions were assigned ) . posttreatment assessments were completed on the final day of treatment or within 1 week of termination ; postwaitlist assessments were conducted 3 months after the baseline assessment but before initiating cbt . families in it also participated in a follow - up assessment 3 months after termination of treatment . families received $ 20 for participating in the assessments . the sample included 19 children , ranging in age from 711 years ( m = 9.37 , sd = 1.42 ) , and their primary parent(s ) ( defined as parent(s ) who were responsible for overseeing the child s daily activities ) living in a major metropolitan area of the western united states ( see wood et al . the 19 children in this sample include the final 11 participants from the wood et al . ( 2009 ) trial as well as eight additional children subsequently randomized into the same treatment protocol : ( a ) beginning with the 29th child enrolled in the wood et al . ( 2009 ) trial , the social responsiveness scale ( srs ; constantino and gruber 2005 ) was added as an outcome measure . once the 40th child completed treatment , anxiety disorder outcomes were evaluated as planned and the paper ( wood et al . ( 2009 ) paper was submitted , we randomized eight additional children into the same study protocol and collected srs data from their parents . ( c ) hence , the final 19 of the 48 total participants who had been randomized to condition had srs data . the cbt intervention was identical for all 48 participants and there were no extra treatment components administered to children whose parents completed the srs . children were referred by a medical center - based autism clinic , regional centers , parent support groups , and school personnel such as inclusion specialists . children met research criteria for asd ( see below ) and at least one anxiety disorder . psychiatric medication , if used , was at a stable dose prior to intake and throughout the duration of the trial . children with verbal iqs less than 70 or who were in concurrent psychotherapy were excluded . most children were boys ( n = 16 ; 84% ) and most primary parents had a college degree ( n = 15 ; 79% ) . children s race / ethnicity varied : caucasian ( n = 10 ; 53% ) ; asian ( n = 4 ; 21% ) ; native american / caucasian ( n = 2 ; 11% ) ; latino ( n = 1 ; 5% ) ; latino / asian ( n = 1 ; 5% ) ; african american / caucasian ( n = 1 ; 5% ) . therapists included four doctoral students in clinical or educational psychology and one postdoctoral fellow in psychology . therapists received at least 8 h of initial training on the intervention , read the treatment manual and attended weekly hour - long supervision with the treatment developers . therapists worked with families for 16 weekly sessions , each lasting 90 min ( about 30 min with the child and 60 min with the parents / family ) , implementing a version of the building confidence cbt program ( wood and mcleod 2008 ) modified by the study authors for use with children with asd . as with other cbt programs for child anxiety disorders , the manual includes coping skills training followed by in vivo exposure . a hierarchy is created in which feared situations are ordered from least to most distressing . children work their way up the hierarchy and are rewarded as they attempt increasingly fearful activities . in asd - specific treatment modules , children and parents are taught friendship skills ( e.g. , giving compliments , acting like a good sport , hosting peer get - togethers successfully , etc . ) and children are given social coaching by the therapist , parents , and available school providers on appropriate ways to enter interactions and ( later in treatment ) maintain conversations with peers . unlike traditional social skills training , social coaching is provided on - site immediately before attempting to join a social activity at school or home or in public and discussed in terms of others thoughts and feelings . these skills are practiced in session , at school , and during play dates and are reinforced with a comprehensive reward system that relies on both daily privileges and longer - term incentives . to address the social isolation that many children with asd experience at school , peer buddy and mentoring programs are set up during the school meeting . two meetings are scheduled at the child s school to teach the social intervention techniques to relevant school providers ( e.g. , aides , teachers ) . all skill development and practice efforts are supported by guided conversations in which the therapist uses socratic questioning to promote conceptual development and particularly , perspective taking ( e.g. , immediately before entering a playground interaction : if you offered her a turn , can you think of a nice thought she would have about you ? children s circumscribed interests and stereotypies are incorporated into the intervention in two ways . to enhance rapport , therapeutic concepts ( e.g. , emotion recognition , cognitive restructuring ) are taught using children s special interests as examples ( e.g. , for a child primarily interested in a particular cartoon character , the character s feelings and thoughts in socially awkward situations could serve as the basis of discussion ) and as rewards ( e.g. , granting access to the preferred stimulus ) . later in treatment , after rapport has been established ( as evidenced by the therapist s and supervisor s observation of at least three consecutive sessions of high child involvement during therapy ) , a suppression approach is introduced , in which increasing amounts of time per day are devoted to consciously refraining from discussing or engaging in activities related to the circumscribed interest or engaging in stereotypies such as flapping . to help children understand the rationale for suppression , information about social expectations and acceptance is provided during these modules ( e.g. , that these behaviors are fine in private but tend to confuse peers and get in the way of friendship ) . asd diagnoses were assigned with an algorithm using the autism diagnosis interview - revised ( adi - r ; le couteur et al . 2002)module 3 , a checklist regarding circumscribed interests , and a review of previous assessment records ( also see wood et al . the adi - r and ados were administered by doctoral students and doctoral - level psychologists who received standardized training and assessment certification . the srs ( constantino and gruber 2005 ) is a standardized , 65-item , 4-point likert style parent - report form assessing children s autism - specific characteristics such as social communication deficits and repetitive behaviors . the srs provides a total score and five subscales , and has demonstrated robust reliability and validity . the anxiety disorders interview schedule : child and parent versions ( adis - c / p ) ( silverman and albano 1996 ) is a semi - structured diagnostic interview with favorable psychometric properties that generates diagnoses as well as clinician s rating scale ( crs ) scores ranging from 0 to 8 ( with higher scores representing more severe anxiety ) . parents gave written informed consent and children gave written assent to participate in the study . children who met all inclusion / exclusion criteria were block randomized by a research assistant to either immediate treatment ( it ) or a 3-month waitlist ( wl ) using a computer randomization program ( the randomization sequence was concealed from investigators until interventions were assigned ) . posttreatment assessments were completed on the final day of treatment or within 1 week of termination ; postwaitlist assessments were conducted 3 months after the baseline assessment but before initiating cbt . families in it also participated in a follow - up assessment 3 months after termination of treatment . for the participating sample ( n = 19 ) , recruitment began in 3/2006 and ended in 8/2007 ; posttreatment assessments were completed by 12/2007 . there were no statistically significant pretreatment group differences on the demographic variables or srs scores . evidence of intervention adherence is excellent ( 94% of core elements of each session detailed in the treatment manual , as rated by a blind coder listening to audiotapes of sessions , were implemented per protocol ) and is described in detail elsewhere ( wood et al . ancova was used to test group differences at posttreatment / postwaitlist on srs scores , with the children s baseline srs scores included as a covariate . there was a statistically significant group difference , f ( 1 , 16 ) = 5.39 , p < .05 . posttreatment / postwaitlist total srs scores were lower in the it group than in the wl group ( d = .77 , approaching a large effect ; cohen 1988 ) ( see table 1 ) . in additional ancovas , posttreatment / postwaitlist group differences favoring cbt were found on three of five srs subscales : social communication , social motivation , and social awareness ( ps < .05 ) ; a trend favoring cbt was also found for social cognition ( p = .10 ) . no group difference was found on the autistic mannerisms subscale.table 1total social responsiveness scale scores for the it and wl groupsbaselineposttreatment / postwaitlistitwlitwlm113116.4089110.30sd18.2730.1926.3929.22range87145611515314053146for it , n = 9 ; for wl , n = 10 . raw scores are reported total social responsiveness scale scores for the it and wl groups for it , n = 9 ; for wl , n = 10 . raw scores are reported follow - up data was collected on four of the nine children in the it group ( the remaining five children were lost to follow - up ) . all four children had lower srs total scores at follow - up than they had at posttreatment , suggesting treatment gains had been maintained or augmented . a t - score of 70 or greater is considered a clinically significant level of autism symptoms . at baseline all but one child in the sample had a total srs t - score above 70 ( the exception was in wl , t = 63 ) . of the nine wl children who began the trial with a t - score in the clinical range , all remained at or above a t - score of 70 at postwaitlist . at immediate posttreatment , two of nine children in it had t - scores below 70 . at 3-month follow - up , three of four it children had t - scores below 70 ( as noted above , the remaining five it children were lost to follow - up ) . hence , using a last - observation - carried - forward ( locf ) approach , five of nine it children ( 56% ) were below a t - score of 70 at their last available assessment ( i.e. , posttreatment or follow - up ) . two ols linear regression models were fitted to evaluate the correspondence of anxiety reduction and autism symptom improvement . in the first model , predictors included adis - c / p crs change scores and baseline srs total scores . in the second model , srs change - scores and baseline adis - c / p scores both models had significant effects ( model 1 adis - c / p change score = .53 , and model 2 srs change score = .63 , ps < .01 , respectively ) , suggesting that when anxiety decreased over time , so did autism symptoms ( and vice versa ) . these results suggest that autism symptoms such as social communication deficits may be positively influenced by cbt . if this finding proves to be robust in larger - scale trials , it would represent a major advance in the treatment of core autism symptoms in high - functioning school - aged children . a key finding was that parent - reported autism symptoms were lower in the cbt group than the waitlist group at the post assessment , with a medium to large effect size . although reliance on parent report is in some ways a limitation , parent report does have the potential advantage of capitalizing on a parent s extensive observations of child behavior at home and in the community . generalization and maintenance of treatment effects in these settings has generally not been attained in social skills training ( rao et al . 2008 ) . furthermore , few if any clinical trials of non - pharmacological behavioral treatments for school - aged children with asd have used diagnostic measures of autism symptoms as an outcome measure ; social skill training studies have tended to use diagnostically nonspecific measures of social skill mastery , which may not be capable of documenting improvement in autism - specific social communication deficits . a second major finding in this study was that improvement in anxiety was associated with improvement in autism symptoms and vice versa , suggesting that for children with comorbid asd and anxiety disorders , the two symptom domains could be interconnected . the potential role of anxiety in exacerbating asd symptoms for some children has been documented in various descriptive studies ( e.g. , sukhodolsky et al . 2008 ) . hypothetically , after experiencing a reduction of the aversive affective experience of anxiety , children with asd may feel less need to engage in maladaptive coping behaviors such as social avoidance and repetitive behaviors to regulate their mood state , permitting interventions in the core autism symptom domain ( e.g. , social reciprocity ) to be more effective . this study was limited by a small sample and reliance on parent reports of symptomatology , which are vulnerable to bias . future research could expand upon these initial findings by using a larger sample of participants and by including objective measures of outcome based on independent evaluators ratings and direct observations of children s behavior . given the limited number of empirical studies on effective intervention modalities for core autism symptoms in this population , further investigation of cbt for the treatment of core autism symptoms appears indicated . this study was strengthened by its use of methods that are consistent with contemporary guidelines for clinical trials research ( chambless and hollon 1998 ) . if the present findings are representative of the effects of cbt on autism symptoms in high - functioning school - aged children , this intervention modality could represent an important step towards developing more efficacious practices for the treatment of asd .

background and aims . minimal hepatic encephalopathy ( mhe ) is diagnosed using neuropsychometric tests or neurophysiological tests that are either inapplicable to illiterate patient population in resource - poor settings or require sophisticated and expensive equipment . the available tests assess discrete domains of mental impairment . our aim was ( a ) to design a neuropsychometric test that measures all domains of mental impairment in mhe using one metric ; ( b ) to evaluate its sensitivity , specificity , and reproducibility . methods . the mind and liver test ( malt ) , a psychometric test assessing cognition , memory , and psychometric impairment , each on a scale of 20 , was designed keeping in mind the requirements of a universal test . 40 cirrhotics and 36 controls were subjected to critical flicker frequency ( cff ) and malt in same sitting . roc curve was plotted for malt using cff as gold standard . bland - altman plot was used to find test - retest agreement . results . cff values and malt scores varied significantly between the cases and the controls ( p < 0.05 ) . malt was 94% sensitive and 83% specific . using roc with cff as gold standard , the auc for diagnosis of mhe using malt score was 0.89 . test - retest agreement was high ( icc = 0.89 ) . conclusion . in this pilot study , malt proved to be highly sensitive , specific , inexpensive , and reproducible .

it is characterized by neuropsychiatric manifestations . in minimal hepatic encephalopathy ( mhe ) , patients have alterations in sleep pattern , and there is difficulty in performing even day - to - day routine activities like driving a vehicle , doing simple calculations , and so forth [ 13 ] . initially there is a mild impairment in cognition and psychomotor skills which then progress to a gross impairment in orientation and general consciousness . these manifestations are potentially reversible , especially , if the diagnosis is made early in the course of the disease [ 4 , 5 ] . the diagnosis of minimal hepatic encephalopathy ( mhe ) is made using a battery of neuropsychometric or neurophysiological tests . the existing neuropsychometric tests include paper - pencil tests like the number connection tests a and b , the figure connection test ( fct ) , trail tracing test , block design test , digit symbol test , and the comparatively recent inhibitory control test . inaccuracies in the paper - pencil tests have been widely reported [ 7 , 8 ] . also , there is a lack of consensus on the diagnostic criteria and a limited correction for educational level and age in these studies . the fct was designed for the indian population and has been widely accepted as a sensitive test to detect mhe in illiterate individuals . however , this test assesses only a few discrete domains of impairment ( attention and psychomotor skills ) . much of the tests hitherto mentioned are seldom used in day - to - day clinical practice . more recent objective neurophysiological tests include critical flicker frequency ( cff ) and , auditory and visual evoked potentials . all these tests involve expensive specialized equipment and trained experienced personnel [ 7 , 11 ] . there is therefore a need to design an inexpensive test which would measure all the impaired domains of the mind using the same metric . we designed a test that would examine the three important domains cognition , memory , and psychomotor skills and used this test in patients with chronic liver disease to detect mhe . the impaired domains of mental function in mhe have an impact on the quality of daily life . the mind and liver test ( malt ) was conceptualized on a scientific basis and was designed to measure the daily mental needs of an individual as a whole , and not for just one specific domain . malt was designed such that it served as a single metric measure for all impaired domains like cognition , memory , and psychometric skills . in order to design the specific tasks which make up the malt , a questionnaire investigating the specific daily mental demands encountered by the target population was administered to 100 individuals attending the out - patient liver clinic of institute of gastroenterology and hepatology , stanley hospital , chennai , india . except for 5 patients who were either professionals or businessmen , the rest belonged to lower socioeconomic status and were laborers , unskilled workers , or farmers ( kuppuswamy 's socioeconomic status scale ) . based upon the results of the questionnaire , the questions were designed to identify certain animals , ability to follow the sequence of a natural phenomenon ( e.g. , morning - noon - evening - night ) , and ability to perform simple mathematical calculations needed during daily transactions ( e.g. , transaction at the shop ) . for memory we also tested the ability to recall a visual as well as an auditory experience . the psycho - motor abilities were tested by asking the patient to copy certain figures on a calibrated graph paper , to build a three - dimensional structure of a wall using building blocks , to cut along a drawn line using a scissor , and to trace along a narrow path ( analogous to a line tracing test ) . all the three tests were designed using a colorful board , set of printed question cards , plastic building blocks , animal models , an audio device , and an instruction cum scoring sheet . for convenience , red steps tested memory , green steps tested cognition , and yellow steps tested psycho - motor skills . these tasks were grouped into three different sections : section 1 : figure the field , section 2 : home sweet home , section 3 : road not taken . section 1 : figure the field , section 2 : home sweet home , section 3 : road not taken . section 1 included steps like identification of animals and vegetables on the countryside / neighborhood , and identification of well - known sequences of events like sowing seeds , transplanting the saplings , harvesting , and finally transporting to the city . in this section the failure to perform this task was considered a sign of psycho - motor impairment . section 2 included steps like remembering the arrangement of things on the shelves at home and ability to narrate the steps of a task performed at home daily , such as washing a cloth or making tea . impairment in memory and organization reflected directly on the performance in these steps . in the same section we tested the ability of the patient to identify and remember sounds by using different stimuli , such as a church bell or an ambulance siren . examinees were also asked to cut paper using a pair of scissors , a measure of psychomotor abilities . section 3 included tasks that tested the numerical abilities of the patient ( like we asked the patient to perform small transactions at the shop ) and tasks that asked them to identify places that they may visit on a daily basis , like a temple , a water pump , or a market place . short term memory was tested by asking the patient to recall the short list of things to buy from the shop that was narrated to him in section 2 . we included a test analogous to the trail tracing test in section 3 where the patient had to negotiate a narrow path from the shop back to home , using his index finger along the trail . two other tests were performed on the malt board , which could affect the daily activities in mhe . these included ( a ) divided attention : ability to observe images on the board that were not included as a part of the task ; ( b ) judgment ability : to select the shortest of the three shown paths . table 1 shows the steps included in the three sections of the test that appear on the malt board ( figure 1 ) and the corresponding mental function that each step evaluates . based upon the performance of initial 10 controls in the test , we fixed an upper limit of 1 minute for each of steps 3 , 5 , and 6 . the maximum scores on the entire test was 60 and 20 each on cognition , memory , and psychomotor skills . informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki as reflected in a priori approval by the ethics committee of stanley medical college and hospital . forty proven cirrhotic patients were recruited as cases from among the out - patients and in - patients registered in our department . patients with neurological deficit , obvious overt encephalopathy , alcohol abuse 72 hours prior to the test , or those on sedatives or psycho - active drugs in the preceding 24 hours were excluded . patients with cardiac failure , renal disease , and chronic obstructive airway disease were also excluded . it was ensured that the patients had not received lactulose , lactitol , or antibiotics like rifaximin , neomycin , or quinolones in the past three months . all controls and patients had critical flicker frequency test using portable battery - powered hepatonorm analyzer , accelab gmbh , kusterdingen , germany . the analyzer evoked an intrafoveal light stimulus with defined pulses [ 7 , 16 ] . the patient was asked to press a hand held switch when the change was apparent . the procedure was repeated 5 times to make the subject familiar with the process and 9 readings were recorded . the mean for each patient was calculated . a value below the 38 hz mark was considered diagnostic of mhe [ 5 , 7 , 16 ] . the subjects ( cases and controls ) were then brought into a well lit room and were allowed a relaxation time of 15 minutes after which malt was administered by two trained medical professionals ( sd and sm ) . the patient and the tester are seated across a table with the test board , question cards , an audio device , and a table clock . the performance of the patient was noted on the instruction / scoring sheet , including time to completion . both tests were executed by two different persons ( sd and sm ) who were blinded to the other examiner 's results . the performers administering the tests were trained by experienced clinicians so as to minimize the intra- and interobserver variations . student 's t - test was used to compare the mean cff values and mean malt scores between the cases and the controls . preliminary analyses provided data for receiver - operating characteristic curves to determine the optimum cut - off value for malt score for the diagnosis of mhe . the best sensitivity and specificity was found at a cut - off score of 20 . using the cff performance as the reference standard , statistical analysis was performed using student 's t - test and the receiver - operating characteristic ( roc ) curve . cirrhotics with mhe and cirrhotics without mhe , based upon their cff readings . spss version 10 was used for all statistical analyses and in the generation of the plots . twenty subjects including 10 cases and 10 controls were administered malt twice at an interval of a six hours . it was ensured that patients being evaluated were not on lactulose , lactitol , neomycin , or any sedative meanwhile . the report was prepared in accordance with standards for reporting diagnostic accuracy , stard guidelines ( figure 2 ) . the study comprised of 40 patients with cirrhosis ( hereinafter referred to as cases ) and included 25 males ( 62.5% ) . there were 36 healthy controls ( hereinafter referred to as controls ) and included 16 males ( 44.44% ) . the mean age of the cases and controls was significantly different ( p = 0.04 ) . there was no difference in sex distribution ( p = 0.18 ) , educational status ( p = 0.93 ) , and monthly income ( p = 0.09 ) among the cases and the controls . the etiology of cirrhosis was alcohol in 15 patients ( 37.5% ) , hcv in 13 ( 32.5% ) , nonalcoholic fatty liver disease in 6 ( 17.5% ) , autoimmune in 1 ( 2.5% ) , and cryptogenic in 8 ( 20% ) . there were 22 patients ( 55% ) belonging to child - pugh class a and 18 ( 45% ) to class b. among the cirrhotics , there were 18 variceal bleeders ( 45% ) and 2 patients ( 5% ) had coagulopathy . 4 patients ( 10% ) had refractory ascites , 2 patients ( 5% ) had spontaneous bacterial peritonitis , and 8 others ( 20% ) had urosepsis . using 38 hz as cff cut - off for mhe , 16 out of the 40 cirrhotics had mhe ( 40% ) , 8 each ( 50% ) belonged to child class a and child class b. among the 24 cirrhotics without mhe , 14 ( 58.33% ) belonged to child a and 10 ( 41.66% ) to child b at the time of mhe testing . the mean cff for the cases was 39 4.2 hz compared to 42 1.9 hz among the controls ( p = 0.00 ) ( figure 3(a ) ) . the demographic characteristics and the lab parameters of the mhe and non - mhe groups among the cirrhotics or the cases are compared in table 4 . the controls scored 10 5 compared to the cases , who scored 18 8 ( figure 3(b ) ) . on plotting the roc curve ( figure 4(a ) ) , the sensitivity and the specificity were found to be 94% and 83% , respectively , for a cut - off value set at 20 . using roc with cff as the gold standard , the auc for diagnosis of mhe using malt score was 89% . figure 3(b ) compares the mean malt scores of mhe , non - mhe cirrhotics , and controls . mhe patients scored higher than the non - mhe and controls using the 20 mark as cut - off . the test - retest variability was assessed using the intraclass correlation coefficient , icc = 0.894 ( p = 0.000 ) . the bland - altman plot ( figure 4(b ) ) shows that the malt retest scores of the 10 cases and 10 controls ( who were subjected to the same test at an interval of 6 hours ) was in agreement with the initial test scores . the hepatonorm analyzer , manufactured in germany , is an electronic device which runs on a battery that needs to be regularly changed . the mean time of administration for both malt and cff was 10 2 minutes . the highlights of the study are ( a ) a significant correlation between cff and malt scores in the population under study ( p < 0.05 ) ; ( b ) the roc curve and the box - whisker plots indicate that malt is a highly sensitive and specific test considering cff as the standard ; and ( c ) the intraclass correlation coefficient was 0.894 on assessment of test - retest variability . the sensitivity and specificity of malt was found to be 94% and 83% , respectively , compared to earlier reported 72.4% and 77.2% for cff when the cut - off is set at 38 hz . though malt may be seen to be a time taking procedure , the mean time taken for malt and cff did not vary significantly . a number of neuropsychological tools have been used to diagnose cognitive deficits in patients with cirrhosis . the extended neuropsychological assessment is based upon the expert judgment and is difficult to validate . the two shorter batteries rbans ( repeatable battery for assessment of neuropsychological status ) and phes ( psychometric hepatic encephalopathy score ) are primarily paper - pencil tests . the performance on these tests is affected by the level of literacy and on the familiarity of the subject to paper and pencil among the literates . however , these tests have their own advantages . rbans is validated on a large normative data , it is available in multiple languages and has parallel versions available for repeated testing . however , most studies are in alzheimer 's patients and the studies in cirrhotics are only few . phes was originally validated in a series of nonalcoholic patients in germany and has normative data available from some countries . however , our knowledge of mhe has grown since those studies and , now , it is understood that an ideal diagnostic tool should reflect on those daily activities which affect the quality of life , the ability to drive , and so forth , which are often impaired in mhe [ 21 , 22 ] . to overcome the shortcoming of these short batteries for the use in resource - limited settings including their dependence on the literacy level , the figure connection test ( fct ) was reported by a group in india . however , fct only tests a discrete cognitive domain impaired in mhe . computerized neuropsychological tests include cognitive drug research ( cdr ) , inhibitory control test ( ict ) , and the scan test which have been validated in the uk , usa , and italy , respectively [ 2325 ] . the introduction of malt has been based on the fundamental assumption that the currently available tests for the diagnosis of mhe are designed to test for one or two domains of mental impairment , and are expensive or not widely available . malt was designed to include tasks performed on a day - to - day routine basis . in the population under study , more than 36% of the participants were illiterate and the remaining who were literate were not used to paper and pencil . this is in line with the demise of the pencil reported by iduru and mullen . moreover , age and education correction factors are absent for psychometric hepatic encephalopathy score ( phes ) in our population . it is in agreement with the prevalence of 41% reported in a recent study involving 200 cirrhotics from north india while on the higher side compared to 30% reported in the study from spain using phes [ 18 , 26 ] . it would either signify that malt included more true positives or that the prevalence is different in our population [ 27 , 28 ] . malt is a psychometric test which displays day - to - day activity on a colorful board and disagreement between administrators of the test is significantly less ( 85% agreement in interobserver studies ) . though the tasks on the game board remain the same , the elements of the tasks are not printed on the board but on multiple cards . malt includes steps like build the wall and trace the path which are analogous to block design and trail tracing test . in malt , the subject is not asked to connect numbers , but rather to trace the sequence of a day - to - day event from morning to night , which in true sense does not need a level of literacy . although the majority of surveyed hepatologists in spain and the united states agreed that mhe was a significant problem requiring testing during out - patient visits , only a few were able to actually perform the test during their day - to - day clinical practice [ 29 , 30 ] . when the test is available in the form of a touch screen audio - visual videogame , the test could be self - administered by the patient during the wait period in the clinic . . it might be interesting to note if administration of such a test has any effect on the mood and affect of the depressed cirrhotic . it will also be of interest to follow up malt scores of patients on therapy like lactulose for hepatic encephalopathy . also , to see a head to head analysis of malt compared to other psychometric tests in future . however , these were beyond the scope of the present study . the choice of cff as the gold standard in the current study was also to circumvent many problems known to be encountered with administering multiple psychometric tests [ 2 , 31 ] . this study largely aimed to design a psychometric battery to diagnose mhe in our population , which is useful for the illiterate patients and to check its reliability . the high sensitivity and specificity of malt , its ease of administration , reproducibility , and cost - effectiveness seen in this pilot beckon further validation studies involving larger sample sizes .

a 47-year - old turkish male was scheduled for laparoscopic cholecystectomy under general anesthesia . the patient had 2 operations 28 and 19 years ago under general anesthesia . it was learned that the patient was administered succinylcholine during both of these previous operations and that he did not have a history of prolonged recovery or postoperative apnea . the patient had been using sertraline for 3 years before the operation . pseudocholinesterase is a drug - metabolizing enzyme responsible for hydrolysis of the muscle - relaxant drugs mivacurium and succinylcholine . deficiency of this enzyme from any cause can lead to prolonged apnea and paralysis following administration of mivacurium and succinylcholine . the diagnosis of pseudocholinesterase enzyme deficiency can be made after careful clinic supervision and peripheral nerve stimulator monitoring . a decrease in the activity of pseudocholinesterase enzyme and a decline in the block effect over time will help verify the diagnosis . our patient s plasma cholinesterase was found to have low activity . instead of pharmacological interventions that may further complicate the situation in such cases , the preferred course of action should be to wait until the block effect declines with the help of sedation and mechanical ventilation . in our case , the prolonged block deteriorated in the course of time before any complications developed .

pseudocholinesterase ( pche ) is an enzyme with a complex molecular structure.1 it is synthesized in the liver and immediately released into the plasma.2 the plasma half - life has been estimated to be approximately 12 days.3 deficiency or reduced activity of this enzyme results in significant prolongation of mivacurium- or succinylcholine - induced neuromuscular blockade.4 in addition , a number of disease states or concomitant drug administration may reduce pche activity . mivacurium , which is a nondepolarizing neuromuscular blocking drug administered in doses of 0.1 to 0.2 mg / kg , also produces rapid onset of neuromuscular blockade lasting for 15 to 30 minutes.5 the rapid ester hydrolysis of mivacurium by pche results in the short duration of action of this drug , which is ideal for providing muscle relaxation for brief surgical procedures.6 but the duration of mivacurium of action in adults is inversely related to serum pche activity.7,8 sertraline is an antidepressant and antipanic agent that is a potent and selective inhibitor of serotonin reuptake into the presynaptic terminals . it has relatively weak effects on the cytochrome p450 system , a tolerability profile that is positive compared with antidepressants of other classes , and it is relatively safe in overdose . these findings contribute to sertraline being the first - line agent for the treatment of several psychiatric disorders.9 it was shown , however , that sertraline and other antidepressants inhibited acetylthiocholine hydrolysis by cholinesterases in human serum and erythrocyte membrane.10 in this article , we share our experience of a case with acquired pseudocholinesterase enzyme deficiency due to sertraline use , and provide a literature review . a 47-year - old turkish male , 83 kg in weight and 175 cm in height , was scheduled for laparoscopic cholecystectomy ( due to cholecystitis with an acute stone ) under general anesthesia . under general anesthesia , the case had an appendectomy 28 years ago and a right inguinal hernia operation 19 years ago . it was learned that the patient was administered succinylcholine during both these operations and that he did not have a history of prolonged recovery or postoperative apnea . except for alkaline phosphatase ( alp ) ( 176 u / l ) , alanine aminotransferase ( alt ) ( 52 u / l ) and a minimal increase in aspartate aminotransferase ( ast ) ( 61 u / l ) , and a slight leucocytosis ( 9.8 10/l ) , no other pathologies were identified during preoperative laboratory investigation . the patient had been using sertraline ( 100 mg / day ) for 3 years for major depressive disorder . after surgery , the inhalation agent was discontinued , the patient received 100% oxygen but there was no clinical evidence of neuromuscular function recovery and there was no response to train - of - four stimulation . anesthesia was therefore maintained with 0.5% isoflurane in 50% nitrous oxide and oxygen because there was no evidence of improved neuromuscular function 50 minutes after administration of mivacurium . five milligrams of midazolam was administered intravenously for its sedative and amnestic effects . later the patient was transferred to the post - anesthesia care unit ( pacu ) for observation and ventilator support . train - of - four response ratio was 75% 2 hours and 55 minutes after mivacurium administration . the patient regained sufficient motor function to meet extubation requirements 3 hours and 15 minutes after the initial use of mivacurium . the patient was extubated for 205 minutes following the administration of mivacurium and further recovery was uneventful . iu / l ) . the patient was transferred to a hospital ward for the evening and was discharged 4 days later . a recommended intravenous dose of 0.15 to 0.2 mg / kg provides tracheal intubating conditions within 2 to 2.5 minutes , with a predicted duration of action of 15 to 25 minutes , making it an ideal drug for short procedures requiring tracheal intubation.11 it is a structural relative of atracurium , but it does not undergo hofmann elimination and is rapidly hydrolyzed by pche . the activity of pche affects its duration of action.4 pche is a tetrameric glycoprotein enzyme produced by the liver , which hydrolyzes choline esters such as those found in succinylcholine , mivacurium , procaine , chloroprocaine , tetracaine , cocaine , and heroine.12 in patients with a normal genotype for pche , mivacurium s duration of action is inversely related to pche activity and duration of action is slightly prolonged if activity is low.13 when there is a deficiency of this enzyme due to the presence of one or more atypical alleles , mivacurium is not properly metabolized and thus muscle paralysis can last for several hours . the heterozygous atypical form affects from 1 in 25 , to 1 in 480 individuals ( depending on the severity of the condition).12 patients heterozygous for an abnormal enzyme may show up to 50% prolongation of block.13 in patients homozygous for an abnormal enzyme , duration may be significantly prolonged , and even a small dose ( eg , 0.03 mg / kg ) can result in complete paralysis for up to 128 minutes.14 there are now several case reports in the literature of prolonged block following mivacurium administration . physiological reductions may occur with extremes of age and during pregnancy.15 other acquired causes of decreased activity include renal and liver disease , malignancy , burns , chronic debilitation / malnutrition , myocardial infarction / cardiac failure , collagen diseases , myxedema , and organophosphate poisoning.16 plasma cholinesterase levels in human subjects with protein energy malnutrition have been investigated.17 drugs that inhibit the enzyme s activity include acetylcholinesterase inhibitors ( neostigmine , pyridostigmine , physostigmine , and edrophonium ) , anticholinesterases ( especially echothlophate ) , cytotoxic agents ( such as cyclophosphamide ) , steroids , ester - type local anesthetics , hexafluorenium , pancuronium , and oral contraceptives.18 our patient previously had 2 operations under general anesthesia with succinylcholine . however , a prolonged apnea and low enzyme activity ( 788 although the patient had not had any problems during the previous operations , absence of a medical history proving the existence of the same problem in close relatives led us to think that this was an acquired pche deficiency . at this stage , we at once started to implement an algorithm which is usually used in cases of pche deficiency . at this stage however , we noted that the medical conditions ( such as extremes of age , pregnancy ) and problems ( such as renal and liver disease , malnutrition ) reported in the literature that we have tried to summarize above did not match our case . therefore , we considered the drugs known to affect pche activity that the patient may have been taking . we discovered that the patient had been using sertraline ( 100 mg / day ) for 3 years due to depression . sertraline is a serotonin reuptake inhibitor ( ssri ) that is indicated for the treatment of depression . with therapeutic doses , sertraline has a wide therapeutic index and appears to be safer than the tricyclic antidepressants in overdose.19 the usual clinical antidepressants ( fluoxetine , sertraline , and amitriptyline ) are , however , inhibitors of the cholinesterases in human serum and erythrocyte membrane . antidepressants exhibit this inhibiting effect by reversibly attaching to the enzyme s bottom of the active site gorge , and the collapse of the product ( eg , mivacurium or succinylcholine ) being hydrolyzed by the enzyme decreases during this process of attachment.10 the order of inhibitory potency was sertraline > amitriptyline > > fluoxetine . the concentrations of antidepressants necessary to inhibit serum cholinesterase are relatively close to the clinical levels found after pharmacological use of sertraline and amitriptyline . consequently , undesirable pharmacological and toxicological side effects of these antidepressants can be related , at least in part , to inhibition of serum and erythrocyte cholinesterases.10 therefore , chronic enzyme inhibition which might have developed due to 3 years of use of sertraline in our patient led to a significant and marked prolongation in the activity time of the neuromuscular blocking agent metabolized by this enzyme . the results achieved by mller et al10 corroborated our suspicions about sertraline . also , the retested ( 3 months after the operation ) pche value ( 2762 iu / l ) was seen to have returned to normal after the patient gave up using sertraline under the supervision of a psychiatrist . we did not discover any case that implied an interaction between sertraline and pseudocholinesterase in our literature review , and we believe this is the first presentation of such a case . when we understood that we were dealing with a case of acquired pche deficiency , we made our treatment plan . our main aim was to administer a supportive treatment to the patient until enzyme activity returned to normal . therefore we did not consider a blood transfusion ( considering the risks of blood transfusion , such as allergic reactions , infection , fever reaction ) . we also avoided administering drugs ( such as calcium , neostigmine ) that might complicate the present condition . the clinician should be especially aware that administering neostigmine to a patient with prolonged mivacurium - induced block will inhibit plasma cholinesterase activity . therefore , blood should not be drawn for cholinesterase activity until the effect of neostigmine dissipates . in our case , when we suspected pche deficiency , together with pns findings , we sent the blood samples to the laboratory for enzyme analysis . consequently , we supported our case solely with sedation , mechanical ventilation , and pns monitoring , and without neostigmine application . prolonged blocks may be encountered as a result of pche enzyme deficiency when mivacurium is used . in our case , this situation was due to sertraline , which is commonly administered for various psychiatric disorders , particularly depression . in this context , we believe two issues should be highlighted . firstly , while examining the drugs used by patients during preoperative anesthesia anamnesis , next - generation antidepressants ( especially ssris ) should not be ignored . as in the case reported , this group of drugs , known to have a wide safety margin , may cause casual and unexpected block prolongation when used along with drugs ( mivacurium and succinylcholine ) that are hydrolyzed especially by pche activity during anesthesia . secondly , in these situations , attempted reversal of the block was at best only partially successful , and the patients were partially paralyzed and minimally sedated while attempts were made to have them breathe a very unpleasant experience . in cases of prolonged block the patients were kept sedated , and their lungs mechanically ventilated until the block recovered spontaneously , and then extubated without problem .

abstractintroductioncollagen crosslinking is a key parameter in extracellular matrix ( ecm ) maturation , turnover , and stiffness . we examined aspects of collagen crosslinking in dystrophindeficient murine , canine , and human skeletal muscle.methodsdmd patient biopsies and samples from mdx mice and golden retriever muscular dystrophy dog samples ( with appropriate controls ) were analyzed . collagen crosslinking was evaluated using solubility and hydroxyproline assays . expression of the crosslinking enzyme lysyl oxidase ( lox ) was determined by realtime polymerase chain reaction , immunoblotting , and immunofluorescence.resultslox protein levels are increased in dystrophic muscle from all species evaluated . dystrophic mice and dogs had significantly higher crosslinked collagen than controls , especially in the diaphragm . distribution of intramuscular lox was heterogeneous in all samples , but it increased in frequency and intensity in dystrophic muscle.conclusionthese findings implicate elevated collagen crosslinking as an important component of the disrupted ecm in dystrophic muscles , and heightened crosslinking is evident in mouse , dog , and man . muscle nerve 54 : 7178 , 2016

six and 18monthold male c57 and mdx mice were euthanized , and the tibialis anterior ( ta ) muscle and the diaphragms were dissected . they were either flash frozen in liquid nitrogen for biochemical analysis or embedded in oct and frozen in melting isopentane for histological evaluation . portions of ta and diaphragm were collected from 1yearold golden retriever muscular dystrophy ( grmd ) dogs ( n = 3 ) and an unaffected dog ( n = 1 ) following unrelated terminal endpoints and were either flash frozen or frozen in oct . samples of human quadriceps muscle ( n = 3 ) were obtained from the wellstone muscular dystrophy cooperative research center , tissue and cell repository at the university of iowa , carver college of medicine ( iowa city , iowa ) . the dmd patients were between 5 and 8 years of age , and control patients between 8 and 13 years of age at the time of biopsy . total rna was isolated from murine and canine muscles using trizol reagent ( invitrogen ) treated with dnase ( promega ) and reverse transcribed to cdna using the geneamp rna polymerase chain reaction ( pcr ) kit ( applied biosystems ) using the manufacturer 's directions . realtime pcr ( model 7300 ; applied biosystems ) was conducted on the resulting cdna with power sybr green master mix ( applied biosystems ) . expression levels of lox ( accession numbers : mouse , nm_010728 ; dog , xm_538599 ) , loxl2 ( mouse , xm_006519744 ) , and col1a2 ( mouse , nm_007743 ; dog , nm_001003187 ) were determined with 18s expression serving as the reference housekeeping gene . flashfrozen murine and canine muscle tissue was finely crushed and homogenized in tissue protein extraction reagent ( tper ; thermo scientific ) supplemented with protease and phosphatase inhibitor cocktails ( thermo scientific ) . quadriceps biopsy samples from healthy males and dmd patients ( pooled from 25 10m frozen sections from each patient ) were lysed in radioimmunoprecipitation assay ( ripa ) buffer supplemented with protease and phosphatase inhibitor cocktails ( sigmaaldrich ) . polyacrylamide gel electrophoresis ( sdspage ) on trishclpolyacrylamide gels ( biorad ) and transferred to polyvinylidene fluoride ( pvdf ) membranes . membranes were blocked with 5% milk in trisbuffered saline and tween 20 ( tbst ) and incubated in primary antibody diluted in 2% milk tbst overnight at 4 c , using antilox ( abcam ab31238 ; 1:500 ) for human and canine samples and antilox ( novus nbp224877 ; 1:500 ) for mouse samples . the membranes were washed , incubated in antirabbit horseradish peroxidase ( hrp)conjugated secondary antibody ( cell signaling technologies ) , and visualized using ecl plus reagents ( thermo scientific ) and autoradiography film ( bioexpress ) . to provide loading controls , membranes were stripped and either reprobed for glyceraldehyde 3phosphate dehydrogenase ( gapdh ; sc25778 ; 1:2,000 ; santa cruz biotechnology ) or stained with coomassie blue ( biorad ) or ponceau s ( sigmaaldrich ) solution for visualization . a collagen solubility assay was adapted22 , 23 to quantify the proportion of mature crosslinked collagen from non crosslinked and immature crosslinked collagen . briefly , flashfrozen muscle tissue was ground using a mortar and pestle over dry ice and then weighed . the tissue was washed in 1 ml of pbs and stirred for 30 minutes at 4 c before centrifuging at 16,000 g for 30 minutes at 4 c . crosslinked collagen was digested in a 1:6 ( weight : volume ) solution of 0.5 m acetic acid with 1 mg / ml pepsin , stirring overnight at 4 c . after centrifuging at 16,000 g for 30 minutes at 4 c , the supernatant was collected as the pepsinsoluble fraction ( psf ) , and the pellet was kept as the pepsininsoluble fraction ( pif ) . for the psf , a 1:1 volume of 4 m nacl was added and stirred for 30 minutes at 4 c . the psf was then centrifuged at 16,000 g for 30 minutes at 4 c , and the supernatant was discarded . the collagen contents of the psf and pif were then determined by a hydroxyproline assay . a hydroxyproline assay was performed to quantify collagen content in both the psf and pif , as described in previous studies.12 , 24 the samples were hydrolyzed overnight in 0.5 ml of 6 m hydrochloric acid at 105 c . 10 l of hydrolysate were mixed with 150 l isopropanol followed by 75 l of 1.4% chloraminet ( sigma , st . louis , missouri ) in citrate buffer and oxidized at room temperature for 10 minutes . the samples were then mixed with 1 ml of a 3:13 solution of ehrlich reagent [ 1.5 g of 4(dimethylamino)benzaldehyde ( sigma ) , 5 ml ethanol , and 337 l sulfuric acid ] to isopropanol and incubated for 45 minutes at 55 c . a standard curve ( 01,000 m , trans4hydroxylproline ; sigma ) was included in each assay . frozen octembedded grmd samples were sectioned at 10 m , and picosirius red staining was performed as previously described.12 briefly , sections were fixed in 4% paraformaldehyde for 10 minutes , rinsed , air dried , stained for 1 hour in 0.1% ( wt / vol ) sirius red ( sigmaaldrich ) , dissolved in saturated aqueous picric acid ( sigmaaldrich ) , washed in 2 changes of 0.5% acetic acid , dehydrated in 3 changes of 100% ethanol , cleared with citra solv , and mounted with cytoseal . the resulting slides were viewed under circularly polarized light scope ( dmlp ; leica ) , rotating polarizer , rotating analyzer , and dual quarter wave plates on a camera system ( micropublisher 5.0 ; q imaging ) . acetonefixed 10m sections of mouse , dog , and patient muscle were incubated overnight at 4 c with antilox ( abcam ab31238 ; 1:500 ) and antivimentin ( novus mb300 ; 1:800 ) primary antibodies using 5% bovine serum albumin ( bsa)pbs with 0.1% triton x100 as a blocking solution / diluent . alexa 568conjugated antirabbit igg ( molecular probes ; 1:500 ) and alexa 488conjugated antichicken igy ( jackson ; 1:500 ) secondary antibodies were used . slides were mounted using vectashield with 4,6diamidino2phenylindole ( dapi ; vector labs ) and imaged using the confocal microscope ( model tscsp8 ; leica ) . quantification of human lox immunoreactivity was achieved by analysis of 5 independent fields of view ( 20 ) from control ( n = 3 ) and dmd patient ( n = 3 ) quadriceps sections using smash muscle image analysis software.25 all results are presented as mean sem unless otherwise stated . statistical analysis of the mouse data was performed by 2way analysis of variance using genotype and muscle followed by a bonferroni post hoc test comparing genotypes within muscles and muscles within a genotype . for the human samples , a student ttest was used to compare muscles from unaffected to dmd subjects . for the dog samples there was only a single control dog , so only a paired student ttest was performed between muscles of grmd dogs . six and 18monthold male c57 and mdx mice were euthanized , and the tibialis anterior ( ta ) muscle and the diaphragms were dissected . they were either flash frozen in liquid nitrogen for biochemical analysis or embedded in oct and frozen in melting isopentane for histological evaluation . portions of ta and diaphragm were collected from 1yearold golden retriever muscular dystrophy ( grmd ) dogs ( n = 3 ) and an unaffected dog ( n = 1 ) following unrelated terminal endpoints and were either flash frozen or frozen in oct . samples of human quadriceps muscle ( n = 3 ) were obtained from the wellstone muscular dystrophy cooperative research center , tissue and cell repository at the university of iowa , carver college of medicine ( iowa city , iowa ) . the dmd patients were between 5 and 8 years of age , and control patients between 8 and 13 years of age at the time of biopsy . total rna was isolated from murine and canine muscles using trizol reagent ( invitrogen ) treated with dnase ( promega ) and reverse transcribed to cdna using the geneamp rna polymerase chain reaction ( pcr ) kit ( applied biosystems ) using the manufacturer 's directions . realtime pcr ( model 7300 ; applied biosystems ) was conducted on the resulting cdna with power sybr green master mix ( applied biosystems ) . expression levels of lox ( accession numbers : mouse , nm_010728 ; dog , xm_538599 ) , loxl2 ( mouse , xm_006519744 ) , and col1a2 ( mouse , nm_007743 ; dog , nm_001003187 ) were determined with 18s expression serving as the reference housekeeping gene . flashfrozen murine and canine muscle tissue was finely crushed and homogenized in tissue protein extraction reagent ( tper ; thermo scientific ) supplemented with protease and phosphatase inhibitor cocktails ( thermo scientific ) . quadriceps biopsy samples from healthy males and dmd patients ( pooled from 25 10m frozen sections from each patient ) were lysed in radioimmunoprecipitation assay ( ripa ) buffer supplemented with protease and phosphatase inhibitor cocktails ( sigmaaldrich ) . polyacrylamide gel electrophoresis ( sdspage ) on trishclpolyacrylamide gels ( biorad ) and transferred to polyvinylidene fluoride ( pvdf ) membranes . membranes were blocked with 5% milk in trisbuffered saline and tween 20 ( tbst ) and incubated in primary antibody diluted in 2% milk tbst overnight at 4 c , using antilox ( abcam ab31238 ; 1:500 ) for human and canine samples and antilox ( novus nbp224877 ; 1:500 ) for mouse samples . the membranes were washed , incubated in antirabbit horseradish peroxidase ( hrp)conjugated secondary antibody ( cell signaling technologies ) , and visualized using ecl plus reagents ( thermo scientific ) and autoradiography film ( bioexpress ) . to provide loading controls , membranes were stripped and either reprobed for glyceraldehyde 3phosphate dehydrogenase ( gapdh ; sc25778 ; 1:2,000 ; santa cruz biotechnology ) or stained with coomassie blue ( biorad ) or ponceau s ( sigmaaldrich ) solution for visualization . a collagen solubility assay was adapted22 , 23 to quantify the proportion of mature crosslinked collagen from non crosslinked and immature crosslinked collagen . briefly , flashfrozen muscle tissue was ground using a mortar and pestle over dry ice and then weighed . the tissue was washed in 1 ml of pbs and stirred for 30 minutes at 4 c before centrifuging at 16,000 g for 30 minutes at 4 c . crosslinked collagen was digested in a 1:6 ( weight : volume ) solution of 0.5 m acetic acid with 1 mg / ml pepsin , stirring overnight at 4 c . after centrifuging at 16,000 g for 30 minutes at 4 c , the supernatant was collected as the pepsinsoluble fraction ( psf ) , and the pellet was kept as the pepsininsoluble fraction ( pif ) . for the psf , a 1:1 volume of 4 m nacl was added and stirred for 30 minutes at 4 c . the psf was then centrifuged at 16,000 g for 30 minutes at 4 c , and the supernatant was discarded . the collagen contents of the psf and pif were then determined by a hydroxyproline assay . a hydroxyproline assay was performed to quantify collagen content in both the psf and pif , as described in previous studies.12 , 24 the samples were hydrolyzed overnight in 0.5 ml of 6 m hydrochloric acid at 105 c . 10 l of hydrolysate were mixed with 150 l isopropanol followed by 75 l of 1.4% chloraminet ( sigma , st . louis , missouri ) in citrate buffer and oxidized at room temperature for 10 minutes . the samples were then mixed with 1 ml of a 3:13 solution of ehrlich reagent [ 1.5 g of 4(dimethylamino)benzaldehyde ( sigma ) , 5 ml ethanol , and 337 l sulfuric acid ] to isopropanol and incubated for 45 minutes at 55 c . a standard curve ( 01,000 m , trans4hydroxylproline ; sigma ) was included in each assay . frozen octembedded grmd samples were sectioned at 10 m , and picosirius red staining was performed as previously described.12 briefly , sections were fixed in 4% paraformaldehyde for 10 minutes , rinsed , air dried , stained for 1 hour in 0.1% ( wt / vol ) sirius red ( sigmaaldrich ) , dissolved in saturated aqueous picric acid ( sigmaaldrich ) , washed in 2 changes of 0.5% acetic acid , dehydrated in 3 changes of 100% ethanol , cleared with citra solv , and mounted with cytoseal . the resulting slides were viewed under circularly polarized light scope ( dmlp ; leica ) , rotating polarizer , rotating analyzer , and dual quarter wave plates on a camera system ( micropublisher 5.0 ; q imaging ) . acetonefixed 10m sections of mouse , dog , and patient muscle were incubated overnight at 4 c with antilox ( abcam ab31238 ; 1:500 ) and antivimentin ( novus mb300 ; 1:800 ) primary antibodies using 5% bovine serum albumin ( bsa)pbs with 0.1% triton x100 as a blocking solution / diluent . alexa 568conjugated antirabbit igg ( molecular probes ; 1:500 ) and alexa 488conjugated antichicken igy ( jackson ; 1:500 ) secondary antibodies were used . slides were mounted using vectashield with 4,6diamidino2phenylindole ( dapi ; vector labs ) and imaged using the confocal microscope ( model tscsp8 ; leica ) . quantification of human lox immunoreactivity was achieved by analysis of 5 independent fields of view ( 20 ) from control ( n = 3 ) and dmd patient ( n = 3 ) quadriceps sections using smash muscle image analysis software.25 . statistical analysis of the mouse data was performed by 2way analysis of variance using genotype and muscle followed by a bonferroni post hoc test comparing genotypes within muscles and muscles within a genotype . for the human samples , a student ttest was used to compare muscles from unaffected to dmd subjects . for the dog samples there was only a single control dog , so only a paired student ttest was performed between muscles of grmd dogs . gene expression of the collagen crosslinking enzyme lox was evaluated at the ends of the mouse phenotypic spectrum by comparing a less affected limb muscle ( ta ) and the more severely affected diaphragm muscle of mdx mice at age 6 months to agematched c57 mice ( fig . although lox expression was not significantly different between the wildtype and dystrophic ta , it was significantly elevated in the diaphragms of mdx mice . accordingly , col1a2 expression was also highly upregulated in the mdx diaphragm ; however , expression of the lox family member , loxl2 , was unchanged across the samples . at the protein level ( fig . 1b ) , lox abundance was similarly increased in the mdx diaphragms at age 6 months compared with those of c57 mice and was also significantly higher in the dystrophic ta . ( a ) lox transcript level is increased in mdx muscle ( n = 7 ) over control ( n = 6 ) . col1a2 is increased in mdx muscles ( n = 7 ) over control ( n = 6 ) , and in mdx diaphragm over ta . loxl2 tends to be upregulated in mdx muscles ( n = 3 ) over control ( n = 3 ) . ( b ) representative portion of immunoblot for lox ( 32 kda ) with ponceau stain for loading control . lox protein is increased in mdx ( n = 3 ) over control ( n = 3 ) , with mdx diaphragm higher than ta . ( c ) soluble hydroxyproline content , representing non crosslinked or immature crosslinked collagen , of mdx ( n = 6 ) muscles are not different from c57 ( n = 6 ) . insoluble hydroxyproline content , representing crosslinked collagen , and total collagen are increased in mdx , with mdx diaphragm higher than control diaphragm or mdx ta for both insoluble and total collagen . ( d ) immunoblot of mdx diaphragm for lox shows no additional increase at 18 months ( n = 2 ) compared with 6 months . insoluble crosslinked collagen is higher at 18 months ( n = 2 ) compared with 6 months in mdx diaphragm . significantly different posthoc test , indicated by * p < 0.05 and * * p < 0.01 , between genotypes of same muscle ; p < 0.05 and p < 0.01 , between muscles of the same genotype ; and p < 0.05 , between ages of mdx diaphragm . the consequence of higher levels of lox was evaluated with a collagen solubility assay , where the soluble , insoluble , and total collagen levels were determined in mdx and control muscles ( fig . the amount of soluble collagen , representing non crosslinked and immature crosslinked collagen , was similar between all muscles analyzed and represented a minor proportion of the total collagen pool . in contrast , 6month mdx diaphragms had significantly more mature crosslinked insoluble collagen and total collagen than the mdx ta and agematched c57 diaphragms . to determine whether there was an agedependent increase in either lox or collagen , muscles from 18monthold mdx mice lox levels were similar between the 2 ages , but the mdx diaphragm continued to accrue insoluble collagen ( fig . 1d ) , whereas the ta insoluble content remained unchanged ( data not shown ) . these data demonstrate that lox is chronically elevated in the dystrophic mouse diaphragm , which results in progressive accumulation of crosslinked collagen . due to the relatively mild phenotype of the mdx mouse compared with dmd patients , we also investigated lox levels and collagen crosslinking in the more severe grmd dog model of dmd . like the mdx mouse , grmd exhibits differential disease severity between limb muscles and the diaphragm . similar to our findings in the mdx mouse , grmd diaphragm displayed high upregulation of both lox and col1a2 gene expression over control dog levels , with no change in the ta ( fig . 2a ) . lox protein , although not detectable in control dog samples by western blotting , was observed in grmd samples , where its abundance among grmd diaphragms was consistently higher than the dogmatched ta ( fig . accordingly , grmd diaphragms also contained more insoluble and total collagen than all other samples ( fig . 2c ) , in agreement with the findings in mice described above . lox expression and collagen crosslinking in the grmd dog model of dmd in limb and diaphragm muscle . ( a ) transcriptional level of lox and col1a2 ( b ) immunoblot for lox ( 32 kda ) using gapdh ( 37 kda ) as a loading control from wildtype and grmd dogs . protein level of lox is not observable in wildtype dogs , but is present in grmd muscles , with diaphragm consistently higher than dog matched ta . crosslinked collagen or immature crosslinked collagen , does not show a consistent difference between wildtype and grmd . ( d ) representative images of grmd dog ta and diaphragm muscles viewed under circularly polarized light . birefringent fibrillar collagen is visible with loosely ( green ) , moderately ( yellow ) , and densely ( red ) packed collagen . as previously performed on mdx mouse muscle,12 the collagen density of grmd muscle sections was visualized under circularly polarized light ( fig . in accordance with the biochemical data described above , the grmd ta contained mostly loosely packed collagen bundles ( visualized as green ) , with an extensive perimysial layer , whereas the diaphragm collagen bundles were largely intermediate ( yellow ) or densely ( orange to red ) packed , verifying that collagen density is increased in grmd diaphragm compared with limb muscle . it is also notable that , in the grmd diaphragm , unlike the ta , the robust endomysial expansion resulted in a lack of distinction between endomysial and perimysial stromal regions . thus , the canine model of dmd also displays heightened indices of collagen crosslinking in the diaphragm as compared with less affected limb muscle . lox crosslinking activity occurs in the ecm , but many different cell types can produce this enzyme , including fibroblasts and muscle fibers.19 , 26 , 27 , 28 to determine the distribution of excess lox in dystrophic muscle , immunofluorescence of lox and vimentin , a marker of mesenchemyl cells , including fibroblasts , was performed . in healthy muscle , lox was evident in sparse puncta associated with the ecm . for the mouse diaphragm , these areas were observed along the epimysium and also in small foci in the perimysium and perivascular areas ( fig . 3a and b ) . the same infrequent lox locations were observed in the wildtype mouse ta ( data not shown ) . for the diaphragm of an unaffected dog ( fig . of particular interest were muscle fibers that were positive for lox both inside and outside of the sarcolemma , suggesting that the muscle fiber itself was producing lox ( fig . hot spots , but these were more extensive both in number and in size ( fig . 3d ) . this included more pronounced distribution of lox along the endomysium , evident in the mdx ta muscle . taken together , the expanded distribution of lox in mdx and grmd muscle sections was consistent with the increased lox transcript and protein levels found in dystrophic muscles . confocal microscopic immunofluorescent images of lysyl oxidase ( lox ; red ) , vimentin ( green ) , and dapi ( blue ) on 10mthick cryosections . ( a ) c57 mouse diaphragm shows epimysial focal regions of lox as well as ( b ) perimysial regions of focal lox , shown in insets . ( c ) unaffected dog diaphragm demonstrates similar focal expression of lox as c57 mice . ( d ) diaphragm and tibialis anterior ( ta ) samples from mdx mice and grmd dogs depict increased frequency of focal lox regions and endomysial lox . higher magnification outsets of ( b ) and ( c ) are indicated by dotted square regions of the lower magnification image . scale bars = 25 m , unless indicated otherwise . to substantiate the translational value of our animal model data , we measured lox protein in quadriceps biopsy samples from unaffected control subjects and dmd patients by western blotting ( fig . similar to the mouse and dog tissue , immunofluorescence of unaffected control human muscle demonstrated infrequent loxpositive loci , which were primarily located near the perimysial regions ( fig . , lox again had a broader distribution that included more frequent foci observed in both endomysial and perimysial regions . quantification of the loxpositive area fraction confirmed our western blotting results , with a 3fold increase compared with control subjects . these data demonstrate that the muscles of dmd patients have increased lox content and distribution , likely contributing to the progressive fibrotic replacement of muscle . ( a ) immunoblot for lox ( 32 kda ) using coomassie staining as a loading control from quadriceps biopsies of control ( n = 3 ) and dmd ( n = 3 ) patients . all lanes are on the same blot with intervening lanes excluded . quantification of lox blots from human patients indicates a significant increase in lox expression of dmd patients compared with unaffected controls . ( b ) immunofluorescent confocal microscopy images of lysyl oxidase ( lox ; red ) , vimentin ( green ) , and dapi ( blue ) on 10mthick cryosections from human quadriceps muscle . area fractions are quantified showing 5 regions of interest ( circles ) from each subject biopsy with averages ( lines ) from dmd ( n = 3 ) and control ( n = 3 ) patients , demonstrating significantly increased lox staining in dmd muscle . significant difference indicated by * p < 0.05 for lox expression between human patient groups gene expression of the collagen crosslinking enzyme lox was evaluated at the ends of the mouse phenotypic spectrum by comparing a less affected limb muscle ( ta ) and the more severely affected diaphragm muscle of mdx mice at age 6 months to agematched c57 mice ( fig . although lox expression was not significantly different between the wildtype and dystrophic ta , it was significantly elevated in the diaphragms of mdx mice . accordingly , col1a2 expression was also highly upregulated in the mdx diaphragm ; however , expression of the lox family member , loxl2 , was unchanged across the samples . at the protein level ( fig . 1b ) , lox abundance was similarly increased in the mdx diaphragms at age 6 months compared with those of c57 mice and was also significantly higher in the dystrophic ta . ( a ) lox transcript level is increased in mdx muscle ( n = 7 ) over control ( n = 6 ) . col1a2 is increased in mdx muscles ( n = 7 ) over control ( n = 6 ) , and in mdx diaphragm over ta . loxl2 tends to be upregulated in mdx muscles ( n = 3 ) over control ( n = 3 ) . ( b ) representative portion of immunoblot for lox ( 32 kda ) with ponceau stain for loading control . lox protein is increased in mdx ( n = 3 ) over control ( n = 3 ) , with mdx diaphragm higher than ta . ( c ) soluble hydroxyproline content , representing non crosslinked or immature crosslinked collagen , of mdx ( n = 6 ) muscles are not different from c57 ( n = 6 ) . insoluble hydroxyproline content , representing crosslinked collagen , and total collagen are increased in mdx , with mdx diaphragm higher than control diaphragm or mdx ta for both insoluble and total collagen . ( d ) immunoblot of mdx diaphragm for lox shows no additional increase at 18 months ( n = 2 ) compared with 6 months . insoluble crosslinked collagen is higher at 18 months ( n = 2 ) compared with 6 months in mdx diaphragm . significantly different posthoc test , indicated by * p < 0.05 and * * p < 0.01 , between genotypes of same muscle ; p < 0.05 and p < 0.01 , between muscles of the same genotype ; and p < 0.05 , between ages of mdx diaphragm . the consequence of higher levels of lox was evaluated with a collagen solubility assay , where the soluble , insoluble , and total collagen levels were determined in mdx and control muscles ( fig . the amount of soluble collagen , representing non crosslinked and immature crosslinked collagen , was similar between all muscles analyzed and represented a minor proportion of the total collagen pool . in contrast , 6month mdx diaphragms had significantly more mature crosslinked insoluble collagen and total collagen than the mdx ta and agematched c57 diaphragms . to determine whether there was an agedependent increase in either lox or collagen , muscles from 18monthold mdx mice lox levels were similar between the 2 ages , but the mdx diaphragm continued to accrue insoluble collagen ( fig . 1d ) , whereas the ta insoluble content remained unchanged ( data not shown ) . these data demonstrate that lox is chronically elevated in the dystrophic mouse diaphragm , which results in progressive accumulation of crosslinked collagen . due to the relatively mild phenotype of the mdx mouse compared with dmd patients , we also investigated lox levels and collagen crosslinking in the more severe grmd dog model of dmd . like the mdx mouse , grmd exhibits differential disease severity between limb muscles and the diaphragm . similar to our findings in the mdx mouse , grmd diaphragm displayed high upregulation of both lox and col1a2 gene expression over control dog levels , with no change in the ta ( fig . 2a ) . lox protein , although not detectable in control dog samples by western blotting , was observed in grmd samples , where its abundance among grmd diaphragms was consistently higher than the dogmatched ta ( fig . accordingly , grmd diaphragms also contained more insoluble and total collagen than all other samples ( fig . 2c ) , in agreement with the findings in mice described above . lox expression and collagen crosslinking in the grmd dog model of dmd in limb and diaphragm muscle . ( a ) transcriptional level of lox and col1a2 ( b ) immunoblot for lox ( 32 kda ) using gapdh ( 37 kda ) as a loading control from wildtype and grmd dogs . protein level of lox is not observable in wildtype dogs , but is present in grmd muscles , with diaphragm consistently higher than dog matched ta . crosslinked collagen or immature crosslinked collagen , does not show a consistent difference between wildtype and grmd . ( d ) representative images of grmd dog ta and diaphragm muscles viewed under circularly polarized light . birefringent fibrillar collagen is visible with loosely ( green ) , moderately ( yellow ) , and densely ( red ) packed collagen . scale bar = 50 m . as previously performed on mdx mouse muscle,12 the collagen density of grmd muscle sections was visualized under circularly polarized light ( fig . in accordance with the biochemical data described above , the grmd ta contained mostly loosely packed collagen bundles ( visualized as green ) , with an extensive perimysial layer , whereas the diaphragm collagen bundles were largely intermediate ( yellow ) or densely ( orange to red ) packed , verifying that collagen density is increased in grmd diaphragm compared with limb muscle . it is also notable that , in the grmd diaphragm , unlike the ta , the robust endomysial expansion resulted in a lack of distinction between endomysial and perimysial stromal regions . thus , the canine model of dmd also displays heightened indices of collagen crosslinking in the diaphragm as compared with less affected limb muscle . lox crosslinking activity occurs in the ecm , but many different cell types can produce this enzyme , including fibroblasts and muscle fibers.19 , 26 , 27 , 28 to determine the distribution of excess lox in dystrophic muscle , immunofluorescence of lox and vimentin , a marker of mesenchemyl cells , including fibroblasts , was performed . in healthy muscle , lox was evident in sparse puncta associated with the ecm . for the mouse diaphragm , these areas were observed along the epimysium and also in small foci in the perimysium and perivascular areas ( fig . 3a and b ) . the same infrequent lox locations were observed in the wildtype mouse ta ( data not shown ) . for the diaphragm of an unaffected dog ( fig . of particular interest were muscle fibers that were positive for lox both inside and outside of the sarcolemma , suggesting that the muscle fiber itself was producing lox ( fig . hot spots , but these were more extensive both in number and in size ( fig . this included more pronounced distribution of lox along the endomysium , evident in the mdx ta muscle . taken together , the expanded distribution of lox in mdx and grmd muscle sections was consistent with the increased lox transcript and protein levels found in dystrophic muscles . confocal microscopic immunofluorescent images of lysyl oxidase ( lox ; red ) , vimentin ( green ) , and dapi ( blue ) on 10mthick cryosections . ( a ) c57 mouse diaphragm shows epimysial focal regions of lox as well as ( b ) perimysial regions of focal lox , shown in insets . ( c ) unaffected dog diaphragm demonstrates similar focal expression of lox as c57 mice . ( d ) diaphragm and tibialis anterior ( ta ) samples from mdx mice and grmd dogs depict increased frequency of focal lox regions and endomysial lox . higher magnification outsets of ( b ) and ( c ) are indicated by dotted square regions of the lower magnification image . to substantiate the translational value of our animal model data , we measured lox protein in quadriceps biopsy samples from unaffected control subjects and dmd patients by western blotting ( fig . similar to the mouse and dog tissue , immunofluorescence of unaffected control human muscle demonstrated infrequent loxpositive loci , which were primarily located near the perimysial regions ( fig . , lox again had a broader distribution that included more frequent foci observed in both endomysial and perimysial regions . quantification of the loxpositive area fraction confirmed our western blotting results , with a 3fold increase compared with control subjects . these data demonstrate that the muscles of dmd patients have increased lox content and distribution , likely contributing to the progressive fibrotic replacement of muscle . ( a ) immunoblot for lox ( 32 kda ) using coomassie staining as a loading control from quadriceps biopsies of control ( n = 3 ) and dmd ( n = 3 ) patients . quantification of lox blots from human patients indicates a significant increase in lox expression of dmd patients compared with unaffected controls . ( b ) immunofluorescent confocal microscopy images of lysyl oxidase ( lox ; red ) , vimentin ( green ) , and dapi ( blue ) on 10mthick cryosections from human quadriceps muscle . area fractions are quantified showing 5 regions of interest ( circles ) from each subject biopsy with averages ( lines ) from dmd ( n = 3 ) and control ( n = 3 ) patients , demonstrating significantly increased lox staining in dmd muscle . significant difference indicated by * p < 0.05 for lox expression between human patient groups . skeletal muscle fibrosis is an excessive deposition of ecm components and accompanies the chronic damage that occurs in muscular dystrophy . little is known about how this aberrant ecm is organized in fibrotic dystrophic muscle , but there is evidence that posttranslational modification of the ecm may be part of the dystrophic pathology . previous measurements of a rarely used chicken model of muscular dystrophy have shown increased proportions of highmolecularweight collagen suggestive of increased crosslinking.29 in addition , studies using mdx mice demonstrated increased lox expression in heart and limb muscle20 and histological association between lox and fibrosis.17 we have now extended these observations to show that lox protein is increased in dmd patient muscle , as well as in mouse and dog dmd models . in dystrophic mouse and dog diaphragm , where pathology is the most severe,10 there is also more collagen crosslinking , as demonstrated by the increased insoluble fraction of collagen . insoluble collagen content increased progressively in the mdx diaphragm , demonstrating the negative implications of chronic lox elevation . we also demonstrated that the increase in lox is conserved across species with muscular dystrophy . taken together , we conclude that increased lox leads to extensive collagen crosslinking , which is an important feature of dmd muscle . although the mdx mouse is the most commonly studied model of dmd , it does not typically develop fibrosis in the limbs at a young age , and it has several differences from the human disease.4 , 30 lifespan is only moderately affected in the mdx mouse , and , although limb muscles undergo cycles of degeneration and regeneration , there is relatively little fibrosis . the mdx diaphragm most accurately reflects the muscle pathology observed in dmd patients,10 thus it is not surprising that our measurements of crosslinking were also most prominent in the mdx diaphragm compared with the ta muscles . this is consistent with our previous observations of increased collagen packing density in the mdx diaphragm.12 to determine whether our results were consistent in a model that more accurately exhibits features of dmd , we used the grmd dog model.31 grmd dogs also exhibit increased crosslinked collagen and increased lox levels , supporting collagen crosslinking as a major feature of the disease that is maintained across species . part of the reason we could not detect this difference , as one would expect for larger mammals , is the presence of a very thick perimysium layer that is found in both unaffected healthy and dystrophic dog ta muscles . this layer is an important structural component of limb muscles in dogs and is crosslinked to a greater extent than the rest of the ecm in this muscle . consistent with this is the localization of more densely packed collagen within the ta perimysium compared with the endomysium . as such , the collagen solubility measurements of muscle homogenates where the perimysium is the overwhelmingly dominant source of matrix likely masks the changes that may have occurred in the endomysial layer around the fibers . in contrast , the grmd diaphragm possessed more densely packed collagen fibrils throughout the muscle , which was accompanied by an increase in collagen insolubility . to verify that the elevation of lox observed in animal models is also a feature of dmd , human biopies were examined . in agreement with the animal models , lox expression was shown to be elevated in quantity as well as in frequency of hot spots with high focal expression . this is also consistent with 3 published microarray studies in the gene expression omnibus database,32 which showed significant upregulation of lox in dmd ( p = 4.9 10 , gds21433 ; p = 6.8 10 , gds302734 ; p = 4.7 10 , gds56335 . although we were not able to measure collagen crosslinking in human samples directly , the increased lox levels suggest that muscle in dmd is more extensively crosslinked as observed in animal models . collagen crosslinking has been described as a major factor in fibrosis of multiple tissues,36 , 37 including the heart.38 the role of collagen crosslinking in the heart is of great clinical significance in patients with dmd , as it also undergoes a fibrotic response . whereas lox mrna is known to be increased in mdx cardiac muscle,20 lox protein levels and collagen crosslinking have not been studied but should be a target of future investigation . increased collagen crosslinking is thought to lead to a stiffer ecm and tissue , as demonstrated in the left ventricle of the fibrotic heart.38 however , recent reports of muscle stiffness in mdx mice have shown relatively more stiffening of limb muscle as opposed to the diaphragm,12 even though crosslinking is most prevalent in the diaphragm . collagen content shows little correlation with passive stiffness,12 but collagen crosslinking does not account for additional variability in muscle fiber bundle stiffness.39 this suggests that , although collagen may be stiffer at the molecular level , other parameters of collagen organization dictate muscle stiffness at the tissue level . however , this does not preclude the microstiffness of the crosslinked collagen fibrils from playing a critical role in the cellular response to collagen crosslinking in fibrosis . lox is critical to the integrity of the developing diaphragm , as mice that lack lox often die perinatally from diaphragmatic hernias.40 further , skeletal muscle development is disrupted in lox knockout mice independent of skeletal or tendon defects , demonstrating a role for lox in this process.28 loxl1loxl4 are also capable of crosslinking collagen , but little is known about their expression in muscle . the loxl1loxl4 family members have a conserved domain responsible for catalyzing the initial reaction in collagen crosslinking . their distinct roles are not well understood , but loxl2 has been shown to be upregulated in fibrotic tissue and responsible for collagen crosslinking in fibrosis.37 our initial investigation showed that loxl2 expression is not significantly altered in dystrophic muscle , providing supporting evidence for lox being the primary enzyme responsible for crosslinking changes in dystrophic muscle . critically , it is well established that crosslinked collagen is much less susceptible to turnover,21 thus implicating collagen crosslinking in the development of progressive scar tissue formation that predominates in muscles from dmd patients . lox is known to be produced by vascular cells , including endothelial cells,26 smooth muscle,19 and fibroblasts,27 which are all muscle resident cells ; however , a recent report by kutchuk et al . suggested skeletal muscle is also a major contributor of lox expression , in which myofiber lox secretion is a negative feedback switch for stromal collagen production and tgf activity.28 although evidence of lox production from a specific cell type was not conclusive in our study , we observed scattered stromal foci of lox immunoreactivity throughout muscle crosssections , as well as evidence of staining within muscle fibers . in healthy muscle , these foci are typically localized to perimysial and epimysial regions , which are points of high mechanical stress in contracting muscle . the increased frequency and intrafascicular presence of lox staining in dystrophic muscle could be due to the increased mechanical burden experienced by dystrophindeficient muscle fibers from reduced attachment to the ecm . taken together , these lines of evidence suggest lox secretion into the ecm region is likely regulated by myofiber mechanical sensing , which leads to appropriate ecm adaptation to stress stimuli in nonpathological situations . unfortunately , dystrophic muscle fibers are impaired in their attachment to the ecm independent of the extent of ecm accumulation and/or crosslinking , thereby preventing the shutdown of collagen and lox production . this scenario potentially results in continuous endomysial ecm accumulation , which leads to replacement of functional muscle with fibrosis , as seen in dmd patients.41 these studies raise important questions regarding the best therapeutic targets for fibrosis . if lox is critical for reducing tgf actions , then targeting it may cause rampant collagen production and lead to increased fibrosis . however , if excessive lox activity on ecm collagen crosslinking is detrimental to muscle function , then lox inhibition may be beneficial to diseased muscles where reduced stiffness may also lessen structural damage to muscle fibers . clinically , therapies that target lox and lox family member activity show promise in preventing lung and liver fibrosis37 and set a precedent for trials to counter muscle fibrosis using this strategy . however , the balance between the extent of fibrosis and the proportion of crosslinking is a major consideration with respect to the therapeutic window for treatments . because crosslinked collagen is relatively stable and refractory to remodeling,18 antifibrotic therapies may not remove crosslinked collagen , yet they may gradually reduce overall levels of fibrosis . this could be important if antifibrotic therapies are applied to latestage disease patients in whom considerable cellular loss has occurred in the heart and diaphragm . at that disease stage , removal of all fibrosis could lead to herniation of the diaphragm and aneurysms in the heart , both lethal consequences . in that case , the crosslinked collagen would safeguard against these potentially lifethreatening events . min liu and zuozhen tian from the physiological assessment core at the university of pennsylvania . steven moore from the wellstone muscular dystrophy cooperative research center for providing human biopsies , and dr . margaret sleeper from the department of small animal clinical sciences , university of florida college of veterinary medicine , for providing dog tissue .

backgroundwith the aging of the population , dementia is increasing worldwide . the objective of this study was to identify risk factors for dementia in an elderly population utilizing a primary health care service in dakar , senegal.methodsthrough a cross - sectional study conducted from march 2004 to december 31 , 2005 , 507 elderly patients aged 65 years who came to the social and medical center of ipres , dakar , senegal , were first screened with the screening interview questionnaire aging in senegal. those who were cognitively impaired underwent a clinical examination to detect dementia . univariate , bivariate , and multivariate logistic regression analyses were done.resultsthe whole population had a mean age of 72.4 years ( 5.2 ) and was mostly male , married , and non - educated . hypertension , arthritis , and gastrointestinal diseases were the main health conditions reported in the past medical history . smoking was important while alcohol consumption was rare . social network was high . forty - five patients ( 8.87% ) had dementia . in the multivariate model , only advanced age , education , epilepsy , and family history of dementia were independently associated with dementia.conclusionthe risk factors identified are also found in developed countries confirming their role in dementia . it is important to take dementia into consideration in senegal and to sensitize the community for prevention .

with the aging of the population worldwide , dementia is a real public health priority . in 2000 , the estimated number of dementia cases was 25.5 million people , representing 0.4% of the worldwide population . nearly two thirds of all people with dementia lived in low- and middle - income countries . the other most important risk factors are low level of education , heredity , low early - life economic conditions , smoking , alcohol , weak social network , hypertension , cardiovascular diseases , stroke , epilepsy , diabetes , anemia , parkinson 's disease , and head trauma . although several population - based studies were conducted in developed countries to better understand the epidemiology of this new epidemic , only a few studies have been done in africa . in senegal , the estimated number of elderly aged 65 years was 421,305 in 2008 and 420,795 in 2009 . considering the economic cost of dementia care , senegal is not able to afford such cost . it is important to have reliable information on the prevalence of dementia to plan for more accurate provision of social and medical services for the elderly population . studies on dementia in the senegalese elderly population are rare [ 20 , 21 ] . thus , a study was conducted in senegal to determine the epidemiology of dementia in an elderly population utilizing a primary health care service for retirees in dakar , senegal . the aim of the present study was to identify risk factors associated with dementia in this population of retirees utilizing the social and medical center ( smc ) of ipres ( institution de prevoyance retraite du senegal ) , dakar , senegal . this is a health center for the retired senegalese elderly population affiliated to ipres ensuring medical and social services . as a primary care center with diverse health personnel , the study population was composed of senegalese elderly patients aged 65 years who came to the smc of ipres for health problems . those patients who were either < 65 years old or were not able to fulfill the interview ( due to aphasia , delirium , coma , extreme visual and auditory impairment , or cancer at terminal phase ) were excluded . 507 elderly patients aged 65 years or their relatives who consulted a doctor for a medical problem at the smc of ipres were first assessed with a screening interview questionnaire . those who were cognitively impaired , with a sex - matched sample , underwent a clinical exam with neuropsychological testing . this study was the first step of a research on the validation of a screening tool to screen for dementia in a senegalese elderly population called the test of senegal . two screening instruments were used : the interview questionnaire called aging in senegal and the clinical assessment instrument . the screening interview questionnaire aging in senegal contained the following : sociodemographic variables ( age , sex , marital status , education ) , medical history [ vascular diseases ( hypertension , heart diseases , vascular peripheral disease , stroke , diabetes ) , respiratory diseases , arthritis , cancer / benign tumor , parkinson 's disease , epilepsy , genitourinary disease , cataract , glaucoma , hearing impairment , digestive disease ( gastritis , constipation ) , anemia , thyroid disease , head trauma , bone fracture ] , familial history of memory impairment , lifestyle ( smoking , alcohol consumption , walking ) , social network ( social ties with spouse , children , brothers / sisters , friends ; frequency of weekly contact with children , brothers / sisters , friends ; members of community association , member of religious association ) , the patient 's functional autonomy , and the neuropsychological tests with the abbreviated mental test and the test of senegal . the clinical assessment instrument had four components : ( 1 ) a historical review of the patient 's cognitive function , i.e. , the onset and progression of any reported symptoms of cognitive impairment ; ( 2 ) a review of the patient 's medical , surgical , and familial history , exposure to toxic products and medications ; ( 3 ) a review of the patient 's functional autonomy , and ( 4 ) a review of the patient 's clinical exam and neuropsychological testing with the mini - mental state examination ( mmse ) . each patient underwent a screening interview with the questionnaire aging in senegal by four medical students at the smc who were trained for this issue . it aimed to collect data related to sociodemographics , lifestyle , past medical history , and social network and to screen for neuropsychological performance with the abbreviated mental test . after the interview , each patient who had a score of 5 on the abbreviated mental test was referred for clinical assessment to the principal investigator ( k.t . ) . at the same time , a sex - matched normal subject was also referred for clinical assessment . the clinical assessment consisted of a complete physical exam followed by a neuropsychological testing with the mmse . if a patient was suspected to have depression , the ces - d scale was administered to him / her to confirm the diagnosis . all clinical assessments were made without knowledge of the screening status of the patient . at the end of the consultation , the team members met in a room to confirm the diagnosis of the patient . on the basis of the examination , appropriate laboratory exams and computerized tomography of the head were ordered and treatment of associated medical conditions proposed . sociodemographic variables [ age in 4 categories ( 6569 , 7074 , 7579 , and 80 years ) , sex , marital status , education ] were collected with the medical history and familial history of cognitive impairment . lifestyle was divided into smoking habit ( yes , no ) , alcohol consumption ( yes , no ) , and walking ( yes , no ) . for the social network , we computed two indexes : diversities of social ties ( score 04 ) and frequency of weekly contact with relatives ( score 06 ) . diversity of social ties was computed by summing having a spouse or husband , children , brothers / sisters , and friends and categorized into 3 levels : 02 ties , 3 ties , and 4 ties . frequency of weekly contact with relatives was obtained by summing the frequency of weekly contact with children , brothers / sisters , and friends and categorized into 4 levels : 03 weekly contacts , 4 weekly contacts , 5 weekly contacts , and 6 weekly contacts . the medical conditions related to medical variables were dichotomized into yes or no. dementia was defined according to the dsm - iv - r criteria [ 27 , 28 ] . this study was approved by the ethical committee of the senegalese ministry of health and university of montreal , que . , this is a health center for the retired senegalese elderly population affiliated to ipres ensuring medical and social services . as a primary care center with diverse health personnel , the study population was composed of senegalese elderly patients aged 65 years who came to the smc of ipres for health problems . those patients who were either < 65 years old or were not able to fulfill the interview ( due to aphasia , delirium , coma , extreme visual and auditory impairment , or cancer at terminal phase ) were excluded . 507 elderly patients aged 65 years or their relatives who consulted a doctor for a medical problem at the smc of ipres were first assessed with a screening interview questionnaire . those who were cognitively impaired , with a sex - matched sample , underwent a clinical exam with neuropsychological testing . this study was the first step of a research on the validation of a screening tool to screen for dementia in a senegalese elderly population called the test of senegal . two screening instruments were used : the interview questionnaire called aging in senegal and the clinical assessment instrument . the screening interview questionnaire aging in senegal contained the following : sociodemographic variables ( age , sex , marital status , education ) , medical history [ vascular diseases ( hypertension , heart diseases , vascular peripheral disease , stroke , diabetes ) , respiratory diseases , arthritis , cancer / benign tumor , parkinson 's disease , epilepsy , genitourinary disease , cataract , glaucoma , hearing impairment , digestive disease ( gastritis , constipation ) , anemia , thyroid disease , head trauma , bone fracture ] , familial history of memory impairment , lifestyle ( smoking , alcohol consumption , walking ) , social network ( social ties with spouse , children , brothers / sisters , friends ; frequency of weekly contact with children , brothers / sisters , friends ; members of community association , member of religious association ) , the patient 's functional autonomy , and the neuropsychological tests with the abbreviated mental test and the test of senegal . the clinical assessment instrument had four components : ( 1 ) a historical review of the patient 's cognitive function , i.e. , the onset and progression of any reported symptoms of cognitive impairment ; ( 2 ) a review of the patient 's medical , surgical , and familial history , exposure to toxic products and medications ; ( 3 ) a review of the patient 's functional autonomy , and ( 4 ) a review of the patient 's clinical exam and neuropsychological testing with the mini - mental state examination ( mmse ) . each patient underwent a screening interview with the questionnaire aging in senegal by four medical students at the smc who were trained for this issue . it aimed to collect data related to sociodemographics , lifestyle , past medical history , and social network and to screen for neuropsychological performance with the abbreviated mental test . after the interview , each patient who had a score of 5 on the abbreviated mental test was referred for clinical assessment to the principal investigator ( k.t . ) . at the same time , a sex - matched normal subject was also referred for clinical assessment . the clinical assessment consisted of a complete physical exam followed by a neuropsychological testing with the mmse . if a patient was suspected to have depression , the ces - d scale was administered to him / her to confirm the diagnosis . all clinical assessments were made without knowledge of the screening status of the patient . at the end of the consultation , the team members met in a room to confirm the diagnosis of the patient . on the basis of the examination , patients with dementia were followed by the principal investigator ( k.t . ) . appropriate laboratory exams and computerized tomography of the head were ordered and treatment of associated medical conditions proposed . sociodemographic variables [ age in 4 categories ( 6569 , 7074 , 7579 , and 80 years ) , sex , marital status , education ] were collected with the medical history and familial history of cognitive impairment . lifestyle was divided into smoking habit ( yes , no ) , alcohol consumption ( yes , no ) , and walking ( yes , no ) . for the social network , we computed two indexes : diversities of social ties ( score 04 ) and frequency of weekly contact with relatives ( score 06 ) . diversity of social ties was computed by summing having a spouse or husband , children , brothers / sisters , and friends and categorized into 3 levels : 02 ties , 3 ties , and 4 ties . frequency of weekly contact with relatives was obtained by summing the frequency of weekly contact with children , brothers / sisters , and friends and categorized into 4 levels : 03 weekly contacts , 4 weekly contacts , 5 weekly contacts , and 6 weekly contacts . the medical conditions related to medical variables were dichotomized into yes or no. dementia was defined according to the dsm - iv - r criteria [ 27 , 28 ] . this study was approved by the ethical committee of the senegalese ministry of health and university of montreal , que . , univariate , bivariate , and multivariate stepwise logistic regression analyses were computed . the odd ratios of the different risk factors were computed with a 95% ci . forty - five patients ( 8.87% ; 95% ci 7.6110.13 ) had dementia . the whole population with a mean age of 72.4 years ( 5.2 ) was mostly male , married , and illiterate . the elderly population had a high diversity of ties and frequency of contact with relatives and friends ( table 1 ) . hypertension ( 58.6% ) , arthritis ( 49.5% ) , gastrointestinal diseases ( 24.1% ) , respiratory diseases ( 14.7% ) , and cataract ( 14.4% ) were the main health conditions reported in the past medical history ( table 2 ) . in the bivariate analysis , age ( p < 0.045 ) , education ( p < 0.02 ) , diversity of ties with relatives ( p < 0.001 ) , frequency of contact with relatives ( p < 0.0001 ) , stroke ( p < 0.002 ) , epilepsy ( p < 0.002 ) , and family history of dementia ( p < 0.0001 ) were associated with dementia . in the multivariate model , advanced age ( 80 years ; or 4.26 ; 95% ci 1.3613.3 ) , illiteracy ( or 2.76 ; 95% ci 1.236.20 ) , epilepsy ( or 11.72 ; 95% ci 2.1065.23 ) , and family history of dementia ( or 7.56 ; 95% ci 3.4216.76 ) were independently associated with dementia ( table 3 ) . in our study , prevalence of dementia was 8.87% with advanced age , illiteracy , epilepsy , and family history of dementia as independent risk factors . these risk factors were either self - reported by the patient or informant - reported . the role of age as a risk factor for dementia has been highlighted in several studies . in fact , the prevalence of dementia is increasing importantly with age as observed in a clinical setting and during population studies . thus , the result we observed confirms the role of ageing ( especially advanced age ) in the occurrence of dementia as described elsewhere in europe [ 3 , 29 , 30 , 31 ] , america [ 32 , 33 , 34 ] , asia [ 35 , 36 , 37 ] , and africa [ 21 , 38 , 39 , 40 , 41 ] . illiteracy was frequent ( 57% ) and was associated with dementia in our elderly population . this result confirms the role of illiteracy as a risk factor for dementia in an elderly population as already seen in studies realized worldwide [ 42 , 43 , 44 , 45 , 46 ] . epilepsy in the elderly is frequent , with an estimated prevalence of 1% after 60 years of age and an incidence increasing with age . in our study population , history of epilepsy this finding has been observed in several studies conducted worldwide , enhancing the association between a history of epilepsy and dementia in an elderly population [ 13 , 49 , 50 , 51 , 52 ] . this association could be related to the disease itself , its cause or risk factor , but also to the treatment prescribed , especially in our country , where most of the epileptic patients are on a first - generation anti - epileptic regimen , which is harmful for cognition . familial history of dementia , which was observed in 9.1% of our study population , has been associated with the occurrence of dementia . in fact , studies in australia , africa [ 55 , 56 ] , europe [ 57 , 58 ] , and the usa have demonstrated that familial history of dementia is a main risk factor for dementia in an elderly population . also , apoe 4 has been associated with alzheimer 's disease in the developed world [ 59 , 60 ] . however , this association is absent in an african study . genetic mutations are also observed in demented persons at chromosome 12 and chromosomes 6 , 9 , 10 , 12 , 19 , and 21 [ 63 , 64 ] . limitations of the study include the underestimation of the prevalence because all the demented patients were not recruited during the study period . medical illnesses were based upon self - report or informant report rather than on direct examination . moreover , memory bias could underestimate risk factors and , thus , their role in the occurrence of dementia . also , this study is a prevalence study , not an incidence study , characterized by temporality of the association between risk factors and dementia . however , it highlights and confirms the role of these risk factors on dementia occurrence in an elderly population . this study has shown that illiteracy , epilepsy , and familial history of dementia were associated with dementia in this study population . additionally , it confirms the role of these risk factors in the occurrence of dementia in an elderly population as described elsewhere . as the senegalese population is aging , it is necessary to take dementia into consideration in the social , political , and medical practice and to sensitize political leaders , the health personnel , and the population for the prevention of this disease .

objectiveadiponectin is an adipocyte - derived hormone that sensitizes insulin and improves energy metabolism in tissues . this study was designed to investigate the direct regulatory effects of adiponectin on lipid metabolism in adipocytes.research design and methodsbasal and hormone - stimulated lipolysis were comparatively analyzed using white adipose tissues or primary adipocytes from adiponectin gene knockout and control mice . to further study the underlying mechanisms through which adiponectin suppresses lipolysis , cultured 3t3-l1 adipocytes and adenovirus - mediated gene transduction were used.resultssignificantly increased lipolysis was observed in both adiponectin gene knockout mice and primary adipocytes from these mice . hormone - stimulated glycerol release was inhibited in adiponectin - treated adipocytes . adiponectin suppressed hormone - sensitive lipase activation without altering adipose triglyceride lipase and cgi-58 expression in adipocytes . moreover , adiponectin reduced protein levels of the type 2 regulatory subunit rii of protein kinase a by reducing its protein stability . ectopic expression of rii abolished the inhibitory effects of adiponectin on lipolysis in adipocytes.conclusionsthis study demonstrates that adiponectin inhibits lipolysis in adipocytes and reveals a novel function of adiponectin in lipid metabolism in adipocytes .

see detailed information on materials , virus construction , quantitative pcr , and adipocyte area measurement in the supplementary methods . adipoq mice were created as previously described ( 5 ) on the 129/svev genetic background and had been back - crossed to c57bl/6 for five generations . the experiments using mouse models were carried out under the association for assessment and accreditation of laboratory animal care guidelines with approval of the animal care and use committee . for some animal studies , 1 10 pfu of purified adenovirus vectors was injected into the mouse tail vein ( 6 ) . 3t3-l1 and 3t3-l1car1 cells and induction of adipocyte differentiation were described in a previous publication ( 7 ) . op9 adipocyte differentiation was induced using knockout sr ( 8) . although both 3t3-l1 and op9 adipocytes secrete adiponectin into the medium , adiponectin in overnight cultured medium accumulated only to 0.1% of mouse serum adiponectin . therefore , co - culture or conditioned medium was used to increase adiponectin protein levels . for co - culture , adipocytes were cultured in the outside well of the bd biosciences ( franklin lakes , nj ) transwell co - culture system , and fao cells were grown on the protein and cytokine - permeable membrane of the insert well . fao cells were transduced with adenovirus encoding mouse adiponectin ( ad - acrp30 ) or green fluorescent protein ( gfp ) for 12 h in a regular 6-well plate , and then the inserts with transduced fao cells were washed and transferred to the co - culture plates with differentiated adipocytes in the bottom wells . this system allows increase of medium adiponectin levels ( 40% of mouse serum adiponectin ) without physical interaction between 3t3-l1 adipocytes and fao cells . in a separate setting , conditioned medium from ad - acrp30transduced fao cells was collected and used for increasing adiponectin levels in some of the studies , as indicated in figure legends . for hormone - stimulated lipolysis , bt2-camp ( 100 mol / l ) was added to culture medium and cells were incubated for 1 h. total protein was used for certain protein or protein phosphorylation measurements using western blots . for pka rii protein half - life measurement , cycloheximide ( 100 g / ml ) was used to inhibit protein synthesis . for the in vivo lipolysis assays , the selective 3-adrenergic agonist brl37344 was injected i.p . into mice at 5 g / g body blood samples were collected 0 , 10 , and 20 min after the injection . for the lipolysis assay of adipose explants , 20 mg of epididymal adipose tissue explants were cultured in dulbecco s modified eagle s medium with 0.5% fatty acid brl37344 was added to the medium at 50 ng / ml . the culture tubes were shaken at 400 rpm . the medium samples were collected 30 , 60 , 120 , and 180 min after adding brl37344 . the level of free fatty acid ( ffa ) or glycerol was normalized to the weight of adipose explants . for primary adipocyte lipolysis assays , adipocytes were prepared from epididymal fat using collagenase with an optimized procedure ( 11,12 ) . briefly , epididymal fat tissue was minced and digested in a krebs - ringer bicarbonate ( krb ) buffer ( ph 7.4 ) supplemented with 3% fatty acid free bsa fraction v , 0.5 mmol / l adenosine , and 1 mg / ml type i collagenase . after 40-min digestion and three washes , isolated adipocytes were resuspended in krb supplemented with 5 mmol / l glucose and 3% fatty acid . suspended adipocytes ( 6,000 cells ) were used for a lipolysis assay in 400 l krb buffer plus 3% fatty acid free bsa , 1 unit / ml adenosine deaminase , and 100 nmol / l ( -)-n-(2-phenyl - isopropyl)-adenosine ( basal only ) with or without brl37344 ( 50 ng / ml ) . after 1-h incubation at 37c , 200 l of infranatant was removed and stored at 20c . serum free glycerol and glycerol released from fat explants were measured using a free - glycerol kit ( sigma - aldrich co. , st . glycerol in the assay media of primary and 3t3-l1 adipocytes was measured using bioluminescence , which is very sensitive and especially well adapted when only small amounts of adipose tissue or cells are available ( 13,14 ) . statistical analysis was performed using the student t test or anova , followed by contrast test . adipoq mice were created as previously described ( 5 ) on the 129/svev genetic background and had been back - crossed to c57bl/6 for five generations . the experiments using mouse models were carried out under the association for assessment and accreditation of laboratory animal care guidelines with approval of the animal care and use committee . for some animal studies , 1 10 pfu of purified adenovirus vectors was injected into the mouse tail vein ( 6 ) . 3t3-l1 and 3t3-l1car1 cells and induction of adipocyte differentiation were described in a previous publication ( 7 ) . op9 adipocyte differentiation was induced using knockout sr ( 8) . although both 3t3-l1 and op9 adipocytes secrete adiponectin into the medium , adiponectin in overnight cultured medium accumulated only to 0.1% of mouse serum adiponectin . therefore , co - culture or conditioned medium was used to increase adiponectin protein levels . for co - culture , adipocytes were cultured in the outside well of the bd biosciences ( franklin lakes , nj ) transwell co - culture system , and fao cells were grown on the protein and cytokine - permeable membrane of the insert well . fao cells were transduced with adenovirus encoding mouse adiponectin ( ad - acrp30 ) or green fluorescent protein ( gfp ) for 12 h in a regular 6-well plate , and then the inserts with transduced fao cells were washed and transferred to the co - culture plates with differentiated adipocytes in the bottom wells . this system allows increase of medium adiponectin levels ( 40% of mouse serum adiponectin ) without physical interaction between 3t3-l1 adipocytes and fao cells . in a separate setting , conditioned medium from ad - acrp30transduced fao cells was collected and used for increasing adiponectin levels in some of the studies , as indicated in figure legends . for hormone - stimulated lipolysis , bt2-camp ( 100 mol / l ) was added to culture medium and cells were incubated for 1 h. total protein was used for certain protein or protein phosphorylation measurements using western blots . for pka rii protein half - life measurement for the in vivo lipolysis assays , the selective 3-adrenergic agonist brl37344 was injected i.p . into mice at 5 g / g body wt ( 9 ) . blood samples were collected 0 , 10 , and 20 min after the injection . for the lipolysis assay of adipose explants , 20 mg of epididymal adipose tissue explants were cultured in dulbecco s modified eagle s medium with 0.5% fatty acid the medium samples were collected 30 , 60 , 120 , and 180 min after adding brl37344 . the level of free fatty acid ( ffa ) or glycerol was normalized to the weight of adipose explants . for primary adipocyte lipolysis assays , adipocytes were prepared from epididymal fat using collagenase with an optimized procedure ( 11,12 ) . briefly , epididymal fat tissue was minced and digested in a krebs - ringer bicarbonate ( krb ) buffer ( ph 7.4 ) supplemented with 3% fatty acid free bsa fraction v , 0.5 mmol / l adenosine , and 1 mg / ml type i collagenase . after 40-min digestion and three washes , isolated adipocytes were resuspended in krb supplemented with 5 mmol / l glucose and 3% fatty acid free bsa . suspended adipocytes ( 6,000 cells ) were used for a lipolysis assay in 400 l krb buffer plus 3% fatty acid free bsa , 1 unit / ml adenosine deaminase , and 100 nmol / l ( -)-n-(2-phenyl - isopropyl)-adenosine ( basal only ) with or without brl37344 ( 50 ng / ml ) . after 1-h incubation at 37c , 200 l of infranatant was removed and stored at 20c . serum free glycerol and glycerol released from fat explants were measured using a free - glycerol kit ( sigma - aldrich co. , st . glycerol in the assay media of primary and 3t3-l1 adipocytes was measured using bioluminescence , which is very sensitive and especially well adapted when only small amounts of adipose tissue or cells are available ( 13,14 ) . statistical analysis was performed using the student t test or anova , followed by contrast test . to study the effects of adiponectin on lipid metabolism in adipocytes , adipoq mice were used . consistent with previous studies ( 5,15 ) , there were no significant differences of body weights between adipoq and wild - type ( wt ) littermate controls when the mice were fed normal chow ( data not shown ) . however , body fat percentage of adipoq mice was significantly lower than that of wt mice ( fig . epididymal fat mass was also significantly smaller in adipoq male mice ( 0.26 0.02 vs. 0.42 0.04 g ; vs. wt , p < 0.05 , n = 10 ) . adipocyte cross - sectional areas of fixed epididymal fat of adipoq mice were 20% smaller than those of wt control mice ( supplementary fig . when mice were fed a 60% ( calorie ) high - fat diet for 8 weeks , adipoq mice gained remarkably less body weight and adipose tissue mass and had smaller adipocyte area ( fig . similar to previous studies ( 5,16 ) , high - fat diet induced more severe insulin resistance in adipoq mice ( data not shown ) . therefore , these studies clearly show that adipoq mice have less fat mass and are resistant to high - fat diet induced obesity . similar phenotypes have been reported by a group using an independent line of adipoq mice ( 17 ) . adiponectin knockout mice have less body fat and are resistant to high - fat diet induced obesity . body composition of chow - fed 2-month - old male adipoq and wt mice was scanned using echomri ( a ) . then the mice were fed a high - fat diet ( 60% of calories from fat ) . body fat was measured after 8 weeks of high - fat feeding ( c ) . our study shows that neither adiponectin gene knockout nor overexpression has any significant effect on gene expression of the key enzymes of triglyceride synthesis in epididymal fat ( supplementary fig . 2 ) , which suggests that adiponectin may regulate lipid metabolism in adipocytes through a pathway other than de novo lipogenesis . although serum ffa levels at the fed state were similar between chow - fed adipoq and wt control mice , prolonged fasting - induced increase of ffa was significantly higher in adipoq mice ( supplementary fig . in addition , high - fat diet further increased fasting blood ffa in adipoq mice ( supplementary fig . these results imply that adiponectin might suppress ffa release and prompted us to study the effects of adiponectin on lipolysis in adipocytes . initially , we used the selective 3-adrenergic receptor agonist brl37344 and adipoq mice to carry out an in vivo lipolysis study , in which increases of serum free glycerol were measured to represent hormone - stimulated lipolysis . as expected , brl37344 treatment robustly increased serum free glycerol and ffa levels in all mice . the magnitudes of increase of serum glycerol in adipoq mice were significantly higher than those in wt mice 10 or 20 min after brl37344 injection ( p < 0.05 , fig . second , to avoid influence from other systems or hormones that may be rapidly altered by brl37344 administration , in vitro lipolysis assays were conducted using epididymal adipose explants . results showed that there were no significant differences in glycerol release between adipose explants from adipoq and wt control in a basal condition ( fig . consistent with the in vivo lipolysis study , brl37344-stimulated glycerol releases were remarkably higher in adipose explants from adipoq mice compared with wt control mice ( p < 0.01 , fig . 2b ) . in the third study , lipolysis was assayed in primary mature adipocytes from adipoq and wt control mice . as shown in fig . 2c , basal medium free glycerol levels were similar between adipocytes from wt control and adipoq mice . however , in bt2-camp treated cells , free glycerol levels in the medium of adipoq adipocytes were twofold higher than those of adipocytes from wt mice ( fig . mouse embryonic fibroblast - derived adipocytes from adipoq mice were treated with or without adiponectin using a transwell co - culture system . results showed that camp - stimulated release of glycerol from adiponectin - treated adipocytes was significantly less than from control adipocytes ( supplementary fig . a : 3-adrenergic receptor agonist brl37344 was injected i.p . into 8-week - old male mice , which had been fasted overnight . n = 6 , * p < 0.05 vs. wt at the same time point . b : epididymal adipose explants were incubated in dulbecco s modified eagle s medium with or without brl37344 stimulation . n = 8 , * p < 0.05 vs. wt adipose explants with brl37344 stimulation . c : primary adipocytes were prepared from epididymal fat of adipoq or wt control mice . adipocytes were treated with or without bt2-camp ( 100 mol / l ) for 1 h. glycerol levels in medium were measured . however , there was no significant difference in serum glucagon and insulin concentrations between chow - fed adipoq and wt mice ( data not shown ) , which prompted us to look at the expression and activation state of the main lipases in wat of adipoq mice . hsl and adipose triglyceride lipase ( atgl ) are two major lipases in adipocytes ( 21,22 ) . we examined the expression and activation status of these lipolytic proteins in epididymal adipose tissues from mice with adiponectin gene deletion or overexpression and from cultured adipocytes . for some animal studies , ad - acrp30 was used to reconstitute serum adiponectin in adipoq mice or to elevate serum adiponectin in c57bl/6 mice . as we previously reported ( 18 ) , 3 days after virus injection , total serum adiponectin protein levels were increased 12-fold in c57bl/6 mice , whereas fasting ffa levels were significantly reduced ( supplementary fig . serum multimeric adiponectin levels were proportionally elevated , and there were no significant changes in insulin sensitivity , as measured by an insulin challenge test ( data not shown ) ( 18 ) . 3a , there were no significant differences of total protein levels of hsl , atgl , or cgi-58 in epididymal fat from adipoq , adiponectin - overexpressing , or adiponectin - reconstituted mice compared with their corresponding controls . in addition , adiponectin treatment did not alter protein levels of hsl , atgl , or cgi-58 in 3t3-l1 adipocytes ( fig . these results suggest that adiponectin - inhibited lipolysis is not through altering hsl , atgl , or cgi-58 protein levels in adipocytes . a : protein samples were prepared from epididymal fat of mice with indicated genotypes or treatment . relative protein levels of hsl , atgl , and cgi-58 , and serine phosphorylation levels of hsl were measured by western blot using specific antibodies . for adiponectin overexpression or reconstitution , ad - acrp30 or ad - gfp adenovirus b : mature 3t3-l1 adipocytes were co - cultured overnight with fao cells transduced with ad - acrp30 or ad - gfp . the insert wells with fao cells were removed , and adipocytes were treated with bt2-camp for 1 h. n = 6 . c : differentiated 3t3-l1 adipocytes were treated with cc ( 10 mol / l ) and co - cultured with ad - acrp30 or ad - gfp transduced fao cells overnight . d : perilipin a protein levels in epididymal fat from adipoq and wt mice were measured using western blotting . e : for measuring pka - phosphorylated perilipin a , perilipin a proteins were immunoprecipitated and then probed with an antibody against phospho - pka substrate . f : after overnight co - culture with adiponectin secreting fao cells and wortmannin ( 200 nmol / l ) , 3t3-l1 adipocytes were stimulated with bt2-camp and insulin ( 25 nmol / l ) for 1 h. glycerol in medium was measured . hsl is the first lipase that was found to respond to lipolytic hormones and hydrolyze triglycerides in adipocytes . pka plays an essential role in mediating hormone - stimulated hsl activation and lipolysis in adipocytes . however , only ser660 phosphorylation activates hsl , whereas ser563 phosphorylation does not correlate with hsl activation ( 24 ) . by using phospho - hsl specific antibodies , our study showed that the levels of hsl ser660 phosphorylation in epididymal fat from adipoq mice were strikingly elevated compared with those in wt control mice ( p < 0.001 , fig . in contrast , phosphorylation of hsl ser660 was significantly suppressed in epididymal fat of the mice with adenovirus - mediated adiponectin overexpression and adiponectin reconstitution ( p < 0.05 , fig . 3a ) , whereas a significantly reduced brl - stimulated glycerol release was observed in epididymal adipose explants of mice with adiponectin overexpression ( supplementary fig . 5a ) . in addition , significantly increased hsl ser660 phosphorylation was observed in the epididymal fat of adipoq mice fed a high - fat diet ( supplementary fig . these data indicate that hsl activity in wat is increased in adipoq mice and suppressed in mice with elevated adiponectin . by using 3t3-l1 adipocytes and co - culture system , our study further revealed that overnight adiponectin treatment significantly decreased both basal and camp - stimulated hsl ser660 phosphorylation ( p < 0.05 , fig . 3b ) . together , these results demonstrate that adiponectin suppresses hsl activation by reducing ser660 phosphorylation . it has been reported that ser565 in hsl can be phosphorylated by amp - activated protein kinase ( ampk ) , resulting in deactivation of hsl ( 21,25 ) . we postulated that adiponectin might increase hsl ser565 phosphorylation via ampk to inhibit hsl . to our surprise , hsl ser565 phosphorylation levels neither decreased in adipoq mice nor increased in adiponectin - overexpressing or -reconstituted mice compared with their controls ( fig . these results exclude the possibility that adiponectin inhibits hsl activation by increasing hsl ser565 phosphorylation . to further determine the role of ampk in adiponectin - suppressed lipolysis however , cc treatment did not abolish the inhibitory effects of adiponectin on glycerol release ( fig . similar results were obtained in 3t3-l1car1 adipocytes transduced with adenovirus encoding dominant negative ampk ( data did shown ) . these results do not support any key role of ampk in adiponectin - suppressed lipolysis . however , our study does not completely exclude the involvement of ampk in adiponectin - suppressed lipolysis . apparently , the underlying mechanisms through which ampk modulates lipolysis are far more complicated than a single serine site phosphorylation in hsl . studies have shown that pka or lipolysis itself induces ampk activation in adipocytes ( 26,27 ) . furthermore , a recent study reported that ampk not only suppresses but also increases lipolysis and that the effects are dependent on duration of ampk activation ( 28 ) . perilipin a is a lipid droplet - binding protein that plays an important role in lipolysis in adipocytes ( 29,30 ) . upon lipolytic hormone stimulation , pka - phosphorylated perilipin a recruits hsl to the lipid droplet surface ( 31 ) . our results showed that perilipin a protein levels in epididymal fat of adipoq mice were similar to those of wt control mice ( fig . these results suggest that pka - mediated perilipin a phosphorylation may be not responsible for adiponectin - inhibited lipolysis in adipocytes . adiponectin sensitizes insulin and insulin inhibits lipolysis ( 32 ) . this raises the question whether adiponectin inhibits lipolysis through insulin and whether there is a synergistic effect . we used the insulin signaling blocker wortmannin , which abolishes the inhibitory effects of insulin on lipolysis in adipocytes ( 32 ) . as expected , wortmannin treatment attenuated the inhibitory effects of insulin on glycerol release 3f ) . in addition , adipocytes treated with both adiponectin and insulin did not exhibit a synergistic reduction in glycerol release . adipor1 is ubiquitously expressed and is the main adiponectin receptor in white adipocytes ( 33 ) . to verify whether adiponectin inhibits hsl activation through the adiponectin receptor , adipor1 or sirna oligo against adipor1 was expressed using adenovirus vectors in 3t3-l1car1 adipocytes , which express adenovirus receptor to facilitate adenovirus - mediated gene transduction in mature adipocytes ( 35 ) . phosphorylation levels of hsl ser660 at both basal level and after camp stimulation were significantly reduced in ad - adipor1treated cells but were remarkably increased in adipor1 sirna treated adipocytes ( supplementary fig . the coordinate changes of hsl ser660 phosphorylation in adipoq mice and adiponectin - treated adipocytes suggest that adiponectin might suppress hsl activation in adipocytes through reducing pka activity . with the use of an antibody that recognizes the consensus of phosphorylated pka substrate , our study showed that there are significant increases of pka substrate phosphorylation of proteins in epididymal fat from adipoq mice ( supplementary fig . 7a ) , which further supports the notion that adiponectin inhibits pka activation in adipocytes . however , adiponectin gene deletion has no significant impact on total camp levels in wat ( p > 0.05 , supplementary fig . pka type ii regulatory subunit ( rii ) has two distinct isoforms , rii and rii. rii is ubiquitously expressed , but rii is predominantly expressed in adipose , brain , and adrenal tissues . rii gene knockout mice are lean and resistant to high - fat diet induced obesity ( 36 ) . we measured the gene expression levels of pka catalytic subunits and regulatory subunits ri and rii at both mrna and protein levels in epididymal fat from adipoq and adiponectin - restored mice . there were no significant changes of mrna levels of the two catalytic and four regulatory subunits of pka in adipoq mice ( data not shown ) . protein levels of two catalytic subunits , pka c and c , were also similar between adipoq and wt controls ( fig . 4a ) . rii but not rii protein levels were significantly elevated in epididymal fat of adipoq mice ( p < 0.05 , fig . 4a ) . in contrast , adiponectin reconstitution in adipoq mice remarkably reduced rii in the epididymal fat ( p < 0.05 , fig . a similar inhibitory effect of adiponectin on rii protein level was observed in 3t3-l1 adipocytes ( p < 0.05 , fig . 4b ) . these results led us to hypothesize that reduced rii mediates the inhibitory effect of adiponectin on hsl activation . a : total protein samples were prepared using epididymal fat from adipoq or ad - acrp30treated mice . b : fully differentiated 3t3-l1 adipocytes were co - cultured with ad - acrp30transduced fao cells , which secrete adiponectin . data are expressed as means sem . to verify this hypothesis , we overexpressed rii in differentiated mature adipocytes . the levels of hsl ser660 phosphorylation were significantly increased in ad - pka riitreated 3t3-l1car1 adipocytes ( p < 0.05 , fig . overexpression of rii even slightly decreased hsl ser660 phosphorylation without statistical significance ( p > 0.05 , supplementary fig . most important , overexpression of rii abolished adiponectin - induced inhibition of hsl ser660 phosphorylation in 3t3-l1car1 adipocytes ( fig . 5a ) . furthermore , knocking down rii in 3t3-l1car1 adipocytes significantly reduced both basal and camp - stimulated hsl ser660 phosphorylation ( fig . these results indicate that adiponectin suppresses hsl activation by reducing rii. pka rii overexpression attenuates the inhibitory effects of adiponectin on hsl ser660 phosphorylation . adenovirus vectors encoding gfp , pka rii ( a ) , or sirna against pka rii ( b ) were added to the medium . a : cells were co - cultured 12 h later with ad - acrp30transduced fao cells , which secrete adiponectin . b : some cells were treated with bt2-camp for 1 h. protein samples were prepared , and the phosphorylation levels of hsl ser660 and other proteins were detected using specific antibodies . * p < 0.05 vs. ad - lacz sirna treated cells at basal state . the above studies show that adiponectin reduces rii protein levels without altering rii mrna level . therefore , we compared the protein half - life of rii between 3t3-l1 adipocytes treated with or without adiponectin . as shown in fig . rii protein levels were reduced by 30% after just 1 h of adiponectin treatment in the presence of cycloheximide ( fig . cycloheximide ( 100 g / ml ) was added to the medium , and the insert wells with fao cells were removed . b : 3t3-l1 adipocytes were treated with adiponectin - enriched conditioned medium from ad - acrp30transduced fao cells . c : proteasome inhibitor mg132 ( 25 mol / l ) was added to the medium 30 min before adiponectin treatment . d : mature 3t3-l1 adipocytes were co - cultured with adiponectin - expressing fao cells for 4 h. the pierce crosslink ip kit ( thermo scientific pierce protein research products , rockford , il ) was used for immunoprecipitation to eliminate nonspecific bands . * p < 0.05 vs. control cells at the same time point ( b ) or without mg132 treatment ( c ) . to further investigate how adiponectin reduces rii , the proteasome - specific inhibitor mg-132 was added into medium 30 min before adiponectin treatment . 6c ) , which indicates that adiponectin enhances rii protein degradation through the ubiquitin - proteasome system . then , we measured ubiquitin levels of immunoprecipated rii protein from adiponectin - treated and control 3t3-l1 adipocytes . 6d shows , the levels of ubiquitinated rii protein were significantly elevated in adiponectin - treated 3t3-l1 adipocytes . together , these results indicate that adiponectin reduces rii protein levels by enhancing ubiquitin - proteasome mediated protein degradation . it should be noted that overall protein ubiquitin levels were not clearly increased in adiponectin - treated cells ( supplementary fig . similar effects of adiponectin on overall protein ubiquitination have also been observed in adiponectin - treated c2c12 myotubes ( data not shown ) . these results suggest that adiponectin - enhanced rii ubiquitination and protein degradation occur in a protein - specific manner . to study the effects of adiponectin on lipid metabolism in adipocytes , adipoq mice were used . consistent with previous studies ( 5,15 ) , there were no significant differences of body weights between adipoq and wild - type ( wt ) littermate controls when the mice were fed normal chow ( data not shown ) . however , body fat percentage of adipoq mice was significantly lower than that of wt mice ( fig . epididymal fat mass was also significantly smaller in adipoq male mice ( 0.26 0.02 vs. 0.42 0.04 g ; vs. wt , p < 0.05 , n = 10 ) . adipocyte cross - sectional areas of fixed epididymal fat of adipoq mice were 20% smaller than those of wt control mice ( supplementary fig . when mice were fed a 60% ( calorie ) high - fat diet for 8 weeks , adipoq mice gained remarkably less body weight and adipose tissue mass and had smaller adipocyte area ( fig . similar to previous studies ( 5,16 ) , high - fat diet induced more severe insulin resistance in adipoq mice ( data not shown ) . therefore , these studies clearly show that adipoq mice have less fat mass and are resistant to high - fat diet induced obesity . similar phenotypes have been reported by a group using an independent line of adipoq mice ( 17 ) . adiponectin knockout mice have less body fat and are resistant to high - fat diet induced obesity . body composition of chow - fed 2-month - old male adipoq and wt mice was scanned using echomri ( a ) . then the mice were fed a high - fat diet ( 60% of calories from fat ) . body fat was measured after 8 weeks of high - fat feeding ( c ) . our study shows that neither adiponectin gene knockout nor overexpression has any significant effect on gene expression of the key enzymes of triglyceride synthesis in epididymal fat ( supplementary fig . 2 ) , which suggests that adiponectin may regulate lipid metabolism in adipocytes through a pathway other than de novo lipogenesis . previous animal studies have clearly demonstrated that adiponectin reduces circulating ffas ( 16,1820 ) . although serum ffa levels at the fed state were similar between chow - fed adipoq and wt control mice , prolonged fasting - induced increase of ffa was significantly higher in adipoq mice ( supplementary fig . in addition , high - fat diet further increased fasting blood ffa in adipoq mice ( supplementary fig . these results imply that adiponectin might suppress ffa release and prompted us to study the effects of adiponectin on lipolysis in adipocytes . initially , we used the selective 3-adrenergic receptor agonist brl37344 and adipoq mice to carry out an in vivo lipolysis study , in which increases of serum free glycerol were measured to represent hormone - stimulated lipolysis . as expected , brl37344 treatment robustly increased serum free glycerol and ffa levels in all mice . the magnitudes of increase of serum glycerol in adipoq mice were significantly higher than those in wt mice 10 or 20 min after brl37344 injection ( p < 0.05 , fig . second , to avoid influence from other systems or hormones that may be rapidly altered by brl37344 administration , in vitro lipolysis assays were conducted using epididymal adipose explants . results showed that there were no significant differences in glycerol release between adipose explants from adipoq and wt control in a basal condition ( fig . consistent with the in vivo lipolysis study , brl37344-stimulated glycerol releases were remarkably higher in adipose explants from adipoq mice compared with wt control mice ( p < 0.01 , fig . 2b ) . in the third study , lipolysis was assayed in primary mature adipocytes from adipoq and wt control mice . as shown in fig . 2c , basal medium free glycerol levels were similar between adipocytes from wt control and adipoq mice . however , in bt2-camp treated cells , free glycerol levels in the medium of adipoq adipocytes were twofold higher than those of adipocytes from wt mice ( fig . 2c ) . to further verify the effects of adiponectin on lipolysis , mouse embryonic fibroblast - derived adipocytes from adipoq mice were treated with or without adiponectin using a transwell co - culture system . results showed that camp - stimulated release of glycerol from adiponectin - treated adipocytes was significantly less than from control adipocytes ( supplementary fig . a : 3-adrenergic receptor agonist brl37344 was injected i.p . into 8-week - old male mice , which had been fasted overnight . n = 6 , * p < 0.05 vs. wt at the same time point . b : epididymal adipose explants were incubated in dulbecco s modified eagle s medium with or without brl37344 stimulation . n = 8 , * p < 0.05 vs. wt adipose explants with brl37344 stimulation . c : primary adipocytes were prepared from epididymal fat of adipoq or wt control mice . however , there was no significant difference in serum glucagon and insulin concentrations between chow - fed adipoq and wt mice ( data not shown ) , which prompted us to look at the expression and activation state of the main lipases in wat of adipoq mice . hsl and adipose triglyceride lipase ( atgl ) are two major lipases in adipocytes ( 21,22 ) . we examined the expression and activation status of these lipolytic proteins in epididymal adipose tissues from mice with adiponectin gene deletion or overexpression and from cultured adipocytes . for some animal studies , ad - acrp30 was used to reconstitute serum adiponectin in adipoq mice or to elevate serum adiponectin in c57bl/6 mice . as we previously reported ( 18 ) , 3 days after virus injection , total serum adiponectin protein levels were increased 12-fold in c57bl/6 mice , whereas fasting ffa levels were significantly reduced ( supplementary fig . serum multimeric adiponectin levels were proportionally elevated , and there were no significant changes in insulin sensitivity , as measured by an insulin challenge test ( data not shown ) ( 18 ) . as shown in fig . 3a , there were no significant differences of total protein levels of hsl , atgl , or cgi-58 in epididymal fat from adipoq , adiponectin - overexpressing , or adiponectin - reconstituted mice compared with their corresponding controls . in addition , adiponectin treatment did not alter protein levels of hsl , atgl , or cgi-58 in 3t3-l1 adipocytes ( fig . these results suggest that adiponectin - inhibited lipolysis is not through altering hsl , atgl , or cgi-58 protein levels in adipocytes . a : protein samples were prepared from epididymal fat of mice with indicated genotypes or treatment . relative protein levels of hsl , atgl , and cgi-58 , and serine phosphorylation levels of hsl were measured by western blot using specific antibodies . for adiponectin overexpression or reconstitution , ad - acrp30 or ad - gfp adenovirus b : mature 3t3-l1 adipocytes were co - cultured overnight with fao cells transduced with ad - acrp30 or ad - gfp . the insert wells with fao cells were removed , and adipocytes were treated with bt2-camp for 1 h. n = 6 . c : differentiated 3t3-l1 adipocytes were treated with cc ( 10 mol / l ) and co - cultured with ad - acrp30 or ad - gfp transduced fao cells overnight . d : perilipin a protein levels in epididymal fat from adipoq and wt mice were measured using western blotting . e : for measuring pka - phosphorylated perilipin a , perilipin a proteins were immunoprecipitated and then probed with an antibody against phospho - pka substrate . f : after overnight co - culture with adiponectin secreting fao cells and wortmannin ( 200 nmol / l ) , 3t3-l1 adipocytes were stimulated with bt2-camp and insulin ( 25 hsl is the first lipase that was found to respond to lipolytic hormones and hydrolyze triglycerides in adipocytes . pka plays an essential role in mediating hormone - stimulated hsl activation and lipolysis in adipocytes . however , only ser660 phosphorylation activates hsl , whereas ser563 phosphorylation does not correlate with hsl activation ( 24 ) . by using phospho - hsl specific antibodies , our study showed that the levels of hsl ser660 phosphorylation in epididymal fat from adipoq mice were strikingly elevated compared with those in wt control mice ( p < 0.001 , fig . 3a , bottom quantified graphs ) . in contrast , phosphorylation of hsl ser660 was significantly suppressed in epididymal fat of the mice with adenovirus - mediated adiponectin overexpression and adiponectin reconstitution ( p < 0.05 , fig . 3a ) , whereas a significantly reduced brl - stimulated glycerol release was observed in epididymal adipose explants of mice with adiponectin overexpression ( supplementary fig . 5a ) . in addition , significantly increased hsl ser660 phosphorylation was observed in the epididymal fat of adipoq mice fed a high - fat diet ( supplementary fig . these data indicate that hsl activity in wat is increased in adipoq mice and suppressed in mice with elevated adiponectin . by using 3t3-l1 adipocytes and co - culture system , our study further revealed that overnight adiponectin treatment significantly decreased both basal and camp - stimulated hsl ser660 phosphorylation ( p < 0.05 , fig . 3b ) . together , these results demonstrate that adiponectin suppresses hsl activation by reducing ser660 phosphorylation . it has been reported that ser565 in hsl can be phosphorylated by amp - activated protein kinase ( ampk ) , resulting in deactivation of hsl ( 21,25 ) . to our surprise , hsl ser565 phosphorylation levels neither decreased in adipoq mice nor increased in adiponectin - overexpressing or -reconstituted mice compared with their controls ( fig . these results exclude the possibility that adiponectin inhibits hsl activation by increasing hsl ser565 phosphorylation . to further determine the role of ampk in adiponectin - suppressed lipolysis , selective ampk inhibitor compound c ( cc ) was used . however , cc treatment did not abolish the inhibitory effects of adiponectin on glycerol release ( fig . similar results were obtained in 3t3-l1car1 adipocytes transduced with adenovirus encoding dominant negative ampk ( data did shown ) . these results do not support any key role of ampk in adiponectin - suppressed lipolysis . however , our study does not completely exclude the involvement of ampk in adiponectin - suppressed lipolysis . apparently , the underlying mechanisms through which ampk modulates lipolysis are far more complicated than a single serine site phosphorylation in hsl . studies have shown that pka or lipolysis itself induces ampk activation in adipocytes ( 26,27 ) . furthermore , a recent study reported that ampk not only suppresses but also increases lipolysis and that the effects are dependent on duration of ampk activation ( 28 ) . perilipin a is a lipid droplet - binding protein that plays an important role in lipolysis in adipocytes ( 29,30 ) . upon lipolytic hormone stimulation , pka - phosphorylated perilipin a recruits hsl to the lipid droplet surface ( 31 ) . our results showed that perilipin a protein levels in epididymal fat of adipoq mice were similar to those of wt control mice ( fig . these results suggest that pka - mediated perilipin a phosphorylation may be not responsible for adiponectin - inhibited lipolysis in adipocytes . adiponectin sensitizes insulin and insulin inhibits lipolysis ( 32 ) . this raises the question whether adiponectin inhibits lipolysis through insulin and whether there is a synergistic effect . we used the insulin signaling blocker wortmannin , which abolishes the inhibitory effects of insulin on lipolysis in adipocytes ( 32 ) . 3f ) . in addition , adipocytes treated with both adiponectin and insulin did not exhibit a synergistic reduction in glycerol release . adipor1 is ubiquitously expressed and is the main adiponectin receptor in white adipocytes ( 33 ) . to verify whether adiponectin inhibits hsl activation through the adiponectin receptor , adipor1 or sirna oligo against adipor1 was expressed using adenovirus vectors in 3t3-l1car1 adipocytes , which express adenovirus receptor to facilitate adenovirus - mediated gene transduction in mature adipocytes ( 35 ) . phosphorylation levels of hsl ser660 at both basal level and after camp stimulation were significantly reduced in ad - adipor1treated cells but were remarkably increased in adipor1 sirna treated adipocytes ( supplementary fig . the coordinate changes of hsl ser660 phosphorylation in adipoq mice and adiponectin - treated adipocytes suggest that adiponectin might suppress hsl activation in adipocytes through reducing pka activity . with the use of an antibody that recognizes the consensus of phosphorylated pka substrate , our study showed that there are significant increases of pka substrate phosphorylation of proteins in epididymal fat from adipoq mice ( supplementary fig . 7a ) , which further supports the notion that adiponectin inhibits pka activation in adipocytes . however , adiponectin gene deletion has no significant impact on total camp levels in wat ( p > 0.05 , supplementary fig . pka type ii regulatory subunit ( rii ) has two distinct isoforms , rii and rii. rii is ubiquitously expressed , but rii is predominantly expressed in adipose , brain , and adrenal tissues . rii gene knockout mice are lean and resistant to high - fat diet induced obesity ( 36 ) . we measured the gene expression levels of pka catalytic subunits and regulatory subunits ri and rii at both mrna and protein levels in epididymal fat from adipoq and adiponectin - restored mice . there were no significant changes of mrna levels of the two catalytic and four regulatory subunits of pka in adipoq mice ( data not shown ) . protein levels of two catalytic subunits , pka c and c , were also similar between adipoq and wt controls ( fig . 4a ) . rii but not rii protein levels were significantly elevated in epididymal fat of adipoq mice ( p < 0.05 , fig . 4a ) . in contrast , adiponectin reconstitution in adipoq mice remarkably reduced rii in the epididymal fat ( p < 0.05 , fig . a similar inhibitory effect of adiponectin on rii protein level was observed in 3t3-l1 adipocytes ( p < 0.05 , fig . 4b ) . these results led us to hypothesize that reduced rii mediates the inhibitory effect of adiponectin on hsl activation . a : total protein samples were prepared using epididymal fat from adipoq or ad - acrp30treated mice . b : fully differentiated 3t3-l1 adipocytes were co - cultured with ad - acrp30transduced fao cells , which secrete adiponectin . data are expressed as means sem . to verify this hypothesis , we overexpressed rii in differentiated mature adipocytes . the levels of hsl ser660 phosphorylation were significantly increased in ad - pka riitreated 3t3-l1car1 adipocytes ( p < 0.05 , fig . overexpression of rii even slightly decreased hsl ser660 phosphorylation without statistical significance ( p > 0.05 , supplementary fig . most important , overexpression of rii abolished adiponectin - induced inhibition of hsl ser660 phosphorylation in 3t3-l1car1 adipocytes ( fig . knocking down rii in 3t3-l1car1 adipocytes significantly reduced both basal and camp - stimulated hsl ser660 phosphorylation ( fig . these results indicate that adiponectin suppresses hsl activation by reducing rii. pka rii overexpression attenuates the inhibitory effects of adiponectin on hsl ser660 phosphorylation . adenovirus vectors encoding gfp , pka rii ( a ) , or sirna against pka rii ( b ) were added to the medium . a : cells were co - cultured 12 h later with ad - acrp30transduced fao cells , which secrete adiponectin . b : some cells were treated with bt2-camp for 1 h. protein samples were prepared , and the phosphorylation levels of hsl ser660 and other proteins were detected using specific antibodies . * p < 0.05 vs. ad - lacz sirna treated cells at basal state . the above studies show that adiponectin reduces rii protein levels without altering rii mrna level . therefore , we compared the protein half - life of rii between 3t3-l1 adipocytes treated with or without adiponectin . as shown in fig . rii protein levels were reduced by 30% after just 1 h of adiponectin treatment in the presence of cycloheximide ( fig . cycloheximide ( 100 g / ml ) was added to the medium , and the insert wells with fao cells were removed . b : 3t3-l1 adipocytes were treated with adiponectin - enriched conditioned medium from ad - acrp30transduced fao cells . c : proteasome inhibitor mg132 ( 25 mol / l ) was added to the medium 30 min before adiponectin treatment . d : mature 3t3-l1 adipocytes were co - cultured with adiponectin - expressing fao cells for 4 h. the pierce crosslink ip kit ( thermo scientific pierce protein research products , rockford , il ) was used for immunoprecipitation to eliminate nonspecific bands . * p < 0.05 vs. control cells at the same time point ( b ) or without mg132 treatment ( c ) . the ubiquitin - dependent proteasome is the most common system for protein degradation . to further investigate how adiponectin reduces rii , the proteasome - specific inhibitor mg-132 was added into medium 30 min before adiponectin treatment . 6c ) , which indicates that adiponectin enhances rii protein degradation through the ubiquitin - proteasome system . then , we measured ubiquitin levels of immunoprecipated rii protein from adiponectin - treated and control 3t3-l1 adipocytes . 6d shows , the levels of ubiquitinated rii protein were significantly elevated in adiponectin - treated 3t3-l1 adipocytes . together , these results indicate that adiponectin reduces rii protein levels by enhancing ubiquitin - proteasome mediated protein degradation . it should be noted that overall protein ubiquitin levels were not clearly increased in adiponectin - treated cells ( supplementary fig similar effects of adiponectin on overall protein ubiquitination have also been observed in adiponectin - treated c2c12 myotubes ( data not shown ) . these results suggest that adiponectin - enhanced rii ubiquitination and protein degradation occur in a protein - specific manner . adipose tissue is the main energy depot in humans and some animals . during fasting and exercise , triglycerides are hydrolyzed to release ffas , which are oxidized by skeletal muscle , heart , and other tissues as fuel . with the discovery of adipocyte - derived hormones , the regulatory role of adipose - derived hormones on lipid metabolism has emerged as an important system in maintaining energy homeostasis . our current study provides strong evidence demonstrating that adiponectin suppresses hormone - stimulated lipolysis in mouse adipocytes . during the revision of this article , the same effects of adiponectin on lipolysis were reported in human adipocytes ( 37 ) . therefore , we propose that adiponectin plays an important role in maintaining energy homeostasis by suppressing lipolysis in wat . it has been reported that leptin , another adipose - derived hormone , directly increases lipolysis and enhances lipid depletion from adipocytes ( 38,39 ) . although it is uncertain whether leptin and adiponectin regulate lipolysis through the same pathway , these studies suggest that adiponectin and leptin exert opposite regulatory effects on lipolysis in adipocytes . in addition , the results from cultured adipocytes clearly indicate that adiponectin suppresses lipolysis through an autocrine loop . the discovery of the inhibitory effects of adiponectin on lipolysis will help us to understand how adiponectin improves circulating lipid profiles . however , it is premature to claim the importance of adiponectin - inhibited lipolysis in the development of obesity . there is a decrease of adiponectin gene expression in obesity ( 2 ) , which should attenuate the inhibitory effect of adiponectin on lipolysis , resulting in less lipid storage in fat . thus , obesity - associated low adiponectin expression may be beneficial in the context of obesity . in addition , despite lipolytic defects that have been observed in obese human and animal models ( 10,40 ) , the role of lipolytic defects in obesity is still uncertain . therefore , more studies are required to elucidate the role of adiponectin - suppressed lipolysis in the development of obesity and in energy metabolism under physiologic conditions . plasma ffa inversely associates with insulin sensitivity , especially in obese subjects ( 41,42 ) . our current study demonstrates that adiponectin inhibits lipolysis in wat , which should decrease the level of circulating ffa . therefore , we postulate that inhibition of lipolysis and reduction of ffa is a mechanism through which adiponectin enhances insulin sensitivity . on the other hand , the insulin - sensitizing effect of adiponectin however , our study demonstrated that insulin signaling is not responsible for adiponectin - inhibited lipolysis in adipocytes . furthermore , although high - fat feeding induces more severe insulin resistance in adipoq mice compared with wt control mice ( 16 ) , the inhibitory effects of adiponectin on lipolysis are still well preserved . therefore , these studies indicate that adiponectin inhibits hormone - stimulated adipocyte lipolysis through a mechanism(s ) other than insulin sensitizing . the essential role of pka in mediating hormone - stimulated lipolysis in adipocytes has been well established . it has been reported that rii gene knockout mice are lean and resistant to high - fat diet induced obesity because of increased locomotor activity , energy expenditure , and lipolysis in wat ( 4345 ) . our study showed that adiponectin has no effect on rii expression and that rii overexpression even slightly reduces hsl ser660 phosphorylation in adipocytes . the current study does not provide any evidence to support the involvement of rii in adiponectin - inhibited lipolysis in adipocytes . in contrast , we found that the protein level of rii was elevated in adiponectin gene knockout mice . ectopic expression of rii abolished the inhibitory effects of adiponectin on hsl ser660 phosphorylation and lipolysis . conversely , knocking down rii reduced both basal and camp - stimulated hsl ser660 phosphorylation in adipocytes . these results led us to conclude that reduced rii mediates the inhibitory effects of adiponectin on lipolysis in adipocytes . it should be pointed out that by comparing the pka substrate phosphorylation profile and pka protein levels between adipoq and wt mice in different tissues ( data not shown ) , we found that the effects of adiponectin on pka subunit expression and pka activation occur in a tissue - specific manner . the mechanism by which rii mediates the inhibitory effect of adiponectin on lipolysis remains unknown . the large number of substrates and diverse downstream signaling pathways of pka , precise localization of pka to its specific substrate by a kinase - anchoring proteins ( akaps ) has been demonstrated to be an important mechanism to restrict pka action ( 46 ) . recent studies revealed that rii binds tightly with some akaps and plays an important role in compartmentalizing camp / pka signal transduction ( 4648 ) . we postulate that adiponectin suppresses rii expression , which may shift akaps / pka away from the compartment where hsl localizes , resulting in decreased hsl phosphorylation and activation . the absence of effect of adiponectin on perilipin a phosphorylation also supports this postulation . in conclusion , our study demonstrates that adiponectin inhibits lipolysis and directly regulates lipid metabolism in adipocytes . our data also suggest that adiponectin suppresses triglyceride hydrolysis by inhibiting pka - induced hsl activation .

this paper outlines botxminer , a publicly available application to search xml - formatted medline data in a complete , object - relational schema implemented in oracle xml db . an advantage offered by botxminer is that it can generate quantitative results with certain queries that are not feasible through the entrez - pubmed interface . after retrieving citations associated with user - supplied search terms , medline fields ( title , abstract , journal , mesh and chemical ) and terms ( mesh qualifiers and descriptors , keywords , author , gene symbol and chemical ) , these citations are grouped and displayed as tabulated or graphic results . this work represents an extension of previous research for integrating these citations with relational systems . botxminer has a user - friendly , intuitive interface that can be freely accessed at .

the national library of medicine 's ( nlm ) medline / pubmed biomedical literature database ( ) is rapidly expanding and as of the 2006 edition , the baseline database contains more than 15 million citations . a number of papers have introduced tools to perform searches more advanced than those available with the current entrez - pubmed user interface to mine the abstracts ( 14 ) . to have more control in conducting searches , investigators have also started to reorganize the medline data into structured relational systems ( 5 ) or to exploit different data formats and create new databases ( ) . these nlm citations are available in several formats ( e.g. medline , asn.1 , and so on ) . importing these citation data into a traditional , relational database limits the query capability to sql ( structured query language ) techniques . the extensible markup language ( xml ) format , however , is emerging as a de facto standard for the hierarchical organization of data in a highly granular structure , and provides a much richer query capability beyond what is available through traditional sql . oracle xml db , available in oracle 9i and 10 g , recognizes this new format . it supports both the necessary massive storage requirements and the extensions to traditional sql that enable a different kind of query which is hierarchical and highly granular . an early study using oracle xml db involved the evaluation of data - form standards and database technologies for medical informatics systems ( 6 ) ( ) . as described by wang et al . ( 6 ) , the set of utilities associated with xml db permits data to be represented both as xml elements from xml documents and as cells within relational tables . a recently developed website that includes a specialized , oracle - based resource for the clostridial neurotoxins , botdb ( 7 ) ( ) , has been extended to conduct literature searches of medline / pubmed using botxminer . the application of xml db for botxminer was based , in large measure , on the documentation and thorough analysis of an original implementation using xml - formatted medline citations ( ) . an advantage offered by botxminer over pubmed is that it can make certain queries possible that can not be performed using the entrez - pubmed interface . the practical example herein illustrates how citations , containing user - supplied search terms , selected medline search fields and terms , are grouped and displayed as hyperlinked tabulated or graphic results . a more detailed technical description of the architecture associated with botxminer and of the more than 15 million medline xml records created through the 2005 production year that were loaded into oracle xml db , a limited reference subset was generated for botxminer using the search words tetanus. this subset has 26 563 citations , a value that represents < 1% of the total number that are available . the search option ( data not shown ) allows the user to query the standard fields of a medline output [ author , terms in the title or abstract , pubmed identification number ( pmid ) and so on ] by entering desired words or terms in the appropriate boxes and by selecting an optional range of publication years . initially , the user - provided search words or terms are searched for within each medline xml file . these results are then grouped by botxminer with terms contained in a select list of medline fields : the medical subject headings ( mesh ) qualifier or descriptor names , author , chemical , keyword and gene symbol . advanced search feature is also available that allows the user to search the common medline fields of journal title , mesh and chemical in addition to the title and abstract . the option to specify a range of publication years is also applicable to the group articles and the advanced search. queries may contain common logical operators for literature searches that are supported by oracle text : and ( & ) , not ( ) , or ( ) and accum ( , ) ( meaning either term is acceptable but both are preferred ) . other operators use context grammar : near ( ;) for proximity searches , and minus ( ) for a lower preference . output graphics can presently be viewed in png , html or svg formats and have so far been tested using microsoft internet explorer ( ie ) v.6 , netscape v.8 and the mozilla firefox v.1 browsers . the html format offers the advantage of being able to hyperlink the graphic points ( rectangles and circles ) to the citation summary information . label information is presented as a pop - up label when moving the mouse over the rectangles and circles . for the svg option , an interactive graphic view of the results is dynamically produced using the aisee graphics package ( ) . the svg format also has interactive hyperlinks while its associated plug - in ( available for ie and netscape ) provides the user with a drop - down feature menu to zoom in and zoom out , pan across the image , save the image , and other graph features . in the svg format under ie , positioning the mouse over the rectangles and circles produces the label information within the window status bar. at present , the firefox browser does support the svg format by using the aisee software but does not support the drop - down feature menu . for these reasons , it is recommended that , at this time , the botxminer graphics be viewed in the svg format using either the ie or the netscape browsers . as a practical example , a search was conducted to find potential interactions between the snare proteins ( three of which are substrates for the proteolytic botulinum neurotoxins ) and other proteins . the substrates of interest [ syntaxin-1a , synaptobrevin ( vamp ) and snap-25 ] are intimately involved in the evoked release of neurotransmitter from synaptic vesicles . protein interactions may , thus , help to predict what other cellular functions might be affected in addition to the toxin - induced blockade of neurotransmission . group articles advanced search was used with the query : near ( ( snare , interact% ) , 5 ) ( figure 1 , back panel ) . snare and interact% ( or interacts , interacting , etc . ) that are separated by no more than five words . this query resulted in a tabulated listing of 83 group ( chemical ) terms that appeared in 18 medline xml files ( figure 1 , middle panel ) . 18 produces a list of all the retrieved citations ( figure 1 , front panel ) . values for the number of articles for a given term are hyperlinked to a second table that lists citations associated with the selected term ( data not shown ) . the pmid numbers are hyperlinked to their corresponding medline pages , which are presented in a modified format , while article titles in this table are hyperlinked to their pubmed abstracts ( data not shown ) . another way to visualize these data is in a network graph format ( figure 2 , upper panel ) with a method analogous to that used in chilibot ( 2 ) and pubnet ( 3 ) . before a graph , there are 83 group terms ( chemical names ) and 859 connections ( circles ) between these terms . all pairs of chemical terms are connected by color - coded lines and circles that show the relative number of articles for two co - occurring chemical terms . each rectangle is hyperlinked to a list of citations containing the group ( chemical ) term , whereas each circle is hyperlinked to a list of citations that contain the pair of terms associated with that connection ( data not shown ) . these graphical views also allow the user to quickly focus on and recognize patterns for the most frequent co - occurring terms . in this example , the cluster of terms near the top of the graph is examined more closely with the zoom in option ( accessed through the drop - down menu with the right - hand mouse button ) that is used with svg ( figure 2 , lower panel ) . in this panel , the pairs are only associated with a single paper ( gray lines and circles ) . protein interactions between the snares and other proteins including calmodulin , neurotransporters and other synaptic proteins were readily identified using this approach . one goal in the development of botxminer was to mine the maximum amount of information from the medline xml files without doing any natural language processing . this development began with l. c. wang 's original documentation ( ) that described how the whole collection of medline data can be stored inside oracle xml db in object - relational tables . since entire medline xml files are stored in this database , the structure of these files is maintained , in contrast to writing new code to parse the data and to subsequently store them into relational tables . the schema - based , structured type data storage strategy is made more efficient in botxminer with oracle 's text indexing capability which makes proximity searches with operators such as near , a context grammar operator , and wildcard searches feasible . the group articles feature of botxminer allows the user to look at a single , tabulated list of relevant grouped terms and to quickly retrieve citations of interest . finally , botxminer has the first user interface for medline xml files that are stored in oracle xml db . in comparison to other publicly available pubmed search applications , the differences exhibited by botxminer provide an opportunity for its descendants to enhance searches of the continually growing biomedical literature . in the tools described by oliver et al . ( 5 ) , medline xml files that have been downloaded are subsequently parsed and stored in schema - defined relational tables . devising appropriate schema to optimally in contrast , pubfinder ( 4 ) stores abstracts in its database and creates a reference dictionary of commonly used words . the user needs to provide pmids of abstracts of interest which are processed for word frequencies and compared to the reference dictionary . applications , such as chilibot ( 1 ) , pubgene ( 8) and medminer ( 9 ) , require , as a minimal user - supplied input , a recognized gene name to conduct its pubmed search . textpresso ( 10 ) , as part of its knowledge retrieval strategy , uses a text - to - xml converter to systematically mark up sentences by its specialized ontology ( that is presently based on caenorhabditis elegans ) from full - length , searchable articles . the example query provided here illustrates the power of botxminer to help the user to efficiently and selectively search for co - occurrences of grouped terms from medline xml files and to focus on the general problem of protein protein interactions . since we have created a database from the entire set of medline xml files , it is anticipated that a variety of other , specialized citation subsets ( e.g. cytoskeletal proteins , signal transduction pathways and diseases such as cancer ) will be developed that are similar in design to botxminer . it is further anticipated that botxminer will serve as a template for future applications that will mine the entire medline and other very large sources of biomedical information . literature search conducted in botdb using botxminer and the group articles ( advanced search option ) . the end - user types in a desired word or term ( words separated by logical or other operators ) and selects one or more of the medline search fields and one of the six presently available terms . middle panel : tabulated results are shown for the query near ( ( snare , interact% ) , 5) , the the resulting list of chemical terms are tallied in the form of an alphabetically sorted table that can be expanded by clicking on a number . 18 associated with the total number of articles ( middle panel ) is hyperlinked to these citations . graphical view link ( figure 1 , middle panel ) produces a dynamic network graph ( upper panel ) that shows the interconnectedness of the chemical terms which , in turn , are linked to references ( color - coded lines ) with the number of co - occurrences labeled in the small circles . this graph displays all the terms from the table ( figure 1 , middle panel ) that are connected by the number of articles in which they co - occur . the lines and circles are color coded depending on the number of articles : gray , the terms co - occur in 1 article ; yellow , co - occurrence in 25 articles ; pink , co - occurrence in 610 articles ; green , co - occurrence in > 10 articles .

lymphangioma circumscriptum ( lc ) , a hamartomatous lymphatic malformation , is a therapeutic challenge for the dermatologist . various modalities like surgical excision , lasers , and sclerotherapy have been used in the past to treat this notorious skin condition . we report the efficacy of a radiofrequency ablation in a patient with lc . the treatment efficacy of radiofrequency was satisfactory in our patient with no recurrence during 1 year follow - up period . the radiofrequency technique is a safe and economic treatment for management of lc .

lymphangioma circumscriptum ( lc ) or microcystic lymphatic malformation is a hamartomatous malformation of the lymphatic channels of the skin . treatment of lc is difficult because of the persistent nature of the disease due to deeper subcutaneous cisternal connections . we report here a case of lc treated effectively with radiofrequency ablation without any recurrence . a 16-year - old male with fitzpatrick skin type v presented to our clinic with partially fluid - filled lesions on left upper arm with slight local swelling . the patient had these lesions since the age of 2 years and had been repeatedly treated with antibiotics for local infection . examination showed a well - defined plaque on left upper arm with grouped vesicular lesions , giving the typical appearance of a a biopsy was taken from the edge of the lesion and histopathology showed multiple thin - walled , dilated lymphatic spaces in the papillary dermis and these spaces contained lymph and few erythrocytes . the overlying epidermis was thin , and there were elongated rete ridges that appeared to surround the lymphatic channels . the patient was given a systemic antiobiotic , cefadroxil 500 mg twice daily for 5 days , to treat the infection including oozing from the lesions and to decrease the inflammation . radiofrequency ablation was done a week later ( basco radiofrequency device , model rf - b2 , high frequency 2 mhz , power < 150 w ) , in cut and coagulate mode with wire loop electrode , under local anesthesia . the patient was advised to clean the area with betadine lotion and apply topical antibiotic mupirocin on the lesions . three sessions were required at weekly intervals for the lesions to clear as some deeper components were left untreated at the first two sessions . one week after last treatment , the area had partially healed with slight depigmentation . at 1 month , the lesions had completely healed and swelling had subsided [ figure 1 ] . no recurrence has occurred in the past 1 year and the patient is still under surveillance . ( a ) lc lesion on the left upper arm ; ( b ) lesions being treated with radiofrequency ablation ; ( c ) immediate post treatment red erythematous area ; ( d ) complete healing of the lesions at 1 month lc is benign ectasia with two components : the clinically obvious , dermal vesicular component , visible on the skin , and the deeper subcutaneous cisternal element . whimster described the pathogenesis and said that lc arises from the subcutaneous muscle - coated lymphatic cisterns which receive lymphatic flow from the surrounding tissue , but this is not drained to the normal lymphatic system . these dilated cisterns conduct the lymph through communicating channels into the dermal thin lymphatics , which balloon out into the epidermis . treatment modalities include surgical excision , lasers and sclerotherapy with varying success.[35 ] we tried the radiofrequency ablation to treat lc , which produced near - complete clinical ablation with coagulation of lesional and perilesional skin leading to fibrosis of the perivesicular lymphatics . the follow up was longer in our case , i.e. , 1 year and we are encouraged to do a case series to determine the efficacy of radiofrequency in this notorious condition .

purposeto report a case of acute visual loss after endoscopic spinal surgery.methodsa patient was immediately referred to our department after epidural endoscopy with severe bilateral visual impairment and underwent complete ophthalmologic examination.resultsvisual acuity was decreased in both eyes . fundus examination revealed the presence of retinal and vitreous hemorrhages bilaterally . four months later , visual acuity increased and the hemorrhages were remarkably resolved.conclusionthe present case represents an extremely rare incidence of intraocular hemorrhage following epiduroscopy .

a patient was immediately referred to our department after epidural endoscopy with severe bilateral visual impairment and underwent complete ophthalmologic examination . a 45-year old man suffered from low back pain unresponsive to pharmacologic treatment , radiating to the left hip and left lower extremity , due to a back injury 4 years ago . other medical history was noncontributory and he was otherwise healthy without hypertension , diabetes mellitus , cardiovascular disease , or blood clotting abnormalities . the patient underwent an epidural endoscopy and endoscopic adhesiolysis in the level of l4l5 and o5s1 by infusion of 120 cc of normal saline . at the end of the procedure the patient received an epidural injection of 80 mg methylprednisolone . after the operation the general condition of the patient was good without clinical signs of elevated cerebrospinal pressure and blood pressure and heart rate remained stable . however , a few hours later , he became aware of a major decrease of vision in both eyes and was immediately admitted to the department of ophthalmology . at presentation , best corrected visual acuity ( bcva ) was 20/400 re and counting fingers le . the ophthalmic examination revealed the presence of extensive scattered preretinal , subhyaloid , and subretinal hemorrhages in both eyes and especially in the le ( figure 1 ) . ocular history before the operation did not reveal any ocular disease and bcva was 20/20 in both eyes . examination of the fundus showed mild vitreous hemorrhage with extensive intraretinal and subretinal hemorrhages involving the macula especially of the le . four months after the operation , bcva was 20/30 in both eyes . despite the improved vision fundus examination revealed a remarkable resolution of the retinal and subretinal hemorrhages bilaterally with mild pigmentary changes of the macula especially of the le ( figure 2 ) . intraocular hemorrhages often occur in association with acute subarachnoid hemorrhage and secondary aneurysms of the anterior communicating and internal carotid arteries ( terson 1900 ) . the present case represents an extremely rare incidence of intraocular hemorrhage following epiduroscopy and endoscopic adhesiolysis ( amirikia et al 2000 ; brian et al 2005 ) . several mechanisms have been proposed to explain the occurrence of intraocular hemorrhage ( purdy et al 1998 ) . it seems that a sudden increase of the epidural pressure after the epiduroscopy causes cerebrospinal fluid to effuse through the communication of the subarachnoid space within the optic nerve sheath and subsequently compresses the optic nerve and its vasculature . specifically , the retinochoroidal anastomosis and the central retinal vein are occluded resulting in venous stasis allowing extravasation of blood through the vessels causing subhyaloid , retinal , and subretinal hemorrhages . the different types of hemorrhages suggest an acute generalized extravasation of blood within the various layers of the posterior segment opposed to the direct tracking of blood from within the optic nerve sheath . more particularly , intracranial pressure can be directly affected if the dura is punctured and fluid is added to the subarachnoid space . the visual prognosis is considered to be good , with recovery occurring within 6 months ( tabandeh 2000 ) . the present case is rare and it represents a rare complication of a rather common operation . however , as endoscopic spinal procedures become widely established , it is anticipated that similar cases will be recognized more frequently in the future . therefore , surgeons should be aware of the possibility of possible vision loss after this procedure .

the human genome is laden with both non - ltr ( long - terminal repeat ) retrotransposons and microsatellite repeats . both types of sequences are able to , either actively or passively , mutagenize the genomes of human individuals and are therefore poised to dynamically alter the human genomic landscape across generations . non - ltr retrotransposons , such as l1 and alu , are a major source of new microsatellites , which are born both concurrently and subsequently to l1 and alu integration into the genome . likewise , the mutation dynamics of microsatellite repeats have a direct impact on the fitness of their non - ltr retrotransposon parent owing to microsatellite expansion and contraction . this review explores the interactions and dynamics between non - ltr retrotransposons and microsatellites in the context of genomic variation and evolution .

the human genome is laden with repetitive sequences in the form of transposable elements ( tes ) and tandem repeats , which comprise 45% and 3% of the human genome , respectively . using new methods capable of annotating repetitive sequences that have substantially diverged from known te sequences , recent analyses indicate that up to 6669% of the human genome may be composed of repetitive sequences , predominately in the form of tes . tes , as their name implies , are able to move about in the genome and create interspersed repeats . tes include dna transposons , long - terminal repeat ( ltr ) retrotransposons and non - ltr retrotransposons . in this review , we will focus on non - ltr retrotransposons , which are the only active tes in the human genome . non - ltr retrotransposons include long interspersed elements ( lines ) and short interspersed elements ( sines ) . both mobilize via a copy and paste mechanism , which requires the transcription of a donor element into an rna intermediate and the subsequent reverse transcription and incorporation of the rna intermediate into the genome as a new dna copy ( i.e. , the retrotransposition process ) . among them , line-1s ( l1s ) are autonomous ; full - length l1s encode two proteins , orf1 and orf2 , which are essential for their mobilization ( fig . in contrast , sines are non - autonomous and their mobilization relies on l1 proteins . 1b ) and mouse b1 , both of which are derived from 7sl rna ; the second class is typified by mouse b2 elements , which are derived from trnas . the quantity and mobility of non - ltr retrotransposons have made them important drivers of genomic diversity and evolution . ( a ) the locations of mononucleotide microsatellite seed sequences ( i.e. , 57 bp proto - microsatellites ) are diagramed along the length of the consensus human l1 sequence . five proto - microsatellites within the sequence are 7 units long , only one nucleotide below the threshold for mononucleotide microsatellites . a long poly(a ) tail is highlighted at the 3 end of the l1 element ( in bold font ; only 7 a s shown due to space limitation ) . ( b ) the locations of 36 bp mononucleotide proto - microsatellites are diagramed along the length of the consensus alu sequence . unlike l1 , alu is g / c rich and only two poly(a ) proto - microsatellites are found in the middle linker region between left and right monomers . over time a long poly(a ) tail is highlighted at the 3 end of the alu element ( in bold font ; only 7 a s shown due to space limitation ) . in contrast to interspersed repeats , tandem repeats consist of sequences that are sequentially repeated . tandem repeats are classified by the size of their repeating unit : those with a repeating unit length of 16 base pairs ( bp ) are commonly referred to as microsatellites ( fig . however , the boundary between microsatellites and minisatellites is often disputed , where the latter typically refer to tandem repeats of greater unit sizes . as they are highly polymorphic , both microsatellites and minisatellites have been instrumental in genetic mapping , evolutionary and phylogenetic studies as well as dna forensics . this review focuses on microsatellites , which encompass mono- , di- , tri- , tetra- , penta- and hexa - nucleotide repeats . notably , mononucleotide a repeats ( i.e. , poly(a ) dna tracts ) represent the most abundant microsatellite class with an overall length of 12 bp in the human genome . due to the repeating nature of these dna tracts , they are prone to rapid and variable contraction and expansion of repeat length ( fig . ( a ) microsatellite anatomy and terminology . a mononucleotide microsatellite and a trinucleotide microsatellite are depicted . the repeat unit length , the number of repeating units , and the overall length are noted for each microsatellite . both microsatellites have the same number of repeating units , but the unit length and the overall length vary among them . the first involves line / sine retrotransposition ; a microsatellite , typically a mature poly(a ) repeat , is born into the genome concurrently upon line / sine integration . the second pathway involves birth from random sequences ( not shown ) and/or proto - microsatellites via base substitution , indel and replication slippage . when a poly(a ) tract reaches 89 a s , it is considered a mature microsatellite . as the length increases , length contractions outweigh expansions , or vice versa , forming an indefinite loop ( thus , bypassing the path to death ) . the interrupted microsatellites may recover and continue expanding ( not shown ) or may be further mutated and eventually die . collectively , the dynamics of these two entities are a major source of genomic variation and serve as the architectural framework for protein - coding genes . recent work has revealed an intimate relationship between non - ltr retrotransposons and microsatellites . on one hand , non - ltr retrotransposons are a major source of new microsatellites as they give birth to microsatellites both concurrent and subsequent to their integration into the genome . on the other hand , the inherent instability of microsatellite repeats has a direct impact on the fitness of their non - ltr retrotransposon parent . this review will focus on the interactions and mutation dynamics between non - ltr retrotransposons and microsatellites as well as the variability and rates of mutation of microsatellite repeats that make them instigators of genomic evolution . it is proposed that each microsatellite locus possesses a lifecycle that begins with its birth into the genome and ends with its death phase , a period that is punctuated by contractions and expansions , which dynamically shorten and lengthen the overall microsatellite length . the most commonly proposed mechanism for microsatellite contractions and expansions is replication slippage by dna polymerases . two other mutation mechanisms , base substitution and indel slippage , are also thought to play an important role in microsatellite birth and death . one is through de novo retrotransposition by non - ltr retrotransposons , while the other involves the birth of proto - microsatellites from random sequences . it is the goal of this review to integrate these two pathways and highlight the role of non - ltr retrotransposons in microsatellite genesis . non - ltr retrotransposons can give birth to microsatellites concurrently to their integration into the genome ( i.e. , the first pathway ; fig . the association between a - rich microsatellites and non - ltr retrotransposons has been repeatedly observed by many independent studies . a recent analysis of microsatellites at orthologous loci in three primate genomes estimated that upon retrotransposition , alus and l1s contribute to 36% of mono- , di , tri- and tetranucleotide microsatellites in human , chimpanzee and orangutan genomes . this pathway is best illustrated by the birth of poly(a ) microsatellites . to mobilize , non - ltr retrotransposons require a poly(a ) tail at the 3 end of their sequence ( discussed later ) . thus , a common feature of alu and l1 insertions that have integrated into the genome is the presence of a 3 poly(a ) dna tail ( fig . 1 ) . these a - rich sequences are born into the genome as mature microsatellites , far above the threshold size ( fig . 3 ) . in humans , 90% of these poly(a ) repeats are derived from poly(a ) tails of l1 and alu sequences . even this may be an underestimate because l1 can integrate a pure poly(a ) dna tract into the genome if the insertion is severely 5 truncated ; such events would typically be undetected . the abundance of l1 and alu sequences in the primate genomes likely accounts for the overrepresentation of poly(a)/poly(t ) repeats compared with poly(c)/poly(g ) repeats . in contrast , dinucleotide microsatellites are most abundant in rodents , and ac repeats are the most frequent dinucleotide repeat in all vertebrates . not surprisingly , 15% of ac repeats are derived from the ac - rich tails of b2 sine elements found in the mouse genome . non - ltr retrotransposons can give birth to microsatellites both concurrently and subsequently to their integration in the genome . ( a ) concurrent birth occurs via the integration of a long poly(a ) tract , which is part of the alu or l1 element . additionally , the integrated element carries proto - microsatellites , depicted by the middle a stretch . here , the waved line represents genomic dna target , and the gray box represents the body of an alu or l1 element . ( b ) subsequent to integration , both the poly(a ) tail microsatellite and the internal proto - microsatellites may expand or contract due to dna polymerase slippage . ( c ) subsequent to integration , the poly(a ) tail microsatellite may also be mutated via base substitution or indel to form a new repeating unit , here depicted as ta at the end of the poly(a ) tract . the combination of mutational forces may eventually lead to the formation a new dinucleotide microsatellite at the 3 end of the retrotransposon . the second pathway posits that microsatellites initially arise from random sequences via mutation as proto - microsatellites , small tandem repeated sequences that are below the length threshold for mature microsatellites ( fig . theoretically , these initiating loci can be found everywhere in the genome . however , the overwhelming abundance of tes in the human genome made it the perfect hotbed for microsatellite births . it has been suggested that the conversion of aaa to naa is a major mechanism for the generation of all a - rich trinucleotide repeats in the human genome as over 60% of gaa , caa and taa repeats with repeat number of 8 or higher are located within alu poly(a ) tails . furthermore , poly(a ) tails have also been implicated in the birth of dinucleotide and tetranucleotide microsatellites . aside from their 3 tails , both alu and l1 have internal proto - microsatellite sequences that can birth new microsatellites subsequent to their integration ( fig . 1 ; fig . 3 ) . an analysis of microsatellites at orthologous loci in three primate genomes indicates that 26% of microsatellite births and it is noteworthy that microsatellites are found in distinct distributions over the length of l1 and alu elements , likely reflecting differences in their sequence compositions . l1 sequences are at - rich and give rise to microsatellites during and after their insertion along their entire length ( fig . in contrast , alus are gc - rich and microsatellite births and deaths are enriched at the 3 poly(a ) tail and the middle a stretch that connects the left and the right monomers ( fig . the middle a - rich region of alu elements tends to expand and mature into poly(a ) microsatellites and can also give rise to gaa repeats . approximately 2% of gaa repeats with a repeat number of 8 or longer are located in the alu middle a tract ; one of such gaa microsatellite is positioned in intron 1 of the frda gene and its expansion is responsible for the neuromuscular disorder friedreich s ataxia . classic studies on microsatellite mutation rates utilize pedigree analysis , usually in cancer patients with microsatellite instability . several model systems , including e. coli , yeast , human and mouse cells have also been described and used to obtain in vivo mutation rates . in addition , computational and mathematical models have been utilized to describe the behavior of microsatellites by comparing genomic sequences from large scale sequencing efforts and from evolutionarily related species . the majority of computational studies and in vivo studies focus exclusively on pure or perfect repeats , which contain only nucleotides pertaining to the repeating unit . imperfect repeats , in contrast , have additional nucleotides that do not belong to the repeating units . overall , the rate of microsatellite mutation is highly variable , and it is influenced by several factors , including the number of repeating units , unit repeat length , sequence composition , genomic loation and the secondary structures of the repeat . specifically , microsatellites become highly mutable when the number of repeating units exceeds a threshold value . although there is disagreement on the exact threshold value , the consensus is that the threshold represents the size that allows the sequence to acquire slippage mutations at a higher rate than the genomic average . the threshold values for mono- , di- , tri- and tetranucleotide repeats are estimated to be 89 , 45 , 3.44 and 4 respectively . in fact , the distinct mutational behaviors manifested by microsatellite before and after they reach the threshold have prompted some researchers to consider microsatellites only as those that exceed their respective threshold . once a tandem repeat has reached its threshold , experimental studies with human and mouse models have shown that the mutation rate increases exponentially as the number of repeating units increases ( table 1 ) . however , due to the differences in the experimental systems used , it is difficult to compare across these studies . the exponential increase in mutation rates is not seen in mismatch repair deficient cells , indicating that mismatch repair proteins are implicated in the expansion process ( table 1 ) . contraction rate ; number in parenthesis is the contraction rate for the same repeat ; trinucleotide studies focus primarily on mismatch repair deficient cells . trinucleotide repeats that cause disease tend to be more unstable in the germline than in somatic cell lines . sequence composition also affects mutability . for similar sized repeats , ga repeats are more mutable than ca repeats and sequences with higher gc content have lower rates of mutation ( table 1 ) . stability increases with the complexity of the repeating pattern , as demonstrated in tetranucleotide repeat studies . despite a doubling of repeat unit length , di- and tetranucleotide repeats have similar stabilities ( table 1;tc vs. ttcc ) . in contrast , mononucleotide microsatellites , such as those associated with retrotransposons , are highly unstable and hypermutable in the germline . for example , the human bat-40 locus is an intronic poly(a ) tract of 40 a s in the 3--hydroxysteroid dehydrogenase gene ( hsd3b1 ) . . five - fold more mutant alleles are found in sperm than in leukocytes of the same individual . the bat-40 poly(a ) microsatellite has not been identified to be associated with non - ltr retrotransposons ; here , we report that it is in fact the poly(a ) tail of an alujb element . despite experimental focus on the expansion dynamics of microsatellites , the mutation dynamics for very long ( > 50 bp ) mononucleotide repeats had been frequently overlooked because of their rarity in the genome . using a transgenic l1 mouse model , we characterized the contraction dynamics of long poly(a ) tracts associated with nascent l1 insertions . these poly(a ) microsatellites show rapid and variable contraction to lengths below 50 bp in both somatic and germ cells at frequencies of 14.8% and 13.3% , respectively . of note , these values represent the highest mutation frequencies hitherto reported for all microsatellite repeats , highlighting the extraordinary mutability of long poly(a ) tracts ( table 1 ) . long poly(a ) microsatellites birthed into the genome via retrotransposition are predicted to shorten in length rapidly within the first few generations . this prediction is consistent with observations from surveys of genomic resident alu and l1 sequences , which demonstrated an inverse correlation of poly(a ) tract length with the evolutionary age of the elements . one prominent form of microsatellite death is interruption by a non - repeat base , which can arise from base substitutions or short indel mutations . in particular , a comparison of human , chimpanzee and orangutan genomes suggests that substitutions are the primary cause of deaths for microsatellites of all lengths . although not widely recognized , non - ltr retrotransposons may also act as a mechanism for microsatellite interruption . such interruption can occur when new copies of non - ltr retrotransposons insert into pre - existing microsatellites . mammalian l1s are members of a diverse and widely distributed group of autonomous non - ltr retrotransposons that encode an endonuclease with homology to apurinic / apyrimidinic endonucleases ( ape ) . a small subset of these ape - type elements insert in a sequence - specific manner into microsatellites : ( 1 ) acay microsatellites are targeted by three waldo elements ( waldoag1,waldoag2 in the african malaria mosquito a. gambiae and walsofs1 in the earwig f. scudderi ) ; ( 2 ) ac microsatellites are targeted by a mino element in a. gambiae ( minoag1 ) ; ( 3 ) tc microsatellites are targeted by four kibi elements ( kibidr1 and kibidr2 in zebrafish d. rerio , kibifr1 in the torafugu f. rubripes , and kibitn1 in the green spotted pufferfish t. nigroviridis ) ; ( 4 ) ttc microsatellites are targeted by two koshi elements ( koshifr1 in f. rubripes and koshitn1 in t. nigroviridis ) ; ( 5 ) taa microsatellites are targeted by three dong elements ( dong in the silkworm b. mori , dongag in a. gambiae and dongbg in the freshwater snail b. glabrata ) . the molecular mechanisms underlying microsatellite targeting by these ape - type non - ltr retrotransposons are not well understood . it is believed that the sequence - specific cleavage by the endonuclease plays a major role in active targeting . in addition , two ape - type elements ( waldo - a and waldo - b in the fruitfly d. melanogaster ) are frequently found within or close to ca microsatellites , most likely as a result of passive accumulation in these low recombination regions . microsatellites may also be targeted by dna transposons ; examples include mine-1 helitrons and micron mites . the former insert at gaaa microsatellites in the genomes of the european corn borer o. nubilalis , the silkworm b. mori and the pink bollworm p. gossypiella , while the latter insert into ta microsatellites in the rice genome . in contrast , mammalian l1s have a weak target site preference with a consensus sequence 5-ttaaaa-3. although they may fortuitously land in or near certain microsatellite sites , they are unlikely to serve as a major factor in disrupting microsatellites . the relationship between microsatellites and non - ltr retrotransposons is not unidirectional . while both l1s and alus give birth to microsatellites , especially poly(a ) mononucleotide microsatellites , these microsatellite sequences can also affect the fitness of their parent due to their unusually high mutation rates . the impact of microsatellite instability on non - ltr retrotransposons depends on the location of the microsatellite , i.e. , whether it is internal or at the 3 terminal of the element . the effect of variation in microsatellites internal to non - ltr retrotransposons is less understood . newly inserted l1 and alu copies carry many mononucleotide proto - microsatellites ( fig . 1 ) . as microsatellites are predicted to mutate faster than the genomic average , expansion and contraction of microsatellite loci internal to l1s may introduce frameshift mutations , abolishing l1 coding capacity ( fig . ( a ) effect of internal microsatellite loci . expansions or contractions of proto - microsatellite loci inside an l1 element can cause frameshift mutations , leading to loss of orf1 and/or ofr2 function . the mutagenized l1 element would be unable to mobilize itself and to support alu retrotransposition . the contraction or expansion of an alu s poly(a ) tail diminishes or stimulates its retrotransposition , respectively , presumably because the length of its poly(a ) rna tail is positively correlated with the efficiency of target - primed reverse transcription ( tprt ) . ( c ) effect of the 3 poly(a ) microsatellite on l1 elements . due to the random nature of integration , an l1 sequence may not have a gt rich region downstream to its polyadenylation signal . however , there is evidence that the l1 poly(a ) tail exerts a crucial role in ensuring proper polyadenylation of l1 mrna in the absence of a strong gt - rich downstream sequence . the relative efficiency in mobilization is depicted by a difference in size of l1 mrna and protein products . the 3 poly(a ) tail of an l1 or alu element is a crucial component of the retrotransposition process and therefore , its length directly affects their retrotransposition potential . distinct cellular processes are responsible for the poly(a ) tail formation in l1 and alu elements . l1 elements are transcribed by rna polymerase ii and poly(a ) polymerases produce a poly(a ) rna tail as the 3 end of an l1 mrna . in contrast , alu elements are transcribed by rna polymerase iii and the resulting transcripts are not polyadenylated . however , active alu elements have a poly(a ) dna tract , which is transcribed as part of the alu rna . the 3 poly(a ) rna tail is predicted to serve two important roles during retrotransposition . first , the initial base pairing of these a s with the t - rich dna sequence at the target site may be required for efficient first - strand cdna synthesis during target - primed reverse transcription ( tprt ) . the l1 orf1 protein is an rna - binding protein with nucleic acid chaperone activity and it may facilitate this strand transfer and annealing process . second , increasing evidence indicates that these poly(a ) rna tails are bound by poly(a ) binding proteins ( pabps ) and that this interaction is critical for the formation of the ribonucleoprotein complex between l1 proteins and l1/alu rnas . indeed , retrotransposition assays demonstrate that the poly(a ) tail is strictly required for alu mobilization and that its retrotransposition activity is positively correlated with the length of poly(a ) tails . in parallel , poly(a ) tail shortening in expressed l1 mrnas impairs rnp formation and retrotransposition . therefore , the high mutability of poly(a ) tails has direct impact on l1 and alu retrotransposition . once captured in genomic dna , the initial long 3 poly(a ) tract undergoes rapid shortening in the first few generations . in fact , genome - wide , the length of the poly(a ) tract is inversely correlated with the evolutionary age of alu and l1 subfamilies . as the length of the poly(a ) tail is a key determinant of alu activity , a - tail expansion due to microsatellite instability is predicted to promote its retrotransposition and , conversely , its contraction would predict the extinction of an alu s ability to mobilize ( fig . indeed , newly generated alu copies typically have longer poly(a ) tails than the original donor elements . additionally , a nascent retrotransposition - competent alu element is able to propagate its newfound mobility to progeny copies through poly(a ) tail expansion during reverse transcription . it is generally accepted that the l1 poly(a ) tail is regenerated during each round of retrotransposition via reverse transcription of the polyadenylated l1 mrna . thus , the length of the poly(a ) tail in the donor l1 element may affect l1 retrotransposition only if it alters transcription and polyadenylation . a canonical polyadenylation signal contains a conserved aataaa hexamer and a downstream gt - rich region . the l1 polyadenylation signal is atypical in that a poly(a ) tail separates the aataaa hexamer from any downstream sequence ; the sequence composition of the latter is determined by chance depending on where the donor element is inserted during the previous round of retrotransposition . such a configuration may predispose an l1 element to bypass its own polyadenylation signal and instead to use a stronger polyadenylation signal fortuitously situated further downstream . nevertheless , the poly(a ) tail appears to exert a critical role in proper polyadenylation of l1 mrna in the absence of a strong gt - rich downstream sequence ( fig . although full length transcription is possible in the context of a weak polyadenylation context , the presence of a string of 17 a s brings about significant increases in the transcriptional efficiency . although its presence and length is not as critical for l1 mobility as for alu mobility , the poly(a ) tail s dynamics still subtly influence the efficiency of its parent in a disadvantageous genomic neighborhood . the availability of complete genome sequences for many organisms has enabled genome - wide analyses of repeat distribution . microsatellites with overall repeat length of 12 bp or longer make up 1.3% of the reference human genome ( i.e. , 13.3 kb per mb genome , excluding hexameric repeats at telomeres ) . although the overall density of microsatellites is relatively uniform in all human chromosomes , the distribution of each repeat class ( mono- , di- , tri- etc . ) varies : ( 1 ) mononucleotide microsatellites show a uniform distribution among exonic , intronic and intergenic regions ( note exonic regions herein include not only protein - coding sequences but also 5 and 3 untranslated regions ) ; ( 2 ) di- , tetra- and pentanucleotide microsatellites are relatively depleted in exonic regions ( 74% , 75% and 87% of the genome average , respectively ) ; ( 3 ) tri- and hexanucleotide microsatellites are enriched in exonic regions ( 191% and 146% of the genome average , respectively ) . the vast majority of human microsatellites that are conserved during vertebrate evolution are found in intergenic and intronic regions , consistent with the prediction that microsatellites in exonic regions are more disruptive to gene function . in comparison , the two major types of non - ltr retrotransposons , l1s and alus , display contrasting distribution patterns relative to genes . alus typically are overrepresented inside genes ( i.e. , residing anywhere in a transcription unit ) as well as within 30 kb from annotated genes . in addition , alus display no orientation bias relative to genes . in contrast , l1s show prominent orientation bias within genes : while antisense l1s are close to the predicted density , sense l1s are strongly depleted within genes . in addition , both sense and antisense l1s are significantly underrepresented within 5 kb from annotated genes . the differential distribution of alus and l1s relative to genes is not a result of target preference because both are retrotransposed by l1 proteins . rather it is consistent with the predicted impact of sense - oriented l1s on premature polyadenylation , cryptic splicing and/or inhibition of transcriptional elongation . this idea is further supported by the random chromosomal distribution of recent endogenous l1 and alu copies in the human genome and de novo insertions from l1 transgenes in mice . furthermore , the large catalog of polymorphic alu and l1 insertions clearly shows that exons can be targeted in proportion to their abundance in the genome . due to their dynamic nature , microsatellite loci are highly polymorphic among individuals . by comparing the reference human genome to a second genome , overall , 2.7% of the microsatellite loci are polymorphic , a frequency that is 45-fold higher than single nucleotide polymorphism ( 0.06% ) . exonic , especially the coding exonic sequences , show the least amount of microsatellite variation as compared with intergenic and intronic sequences , reflecting increased selective constraints on exonic sequences . polymorphism incidence increases with repeat number , an observation that is reproduced by surveying polymorphic microsatellite loci among individual genomes from the 1000-genome project . there is remarkable heterogeneity in the levels of variation at specific microsatellite loci . for example , the trinucleotide repeat in the 5utr of the fragile x mental retardation 1 ( fmr1 ) gene is one of the most studied microsatellite loci with respect to polymorphism in ethnically diverse , healthy individuals . the range of repeat number varies from population to population ; the overall range is 6 to 87 in surveyed populations . a repeat number of > 200 is considered a full mutation , which is typically associated with intellectual and developmental disabilities . whole - genome as well as targeted sequencing has also started to reveal the extent of genetic variations caused by non - ltr retrotransposons . polymorphisms in non - ltr retrotransposons are manifested as indels ( i.e. , insertions in one genome and deletions in another genome ) , a common form of structural variations ( other forms include tandem duplications , inversions and translocations ) . recent whole - genome sequencing efforts have cataloged a large number of structural variations among diverse human populations . detailed analyses of mutational mechanisms indicate 2030% of svs are caused by non - ltr retrotransposons . on average , each human genome contains 10002000 polymorphic non - ltr retrotransposons , with 7985% alu , 1217% l1s and 3% svas . based on the number of polymorphic insertions and the estimated genetic distance between individual genomes , the frequency of retrotransposition has been calculated as one alu insertion in 21 births and one l1 insertion in 108 to 212 births . most intriguingly , an analysis of the 1000 genomes project pilot data set suggests a recent increase in retrotransposition rates in humans . these insertions continuously generate new germline polymorphisms that can be passed across generations ( fig . 5 ) . as compared with humans , tes play an even bigger role in generating structural variations in the mouse genome . this discrepancy can be largely explained by the presence of a large number of active ltr retrotransposons in the mouse genome besides non - ltr retrotransposons . together , retrotransposon insertions contribute to of structural variations between two inbred mouse lines , with 52% , 43% and 5% contribution from ltr retrotransposons , l1s and sines , respectively . in addition to retrotransposition in the germline , non - ltr retrotransposons are active in somatic tissues , including normal brain tissues and most epithelial cancers ( further discussed in the next section ) . despite the significant progress made thus far , it should be noted that the discovery of structural variations mediated by tes remains a challenge because of the technical difficulty in de novo assembling repetitive sequences from short sequence reads as well as the high sequence depth that is required for detecting low - frequency alleles . the schematic illustrates the mutation dynamics of poly(a ) microsatellites across generations , beginning with an individual who has a newly acquired long poly(a ) microsatellite from l1 or alu retrotransposition . color is used to distinguish the size and mutability of the microsatellite , where darker color indicates longer poly(a ) tails that are more mutable . a long poly(a ) microsatellite can expand or contract between generations and give rise to variations in the offspring . however , when a poly(a ) microsatellite mutates below the threshold repeat number , the rate of mutation decreases to the average genomic mutation rate . the impact of a microsatellite or a transposable element and its associated variability is dependent on its genomic location . the most direct phenotypic impact of microsatellites and retrotransposons is manifested as disease causing mutations . microsatellite instability has been implicated in a variety of human diseases with over 40 neurological , neurodegenerative and neuromuscular disorders associated with trinucleotide repeat instability . well known examples include huntington s disease , fragile x syndrome and friedreich s ataxia . unlike static mutations , which can be stably passed from one generation to the other , alleles with expanded microsatellites may continue to expand over the course of the individual s life or through germline transmission . depending on the genomic neighborhood , variation in microsatellite length these mechanisms have been best characterized in trinucleotide repeat expansion disorders and typically consist of one ( or the combination ) of three following three pathways : the loss of function at the protein level , the gain of function at the protein level , or the gain of function at the rna level . the latter is gaining increased attention due to the recognized role of non - coding rna in epigenetic pathways . a noticeable absence among these molecular mechanisms is a loss of function at the rna level , a potential mechanism that should be investigated in future studies . likewise , de novo retrotransposition has been implicated in a variety of sporadic human diseases , including hemophilia , apert syndrome , neurofibromatosis type 1 and congenital muscular dystrophy . a multitude of molecular mechanisms have been proposed to account for the mutagenesis effect for intronic or exonic insertions . a less emphasized impact is post - insertional genome instability mediated by non - ltr retrotransposons , which likely poses even greater hazard to the human genome . for example , nonallelic homologous recombination ( nahr ) between two separate copies of alu repeats may generate structural variations , such as indel , inversion and translocation mutations . in addition , a hallmark of non - ltr retrotransposition is the formation of a pair of target site duplications ( tsds ) that flank the new insertion . these tsds represent pre - insertion l1 endonuclease cleavage sites and may be targeted for subsequent cleavage by other l1 endonucleases . it is proposed that new double strand breaks in tsds may trigger rearrangement events due to their adjacency to non - ltr retrotransposons with abundant homologous copies . these mechanisms highlight the dynamic nature of l1 and alu sequences in the human genome . furthermore , as has been discussed earlier , non - ltr retrotransposons are not static entities after insertion ; they are a significant source for microsatellite genesis . high - frequency microsatellite instability ( msi - h ) is a molecular feature of tumors associated with hereditary nonpolyposis colorectal cancers ( hnpcc ; lynch syndrome ) and has been seen in other sporadic cancers , usually of the gastrointestinal system . these tumors generally arise from defects in the mismatch repair system , leaving dna vulnerable to a high degree of mutation within microsatellites . detection of msi - h tumors is often achieved with a reference panel of five microsatellites , including two mononucleotide and three dinucleotide markers . the two mononucleotide markers , bat26 and bat25 , contain ( a)26 and ( a)25 repeats , respectively ; their length changes can be easily discerned even in the absence of normal tissue . it is thought that msi - h contributes to tumorigenesis mainly through mutating genes with coding microsatellites . a recent genome - wide survey yields the initial glimpse of microsatellite mutational landscapes in msi - h gastric cancers . there are four to 6-fold higher microsatellite mutations in genes in msi - h gastric cancers than microsatellite stable ( mss ) cancers . only a very small fraction of microsatellite mutations ( 0.1% ) are located in protein - coding exonic regions ; the remaining are found in 5utr , 3utr , intronic and intergenic regions of the genome . importantly , genes with significant changes in mrna expression between msi - h and mss samples are enriched for microsatellite mutations in their utrs , indicating a role of non - coding microsatellite instability in regulating gene expression . a role of non - ltr retrotransposons in tumorigenesis several studies have used nextgen sequencing approaches to detect genome - wide structural variations or specifically new retrotransposon insertions in tumor samples . the first study identified 9 tumor - specific ( i.e. , somatic ) l1 insertions in 6 of the 20 lung tumors but none in 10 brain tumors by targeted sequencing . a subsequent study characterized retrotransposon insertions in 43 human tumors of different origins by whole - genome sequencing . tumor - specific somatic insertions were found in all tumors of epithelial origin ( colorectal , prostate and ovarian ) , but not in blood or brain cancers . among epithelial cancers , colorectal cancers had the highest incidence of l1 insertions at an average of 9 insertions per tumor among 4 colorectal samples ; the fifth colorectal tumor sample alone had an astonishingly high number of 102 l1 insertions . interestingly , this tumor was the only colorectal sample that manifested msi - h with altered dna repair pathways , suggesting a common origin to both sources of genomic instability . an independent study mapped tumor - specific l1 insertions in 16 colorectal tumors by targeted l1 sequencing . intriguingly , although two samples with the highest number of somatic insertions showed microsatellite instability , no correlation was found between the number of insertions and the microsatellite instability status . future studies with higher coverage and/or larger sample size may be needed to establish a connection between microsatellite instability and retrotransposon mutagenesis . it should be emphasized that , in addition to somatic insertions , germline insertions may also be an important etiological factor in predisposing the carriers to cancers , as has been recently shown for hepatocellular carcinoma patients . the precise role of retrotransposon insertions in cancer remains to be determined but they can have a significant impact on gene function . consistent with sense - oriented l1 insertions being more disruptive to gene expression , genes targeted by sense insertions , but not those targeted by antisense insertions , showed statistically significant decrease in transcription . although usually highlighted for their negative roles , microsatellites and retrotransposons have evolutionary importance due to their ability to provide genetic novelties in human populations . a salient example is sequence shuffling through 3 transduction . due to the weak nature of the l1 poly(a ) signal , it is possible for l1 to bypass the signal during transcription and use an alternative poly(a ) signal downstream . the latter involves a 3 transduction event that is severely 5 truncated , which leaves behind no l1 sequence to be associated with the mobilized sequence . thus , 3 transduction has the potential to generate new genes and bring new regulatory features to the vicinity of other genes . although no cases have been reported , it is conceivable that l1s could also mobilize microsatellites by the same mechanism . another way of introducing evolutionary novelties is through trans - mobilization of cellular rnas , which form processed pseudogenes . lastly , since the exciting discovery of somatic retrotransposition in the brain , it has been postulated that l1-induced neuronal diversity may be an important source for behavioral phenotypes and confer evolutionary advantage . beyond genome sequence changes , the importance of other epigenetic mechanism , including dna methylation , histone modification and nucleosome occupancy , is coming to light . therefore , the insertion of an l1 into a genomic region may alter the previously existing epigenetic state ( and our unpublished data ) . due to their unique sequence composition , microsatellites can change the physical forms of dna where they occur , which can have implications for gene expression and genome stability . though not all microsatellites meet the criteria to do so , many repeats form non - b dna structures . these non - b forms of dna are often associated with locations of chromosomal breakage . an inverse correlation exists between a repeat s ability to form non - b form dna and its abundance in the genome , suggesting that these unstable structures are selected against due to reduced evolutionary fitness . the presence of poly(a ) tracts within promoter sequences also has implications for nucleosome organization . poly(a ) repeats form shorter and more rigid helix structures and have a narrower minor groove , altering nucleosome positioning in manners which increase the binding ability of transcription factors . thus , the mobilization of retroelements and their associated poly(a ) tails present a direct manner for l1 , alu and sva elements to affect the genome compaction and expression dynamics of the local regions where they land . in summary , microsatellites and non - ltr retrotransposons are dynamic dna sequences that rapidly change from generation to generation . expansion and mutation of microsatellites within l1 and alu sequences may change the size and sequence identity of these non - ltr retrotransposons , perhaps modulating their mobility . future studies to compare the sequence of full length l1s may reveal how the dynamics of microsatellites affect the retrotransposons from which they are born .

variable order markov models and variable order bayesian trees have been proposed for the recognition of transcription factor binding sites , and it could be demonstrated that they outperform traditional models , such as position weight matrices , markov models and bayesian trees . we develop a web server for the recognition of dna binding sites based on variable order markov models and variable order bayesian trees offering the following functionality : ( i ) given datasets with annotated binding sites and genomic background sequences , variable order markov models and variable order bayesian trees can be trained ; ( ii ) given a set of trained models , putative dna binding sites can be predicted in a given set of genomic sequences and ( iii ) given a dataset with annotated binding sites and a dataset with genomic background sequences , cross - validation experiments for different model combinations with different parameter settings can be performed . several of the offered services are computationally demanding , such as genome - wide predictions of dna binding sites in mammalian genomes or sets of 104-fold cross - validation experiments for different model combinations based on problem - specific data sets . in order to execute these jobs , and in order to serve multiple users at the same time , the web server is attached to a linux cluster with 150 processors . vombat is available at .

one important and interesting problem in genome research is the prediction of transcription factor binding sites ( tfbss ) . binding of transcription factors to their dna binding sites in the regulatory region of a gene is a prerequisite for its activation or repression . the combinatorial presence of tfbss controls gene regulation at least partially , and the prediction of tfbss is of importance for unraveling the underlying molecular mechanisms . wet - lab experiments allow the identification of tfbss , but these experiments are expensive and time consuming . computational methods , which are often less accurate but easy to conduct with available resources , are a welcome complementation to wet - lab experiments . many tfbs prediction algorithms employ statistical models for scoring a given sequence as tfbs or non - tfbs . for these algorithms , the family of markov models ( 13 ) is chosen in many tfbs prediction algorithms as well as for a variety of other classification problems . well - known examples are the position weight matrix ( pwm ) model ( 4 ) , which is an inhomogeneous markov model of order 0 , and the weight array matrix ( wam ) model ( 5 ) , which is an inhomogeneous markov model of order 1 . from a statistical point of view , markov models employ assumptions about the statistical independence of nucleotides at different positions of the binding site . the strongest independence assumption is realized by the pwm model , where each position is assumed to be statistically independent of all other positions . as indicated e.g. in ( 6 ) , it is an open question whether this strong independence assumption is reasonable in view of recent results indicating the presence of statistical dependences between positions ( 7,8 ) . markov models of higher order take into account statistical dependences from previous positions , called the context , where the length of the context is equal to the order of the markov model . despite the often unjustified independence assumption , pwm models are often found to outperform markov models of higher order in the prediction of tfbss . this is probably caused by the rather limited amount of training data available from experimentally verified tfbss . as the number of model parameters grows exponentially with the order of the markov model , markov models of higher order tend to be over - fitted , resulting in poor performance . one possibility to circumvent this problem is provided by variable order markov models , which do not require the contexts to be of a fixed length , but allow contexts with variable lengths ( 912 ) . the power of variable order markov models stems from the freedom to include only those contexts into the model for which there are strong statistical dependences . another possibility to capture statistical dependences between non - adjacent positions without the burden of increasing the number of model parameters exponentially are provided by bayesian trees ( bts ) ( 1316 ) . in bts the contexts are not restricted to the previous positions within the binding site , and it could be demonstrated that bts outperform pwm models and wam models in the prediction of splice sites ( 17,18 ) and tfbss ( 6,19 ) . as an extension of variable order markov models and bts , we introduce variable order bts ( 19 ) . the vombat web server is designed to train variable order markov models and variable order bts on user - supplied data and to apply the resulting models to the prediction of tfbss . in addition , the vombat web server provides a cross - validation platform , allowing advanced users to compare different model combinations with different parameter settings on problem - specific datasets . the vombat web server is primarily designed for the prediction of tfbss , but applicable to other types of fixed - length motifs as well . the prediction of tfbss is based on one statistical model for tfbss and one for non - tfbs sequences constituting the background . each statistical model assigns a probability p(x1xl ) to a given dna sequence x1xl of l nucleotides . for any value of m , 1 m l , p(x1xl ) markov models of order m assume that the conditional probabilities pl(xlx1xl1 ) do not depend on all previous nucleotides x1xl1 , but only on the m previous nucleotides xlmxl1 , which are called the context of position l. hence , a markov model of order m is defined by p(x1xl)=p0(x1xm)l = m+1lpl(xlxlmxl1 ) . if the conditional probabilities pl(xlxlmxl1 ) are identical at all positions , the markov model is called homogeneous , otherwise it is called inhomogeneous . in contrast to a markov model of order 1 , where the conditional probabilities at position l depend only on the previous nucleotide xl1 , a bt allows that the conditional probabilities at position l may depend on the nucleotide xpa(l ) at a possibly non - adjacent position pa(l ) , which is called the parent of position l. in a bt , arbitrarily remote positions may be chosen as parents as long as no cycles between positions are induced . this implies that there is one position , called the root position , which must not depend on any other position , and that the statistical dependences of a bt may be graphically represented by a rooted tree , where the positions are represented by nodes and the statistical dependences are represented by edges ( 1316 ) . the probability distribution defined by a bt with root position r decomposes as p(x1xl)=pr(xr)lrpl(xlxpa(l ) ) . in the vombat web server , the so - called motif model pmotif ( used for modeling tfbss ) and the so - called background model pbg ( used for modeling background dna sequences ) can be chosen by the user . an inhomogeneous markov model or a bt should be chosen if statistical dependences are assumed to vary from position to position within the sequence . a homogeneous markov model should be chosen if statistical dependences are assumed to be the same at all positions within the sequence . the appropriate order of a markov model depends on the expected range of statistical dependences and on the amount of available training data . for markov models of fixed order , the number of model parameters grows exponentially with the order , resulting in a sharp transition from under - fitted to over - fitted models . the idea of variable order markov models is to shorten the context in those cases where extending the context does not yield mathematically this is formulated by measuring to which degree the conditional probabilities change when extending or reducing the context . the kullback leibler divergence ( 20 ) is used as a measure of change of the conditional probability distributions . it measures the degree of dissimilarity of the conditional probability distribution given the extended context and the conditional probability distribution given the reduced context . leibler divergence is always non - negative , but small positive values of the kullback the threshold above which the kullback leibler divergences are considered important can be set by an external parameter , c , called pruning constant . for c = 0 , any extension of the context is considered important , and the resulting variable order markov model becomes a traditional markov model of order m. for c , any extensions of the context is considered unimportant , and the resulting variable order markov model becomes a traditional markov model of order 0 , i.e. a pwm model . as c grows from 0 to , more and more contexts are considered unimportant , and the resulting variable order markov models become smaller and smaller , interpolating between a markov model of order m and a pwm model . vombat uses a maximum likelihood estimator with optional pseudo counts , which can be specified by the user , for smoothing the conditional probability distributions and to compensate for zero occurrences of nucleotides and contexts . the motif model pmotif is learned from a user - supplied training set of tfbss , and the background model pbg is learned from a user - supplied training set of background sequences . after training pmotif and pbg , vombat can be used to compute genome - wide predictions of the learned motif in a set of user - supplied sequences . for each position of a sliding window of length l , the log - likelihood ratio of the oligonucleotide y1yl occurring at that position is computed , and y1yl is predicted as tfbs if the log - likelihood ratio is greater than a given threshold t , i.e. if log2pmotif(y1yl)pbg(y1yl)t . specific knowledge , such as the number of tfbss expected in the genome , can be taken into account to adjust the threshold t to achieve tfbs predictions with the desired balance of sensitivity and specificity . for advanced users , vombat allows a cross - validation of the prediction accuracy of a user - defined combination of pmotif and pbg based on a user - supplied set of tfbss and a user - supplied set of background sequences . from each of the datasets , 10% of the sequences are randomly excluded , and the models pmotif and pbg are trained on the remaining 90% . subsequently , both models are used for the prediction of the withheld data , and the sensitivity given a user - defined value of the specificity is computed . this procedure is repeated k times , where k can be specified by the user , and the mean sensitivity and its standard error are reported . vombat is based on a variant of the three - tier architecture , where the presentation layer , the management layer and the execution framework are logically separated and physically located on different servers . the web front - end of vombat is based on standard technologies like javaserver faces for the web forms and servlets for displaying images and providing downloads of results . the user - supplied parameters and input files are stored in a mysql - database . this database is queried by the execution framework . if the execution framework detects a job to be executed , it requests the necessary parameters and input files and submits the corresponding job to the scheduler of the attached linux cluster . after the job is finished , the results are written to the database and can be displayed by the web front - end , which loads numerical results , model descriptions and images from the result - tables of the mysql - database . imagine a user interested in a genome - wide prediction of binding sites of the transcription factor sp1 in gc - poor upstream regions of human refseq ( 21 ) genes . if the user were interested in a prediction based on pre - trained pwm models stored in the transfac database ( 22 ) , he could use the tfbs prediction program match ( 3 ) . if the user were interested in a prediction based on variable order markov models or variable order bts , he could use the vombat web server . the two main functions of the vombat web server are training variable order markov models or variable order bts based on user - supplied sequences and predicting putative tfbss based on user - supplied variable order markov models or variable order bts . the training function allows the user to choose an optimal combination of a motif model and a background model and train these models on problem - specific datasets . for example , the user might choose a variable order bt as motif model and a variable order markov model of order 5 as background model , he might choose that subset of sp1 binding sites available from transfac that are located in gc - poor promoters as motif dataset , and he might choose a representative set of gc - poor promoter regions as background dataset . for training a model from user - supplied input data , the user first selects train a model from data from the selection of tasks . the user can enter a comment on the model to be trained in order to make it easier to identify the corresponding results in a list of all results . the rest of the parameters defines the type of model to be trained as well as the pruning constant and the pseudo count . the default value of the pseudo count is 1 , and we recommend the user to always specify a pseudo count greater than 0 to compensate for zero occurrences . if the model is defined to be inhomogeneous , the user can select between a markov model and a bt as a third parameter . based on ( 6,19 ) and based on systematic cross - validation analyses of different model combinations applied to different sets of tfbss and background sequences , we generally recommend to use a bt as motif model and a homogeneous markov model with an initial order of at least 3 as background model . the user - supplied input file to train an inhomogeneous model must consist of aligned sequences of identical length , separated by line breaks . the vombat web server is designed for , but not restricted to , the prediction of motifs in dna sequences . hence , it is possible to specify the alphabet of the sequences . in case of dna sequences the alphabet is acgt. as a last option the user may select a checkbox that determines if a graphical representation of the trained model shall be displayed as supplementary data . if the job of training a model could be successfully submitted to the scheduler of the attached linux cluster , the subsequently displayed page reports jobs success, and vombat gives the user the possibility to go either to the list of tasks for starting another job or to the job overview . the job overview displays a table of all jobs with the following columns : the time at which the job was started , the comment that was entered by the user , the type of the job ( train , classify , crossval ) , the current state of the job , a button for the already available results and a button to stop a running job . the possible states of a job are view results in the same row to inspect the results of this job . the results of training a model are a link to an xml representation of the trained model , which can be saved and used as input for one of the other tasks presented in the following . if the user selected to obtain a graphical representation of the trained model , this representation is also displayed as a series of images , which may be saved using the corresponding browser command . after a motif model and a background model have been trained and saved , they can be combined to predict putative tfbss in a user - supplied set of input sequences . for this purpose , the first input field of the subsequently displayed form , which is shown in figure 1 , allows to enter a user - defined comment on the job . next , the user uploads two files containing the xml representations of the desired motif and background models , which have been trained using the train a model from data task described above . the classification threshold must be specified , and the file containing the set of sequences in which the motif is to be predicted must be uploaded . after a classification job has been started , the user can monitor the current state of the job in the job overview of vombat . if the job is finished , the predictions can be inspected by clicking on the corresponding view results button . the html file contains a textual description of the putative motifs , which contains for each predicted motif its position , its sequence , its probabilities returned by the two models , and the corresponding log - likelihood ratio . additionally , a profile of the log - likelihoods and the log - likelihood ratio is plotted for each sequence of the input file . the chosen classification threshold is also plotted as a reference for the log - likelihood ratio , allowing the user to judge the influence of the classification threshold on the prediction results at a glance . as an auxiliary function for advanced users , vombat provides a platform for cross - validation analyses of different combinations of user - defined models based on problem - specific data . selecting cross validation from the list of tasks brings up the cross - validation input form . the first input field allows to enter a user - defined comment on the cross - validation job . next , the number of iterations of the cross - validation job can be entered . two files containing xml representations of the motif model and the background model must be uploaded by the user . these files can be obtained by a standard training on the datasets to be used for cross - validation . next , the user must specify the desired specificity for which the mean sensitivity is to be computed . the last option in the form allows to plot a histogram of the log - likelihood ratios for the motif sequences and background sequences , which allows further analyses of the cross - validation results , such as judging the effect of a shifted threshold or inspecting the separation of the samples of both classes . the presented results are the mean ( and standard error ) of the sensitivity for the fixed specificity entered in the cross - validation form , the mean ( and standard error ) of the maximum correlation coefficient , and the corresponding thresholds and optionally the histogram of the log - likelihood ratios as shown in figure 3 . the following prototypical example illustrates how the three functions of vombat may be utilized for the prediction of putative tfbss . typically , the user has no a priori knowledge of which model combination and which model parameters could provide an accurate prediction of these binding sites in gc - poor upstream regions . hence , he would start a series of different cross - validation experiments for different model combinations and different parameter settings using the cross - validation function of vombat . after having established an optimal model combination ( including optimal parameters ) for his specificity requirements and based on his problem - specific datasets , e.g. based on already annotated sp1 binding sites from transfac as motif dataset and representative gc - poor upstream regions as background dataset , he would then use these models to run the training and prediction functions of vombat as outlined above . for training a model from user - supplied input data , the user first selects train a model from data from the selection of tasks . the user can enter a comment on the model to be trained in order to make it easier to identify the corresponding results in a list of all results . the rest of the parameters defines the type of model to be trained as well as the pruning constant and the pseudo count . the default value of the pseudo count is 1 , and we recommend the user to always specify a pseudo count greater than 0 to compensate for zero occurrences . if the model is defined to be inhomogeneous , the user can select between a markov model and a bt as a third parameter . based on ( 6,19 ) and based on systematic cross - validation analyses of different model combinations applied to different sets of tfbss and background sequences , we generally recommend to use a bt as motif model and a homogeneous markov model with an initial order of at least 3 as background model . the user - supplied input file to train an inhomogeneous model must consist of aligned sequences of identical length , separated by line breaks . for homogeneous models the length of the sequences may differ . the vombat web server is designed for , but not restricted to , the prediction of motifs in dna sequences . hence , it is possible to specify the alphabet of the sequences . in case of dna sequences the alphabet is acgt. as a last option the user may select a checkbox that determines if a graphical representation of the trained model shall be displayed as supplementary data . if the job of training a model could be successfully submitted to the scheduler of the attached linux cluster , the subsequently displayed page reports jobs success, and vombat gives the user the possibility to go either to the list of tasks for starting another job or to the job overview . the job overview displays a table of all jobs with the following columns : the time at which the job was started , the comment that was entered by the user , the type of the job ( train , crossval ) , the current state of the job , a button for the already available results and a button to stop a running job . the possible states of a job are if the state of a job is finished, the user can click on the button view results in the same row to inspect the results of this job . the results of training a model are a link to an xml representation of the trained model , which can be saved and used as input for one of the other tasks presented in the following . if the user selected to obtain a graphical representation of the trained model , this representation is also displayed as a series of images , which may be saved using the corresponding browser command . after a motif model and a background model have been trained and saved , they can be combined to predict putative tfbss in a user - supplied set of input sequences . for this purpose , the first input field of the subsequently displayed form , which is shown in figure 1 , allows to enter a user - defined comment on the job . next , the user uploads two files containing the xml representations of the desired motif and background models , which have been trained using the train a model from data task described above . the classification threshold must be specified , and the file containing the set of sequences in which the motif is to be predicted must be uploaded . after a classification job has been started , the user can monitor the current state of the job in the job overview of vombat . if the job is finished , the predictions can be inspected by clicking on the corresponding view results button . the html file contains a textual description of the putative motifs , which contains for each predicted motif its position , its sequence , its probabilities returned by the two models , and the corresponding log - likelihood ratio . additionally , a profile of the log - likelihoods and the log - likelihood ratio is plotted for each sequence of the input file . the chosen classification threshold is also plotted as a reference for the log - likelihood ratio , allowing the user to judge the influence of the classification threshold on the prediction results at a glance . as an auxiliary function for advanced users , vombat provides a platform for cross - validation analyses of different combinations of user - defined models based on problem - specific data . selecting cross validation from the list of tasks brings up the cross - validation input form . the first input field allows to enter a user - defined comment on the cross - validation job . next , the number of iterations of the cross - validation job can be entered . a higher number of iterations produces more reliable results . on the other hand , two files containing xml representations of the motif model and the background model must be uploaded by the user . these files can be obtained by a standard training on the datasets to be used for cross - validation . next , the user must specify the desired specificity for which the mean sensitivity is to be computed . the last option in the form allows to plot a histogram of the log - likelihood ratios for the motif sequences and background sequences , which allows further analyses of the cross - validation results , such as judging the effect of a shifted threshold or inspecting the separation of the samples of both classes . the presented results are the mean ( and standard error ) of the sensitivity for the fixed specificity entered in the cross - validation form , the mean ( and standard error ) of the maximum correlation coefficient , and the corresponding thresholds and optionally the histogram of the log - likelihood ratios as shown in figure 3 . the following prototypical example illustrates how the three functions of vombat may be utilized for the prediction of putative tfbss . typically , the user has no a priori knowledge of which model combination and which model parameters could provide an accurate prediction of these binding sites in gc - poor upstream regions . hence , he would start a series of different cross - validation experiments for different model combinations and different parameter settings using the cross - validation function of vombat . after having established an optimal model combination ( including optimal parameters ) for his specificity requirements and based on his problem - specific datasets , e.g. based on already annotated sp1 binding sites from transfac as motif dataset and representative gc - poor upstream regions as background dataset , he would then use these models to run the training and prediction functions of vombat as outlined above . the form requesting the parameters for a tfbs prediction . the results of a tfbs prediction . the list of putative tfbss are displayed as a link to sites.html, and the profiles for the sequences are plotted . the results of a cross - validation .

a spinal epidural hemangioma is rare . in this case , a 51 year - old female patient had low back pain and right thigh numbness . she was initially misdiagnosed as having a ruptured disc with possible sequestration of granulation tissue formation due to the limited number of spinal epidural hemangiomas and little - known radiological findings . because there are no effective diagnostic tools to verify the hemangioma , more effort should be put into preoperative imaging tests to avoid misdiagnosis and poor decisions ) .

spinal epidural hemangiomas have been reported in the literature , but most of them were cavernous type hemangiomas that enable a preoperative differential diagnosis with relative ease . the limited number of spinal epidural hemangiomas and few radiological findings make an exact diagnosis difficult prior to surgery . a high vascularization of spinal epidural hemangiomas may result in an unexpected surgical situation in the case of preoperative misinterpretation . in the current case , a 51-year - old woman presented with a 3-week history of lower back pain with right anterior thigh numbness . the patient had a magnetic resonance imaging ( mri ) at a local hospital which revealed a lesion at the l3 level , located in the ventral epidural space and connected with l3/4 protruded disc material that demonstrated a heterogeneous signal at t2 weighted images and an iso- to low signal at t1 weighted image ( t1-wi ) . 1 ) . based on a presumed diagnosis of a ruptured disc with possible sequestration or granulation tissue formation , the patient underwent surgery . a right hemilamincetomy of l3 was performed , and a retracting thecal sac revealed a highly engorged vascular structure . near - infrared indocyanine green videoangiography ( icg - va ) showed a delayed mass filling . histological examination revealed a vascular lesion composed of small to medium sized veins with irregular calibers , which is consistent with an arteriovenous hemangioma ( fig . the typical symptoms of epidural lesions other than disc herniation of the lumbar region are low back pain or radiculopathy which are indistinguishable from the clinical symptoms of disc herniation diseases . moreover , spinal epidural hemangiomas are very rare . also , spinal epidural hemangiomas constitute approximately 4% of all epidural tumors and 12% of all intraspinal hemangiomas4 ) . the differential diagnosis for spinal epidural hemangiomas before surgery included schwannoma , lymphoma , meningioma , angiolipoma , disk herniation , synovial cysts , granulomatous infection , pure epidural hematoma , and extramedullary hematopoiesis9,12 ) . the cavernous type displays histologically with large number of sinusoidal channels in collagenous tissue7 ) , whereas the arteriovenous type shows with a cluster of abnormal arteries and veins and vessel walls containing elastin , and smooth muscle5 ) . a complete surgical en bloc removal is the treatment of choice for spinal epidural hemangiomas with mass effect because of the excessive vascularity of hemangioma , piece to piece resection should be avoided6 ) . an indocyanine green videoangiography can help surgeons understand the vasculature surrounding the mass and facilitate the en bloc removal of the hemangioma . fluorescence angiography with indocyanine green provides real - time information regarding the patency of vessels . an icg - va enhances the flow direction delineation capability , flow velocity and sequence of dye filling in different components of complex spinal vascular lesions3 ) . due to the high vascularization of hemangiomas , the incomplete surgical removal of a spinal hemagioma because of diffuse bleeding or minimal exposure during disk surgery might result in the persistence of clinical symptoms or recurrence . reoperation for remnant or recurrent spinal hemangioma is very difficult due to peridural or periradicular adhesion and unclear margins ; as a result , complete resection can not be guaranteed . therefore , proper preoperative planning and complete resection during the operation is essential . for this , but , like this case , an angiography may not always confirm the diagnosis of an artriovenous hemangioma . clinicians should be aware that an angiography can not provide conclusive evidence of the presence of an antriovenous hemangioma . if spinal epidural hemangiomas are unexpected encountered during surgery , an icg - va can be helpful to diagnose and surgery . this technique provides accurate information about the flow dynamics through the anatomy of vascular lesions in real time10,11,13,14 ) . further study is required to recognize and to provide a differential diagnosis of spinal epidural hemangiomas . if the lesion is like a ruptured disc in mri , we should consider spinal epidural hemangioma as one of differential diagnosis .

backgroundto address the long - term problems of bearing surface wear and osteolysis associated with conventional metal - polyethylene ( m - pe ) total hip arthroplasty ( tha ) , metal - metal ( m - m ) , and ceramic - ceramic ( c - c ) bearings have been introduced . these bearing surfaces are associated with unique risks and benefits and higher costs . however the relative risks of these three bearings in an older population is unknown.questions/purposeswe compared the short - term risk of complication and revision tha among medicare patients having a primary tha with metal - polyethylene ( m - pe ) , metal - metal ( m - m ) , and ceramic - ceramic ( c - c ) bearings.methodswe used the 2005 to 2007 100% medicare inpatient claim files to perform a matched cohort analysis in three separate cohorts of tha patients ( m - pe , m - m , and c - c ) who were matched by age , gender , and us census region . multivariate cox proportional - hazards models were constructed to compare complication and revision tha risk among cohorts , adjusting for medical comorbidities , race , socioeconomic status , and hospital factors.resultsafter adjusting for patient and hospital factors , m - m bearings were associated with a higher risk of periprosthetic joint infection ( hazard ratio , 3.03 ; confidence interval , 1.029.09 ) when compared with c - c bearings ( 0.59% versus 0.32% , respectively ) . there were no other differences among bearing cohorts in the adjusted risk of revision tha or any other complication.conclusionsthe risk of short - term complication ( including dislocation ) and revision tha were similar among appropriately matched medicare tha patients regardless of bearing surface . hard - on - hard tha bearings are of questionable value in medicare patients , given the higher cost associated with their use and uncertain long - term benefits in older patients.level of evidencelevel ii , prognostic study . see guidelines for authors for a complete description of levels of evidence .

numerous investigators have reported improved function , reduced pain , and high rates of implant survivorship at up to 20 years followup [ 4 , 29 ] . however , concerns about limitations in implant longevity related to the osteolysis associated with metal - on - polyethylene ( m - pe ) bearing surface wear have led to innovation in tribology [ 10 , 15 , 16 ] and the introduction of new tha bearing couples over the past decade . in particular , second - generation hard - on - hard bearings , including metal - on - metal ( m - m ) and ceramic - on - ceramic ( c - c ) , have been introduced into the us market based on laboratory studies showing lower wear rates when compared with m - pe bearings [ 9 , 10 , 15 ] . lower wear rates associated with hard - on - hard bearings are likely to have the biggest impact on revision rates in younger , more active patients , who are at higher risk for wear - related failures [ 3 , 12 , 17 , 18 , 27 ] . the long - term benefits of more costly hard - on - hard bearings are less obvious in older patients , who are at lower risk for wear - related failures . however , a potential benefit of m - m bearings is the ability to use large diameter femoral heads , which theoretically could result in a lower short - term risk of dislocation when compared with m - pe bearings . this potential benefit could be particularly advantageous in older patients who are reportedly at higher risk for tha dislocation [ 5 , 25 , 28 ] . however , that study employed a cross - sectional study design , and due to the nature of the database used ( the nationwide inpatient sample ) , we were unable to compare longitudinal patient outcomes and differences in the risks of complication and revision tha among patients with different tha bearings . the purpose of this study was to compare the short - term risk of complication and revision tha among medicare patients having a primary tha with m - pe , m - m , and c - c tha bearings . the 2005 to 2007 100% medicare inpatient claim files were used to perform a matched cohort analysis in three separate cohorts of tha patients ( m - pe , m - m , c - c ) . patients in each tha cohort were matched by age , gender , and us census region to the comparison or m - m hip resurfacing arthroplasty procedures ( which have a separate icd-9-cm procedure code ) were excluded from the analysis . each m - m patient and c - c patient ( case ) were matched to three m - pe patients ( control ) , while each c - c patient ( case ) was matched to three m - m patients ( control ) . case patient , the eligible pool of controls consisted of those patients who were within 3 years in age of the case patient and who were at least 65 years old . if more eligible controls were available than the three required per case , controls closest in age were selected first . eligible controls were also of the same gender and resided in the same us census region . all cases and controls had to be enrolled in both part a and part b of medicare . patients who received their medicare health benefits through a health maintenance organization were excluded because their healthcare expenses were not submitted to the centers for medicare and medicaid services ( cms ) for payment and , therefore , claims from these beneficiaries were not available from the database . primary tha patients were identified using international classification of diseases , 9th rev , clinical modification ( icd-9-cm ) procedure code 81.51 from the inpatient claims records and grouped into three bearing cohorts based on their corresponding icd-9-cm optional procedure modifier bearing codes ( 00.74 for m - pe , 00.75 for m - m , 00.76 for c - c ) . using each patient s unique de - identified beneficiary i d , the patients were tracked longitudinally with revision tha or selected complications as end points . the selected complications included deep venous thrombosis ( dvt ) , dislocation , infection , and mechanical loosening . dvt was identified using the icd-9-cm diagnosis codes 451.1 , 451.2 , 451.81 , 451.9 , 453.1 , 453.2 , 453.4 , 453.8 , and 453.9 , while tha dislocation was identified using any occurrence of icd-9 diagnosis codes 718.35 , 835 , or 996.42 . infection and mechanical loosening were identified using icd-9 diagnosis codes 996.66 and 996.41 , respectively . an overall cohort of 36,423 tha patients with m - pe bearings were identified , along with 17,789 tha patients with m - m bearings and 2,835 tha patients with c - c bearings , from which the matched cohorts were derived . the mean age of all tha recipients included in the study was 74.5 years ( sd = 6.4 years ) . multivariate cox proportional - hazards models were constructed to compare complication and revision tha risk among cohorts , adjusting for medical comorbidities , race , socioeconomic status , and hospital factors . the medicare buy - in status was used as an identifier of patients whose medicare premiums and deductibles were subsidized by the state ( eg , medicaid ) and were used as a proxy for the patient s socioeconomic status . to account for the health status of each patient , the charlson comorbidity index was used as a measure of comorbid illness . for this analysis , the charlson index values were grouped into the following previously validated categories : 0 ( none ) , 1 to 2 ( low ) , 3 to 4 ( moderate ) , and 5 or more ( high ) . among younger medicare patients ( aged 6569 years ) , m - m bearings accounted for 35% of tha bearings and c - c bearings accounted for 8% . among older medicare patients ( older than 80 years ) , among female patients , 67% had m - pe bearings , while 29% had m - m bearings , compared to male patients , among whom 60% had m - pe and 35% had m - m bearings ( fig . 2 ) . the highest frequency of m - pe bearing surface use was in the northeast ( 72% ) , and the highest frequency of m - m bearing usage was in the south ( 37% ) , while c - c bearing usage was fairly consistent across the four us census regions ( 4%5% ) ( fig . 1the bearing surface frequency in medicare tha patients by age is shown.fig . 2the bearing surface frequency in medicare 3the bearing surface frequency in medicare tha patients by us census region is shown . the bearing surface frequency in medicare tha patients by age the bearing surface frequency in medicare tha patients by us census region is shown . when comparing the unadjusted risk of complication using the kaplan - meier method , m - m bearings showed an apparent trend toward higher overall risk of dvt ( fig . 5 ) than c - c bearings . however , these differences were not present after adjusting for other covariates in these matched cohorts ( table 1 ) . after controlling for patient and hospital factors , m - m bearings were associated with a higher risk of periprosthetic joint infection ( hazard ratio , 3.03 ; confidence interval , 1.029.09 ) when compared with c - c bearings . there were no other differences among cohorts in the adjusted risk of revision tha or any other complication , including dvt , dislocation , or mechanical loosening ( table 1).fig . 4this graph shows trends of higher overall rates of dvt for m - m versus c - c bearings ( unadjusted ) . ( t ) = percentage of patients without the complication ( dvt ) at the given time points.fig . 5this graph shows trends of higher overall rates of revision for m - m versus c - c bearings ( unadjusted ) . ( t ) = percentage of patients without the complication ( revision ) at the given time points.table 1risk - adjusted comparison of risk of complication and revision tha between m - pe and m - m bearings , m - pe and c - c bearings , and m - m and c - c bearingscomplicationm - pe ( n = 54,431)m - m ( n = 17,789)hazard risk ratio ( compared to m - pe)p valuem - pe ( n = 8073)c - c ( n = 2835)hazard risk ratio ( compared to m - pe)p valuem - m ( n = 7853)c - c ( n = 2835)hazard risk ratio ( compared to m - m)p valuedvt1.05%1.03%1.010.881.08%0.88%0.590.070.90%0.88%0.900.73dislocation0.80%0.74%0.920.490.68%0.78%1.080.800.57%0.78%1.210.57infection0.49%0.53%0.980.890.37%0.32%0.620.360.59%0.32%0.330.045mechanical loosening0.22%0.20%0.870.560.25%0.39%0.970.970.23%0.39%1.100.89revision1.16%1.12%0.940.511.04%1.02%0.670.141.34%1.02%0.670.09m - pe = metal - on - polyethylene ; m - m = metal - on - metal ; c - c = ceramic - on - ceramic ; dvt = deep vein thrombosis . this graph shows trends of higher overall rates of dvt for m - m versus c - c bearings ( unadjusted ) . ( t ) = percentage of patients without the complication ( dvt ) at the given time points . this graph shows trends of higher overall rates of revision for m - m versus c - c bearings ( unadjusted ) . ( t ) = percentage of patients without the complication ( revision ) at the given time points . risk - adjusted comparison of risk of complication and revision tha between m - pe and m - m bearings , m - pe and c - c bearings , and m - m and c - c bearings m - pe = metal - on - polyethylene ; m - m = metal - on - metal ; c - c = ceramic - on - ceramic ; dvt = deep vein thrombosis . tha has become one of the most commonly performed operations in medicare patients , with over 160,000 primary and revision tha procedures reported in medicare patients in 2007 . successful tha allows elderly patients who suffer from disabling hip disease to regain their function and maintain a more active , healthy lifestyle . nevertheless , increased utilization and rising costs have led to concerns regarding the appropriateness of using hard - on - hard bearings are associated with lower wear rates in the laboratory setting [ 15 , 20 ] . however , wear - related failures , such as bearing surface wear , osteolysis , and mechanical loosening , may be less of a concern among medicare patients when compared with younger , more active patients who undergo primary tha . therefore , the higher cost associated with hard - on - hard bearings may not be justifiable in terms of improved patient outcomes and lower reoperation rates in medicare patients , unless they are associated with fewer short - term complications , such as dislocation . one example of a potential short - term benefit of m - m bearings that is often cited is the ability to use large diameter femoral heads , which theoretically could result in a lower risk of dislocation when compared with m - pe bearings . we therefore compared the short - term risks of complication and revision tha among medicare patients having a primary tha with m - pe , m - m , and c - c tha bearings . our findings are limited by the use of an administrative database , where bearing surface type is an optional modifier code that can be reported in conjunction with the primary procedure code ( primary or revision total hip arthroplasty ) , which introduces a potential source of bias into our study . however , this limitation is somewhat mitigated by the matched cohort study design , where patients with known bearing types were matched by age , gender , and u.s . second , other variables of interest which may influence patient outcomes , including surgical approach , surgeon experience , and implant design are not accessible from administrative claims , and we were only able to evaluate complications which are captured in administrative claims data , such as dvt , dislocation , infection , mechanical loosening , and revision surgery , rather than pain and patient reported functional outcomes . third , since the optional bearing surface modifier administrative codes were not introduced until october , 2005 , our study was limited to outcomes and complications that occurred within two years of the index procedure . however , the rationale for our study was that presumably the reason for using alternative bearings in medicare patients is to reduce short - term complication rates ( eg , dislocation ) , rather than long - term complications ( eg , wear / osteolysis ) , which are more of a concern in younger patients than in medicare patients . furthermore , the australian hip registry and other reports in the literature [ 6 , 11 , 19 , 22 , 23 , 26 ] have raised concerns regarding higher than expected short - term complication and revision rates in patients with hard - on - hard bearings . nevertheless , further study is necessary to evaluate differences in long - term clinical outcomes and revision rates as additional data becomes available . we found the adjusted risk of short - term complication and revision tha among medicare tha patients was similar , regardless of bearing surface , with the exception of patients who had m - m bearings , who had a slightly higher risk of periprosthetic joint infection than patients who had c - c bearings . the reasons underlying the increased risk of infection for m - m bearings compared with c - c bearings , even after adjusting for medical comorbidities and hospital factors , remain unclear . it is possible that some of these patients may have been misdiagnosed as having a periprosthetic joint infection when in fact they had a local soft tissue inflammatory reaction related to the m - m articulation [ 6 , 11 ] . the findings in some patients with such reactions mimic periprosthetic joint infection , as reported previously by mikhael et al . . although the higher risk of infection in the m - m cohort compared with the c - c cohort was significant ( hazard ratio 3.03 , ci = 1.02 9.09 ) , the clinical importance of this difference is unclear , especially given the relatively low incidence of infection ( 0.59% versus 0.32% , respectively ) . the relatively high incidence of m - m bearings reported in the medicare population is somewhat surprising , given m - m bearings are thought by some to be primarily indicated for younger , more active patients who are at higher risk for bearing surface wear and osteolysis with m - pe bearings [ 14 , 17 , 20 ] . however , one theoretical advantage of m - m bearings in older patients is improved stability ( eg , lower risk of dislocation ) due to the ability to use larger - diameter femoral heads . our data do not demonstrate an advantage of m - m bearings in terms of lower short - term risk of dislocation when compared with either m - pe or c - c bearings . this could be related to the increasing trend of using 32- and 36-mm heads in m - pe bearings , which may be sufficient to substantially reduce the risk for dislocation . although the database used in this study did not include information about femoral head size , according to industry sources , the proportion of femoral heads implanted in the us sized 32 mm or greater has increased from approximately 12% to 79% between 1998 and 2008 . in summary , we found medicare tha patients with hard - on - hard ( m - m , c - c ) bearings had a similar risk of complications and revision tha compared to patients who had m - pe bearings during the first 2 years after primary tha . these findings provide a basis for additional analyses of the comparative effectiveness of tha bearing surfaces in the medicare population .

hemodialysis patient survival is dependent on the availability of a reliable vascular access . in clinical practice , procedures for vascular access cannulation vary from clinic to clinic . we investigated the impact of cannulation technique on arteriovenous fistula and graft survival . based on an april 2009 cross - sectional survey of vascular access cannulation practices in 171 dialysis units , a cohort of patients with corresponding vascular access survival information was selected for follow - up ending march 2012 . of the 10,807 patients enrolled in the original survey , access survival data were available for 7058 patients from nine countries . of these , 90.6% had an arteriovenous fistula and 9.4% arteriovenous graft . access needling was by area technique for 65.8% , rope - ladder for 28.2% , and buttonhole for 6% . the most common direction of puncture was antegrade with bevel up ( 43.1% ) . a cox regression model was applied , adjusted for within - country effects , and defining as events the need for creation of a new vascular access . area cannulation was associated with a significantly higher risk of access failure than rope - ladder or buttonhole . retrograde direction of the arterial needle with bevel down was also associated with an increased failure risk . patient application of pressure during cannulation appeared more favorable for vascular access longevity than not applying pressure or using a tourniquet . the higher risk of failure associated with venous pressures under 100 or over 150 mm hg should open a discussion on limits currently considered acceptable .

out of the 10,807 patients enrolled for the original survey , access survival data were available for 7058 ( 65% ) patients . these patients resided in portugal , the united kingdom , ireland , italy , turkey , romania , slovenia , poland , and spain . the mean age was 63.515.0 years , 38.5% were female , 27.1% were diabetic , 90.6% had a native fistula , and 9.4% had a graft . median dialysis vintage was 43.2 months ( minimum : 0.1 months ; maximum : 419.6 months ) . access location was lower arm for 51.2% of patients . during the follow - up , prevalent needle sizes were 15 and 16 g for 63.7% and 32.2% of the patients , respectively ( 14 g : 2.7% 17 g : 1.4% ) . in spain , 98% of patients were treated with 15-g needles , and in romania 75% of patients were treated with 16-g needles . cannulation technique was area for 65.8% , rope - ladder for 28.2% , and buttonhole for 6% of patients , with some country preferences clearly visible : area technique was applied in as much as 77% of patients in romania , and rope - ladder was more common in poland than in the total study population ( 44% ) . the direction of arterial puncture was antegrade for 57.3% of patients ; this was the preference for 99% of patients in poland . the bevel orientation was upward for 70.2% of the patients , peaking in poland with 95% . the practice of needle rotation after insertion was practiced for 42% of patients , with a much higher percentage in italy ( 82% ) . the prevalent combination between arterial needle puncturing and bevel direction was antegrade with bevel upward ( 43.1% ) , followed by retrograde with bevel up ( 27.1% ) . the proportion of the two other combinations , that is , antegrade and retrograde with bevel downward , was 14.2% and 15.6% , respectively . the 15.6% with retrograde and bevel down were mainly treated in two countries ( spain and portugal ) . median blood flow was 350400 ml / min . in italy and spain , 40% and 38% of patients conversely , in slovenia and in poland 5455% of patients were treated with blood flows below 300 ml / min . figure 3 shows the distribution of patients according to the prescribed needle size , blood flow , and venous pressure levels . the primary outcome event ( i.e. , surgery for a new va during the follow - up period ) was observed in 1485 patients ( 21% ) . univariate survival analysis revealed a significant benefit for access survival for patients who are younger , nondiabetic , male , have lower body mass index , do not take platelet antiaggregants , do not have heart failure , and are able to assist with compression . a significant benefit was also seen for patients with fistula ( vs. graft ) , smaller needles , distal location of the access , and low venous pressure . with regard to cannulation technique , positive effects were observed for antegrade needle direction ( vs. retrograde ) , bevel up ( vs. down ) , nonalcohol - based disinfection , and application of local anesthesia . although not statistically significant , a potential survival benefit was indicated for higher blood flow ( p=0.056 ) and buttonhole technique ( vs. rope - ladder and area , p=0.11 ) . needle rotation did not affect the access survival ( p=0.81 ) , neither did access vintage ( age < 1 month before baseline vs. 1 month before baseline ; p=0.29 ) . meier access survival curves according to blood flows , venous pressures , needle sizes , and cannulation techniques are presented in figure 4 . in a second step , after adjustment for age , gender , diabetes , va type , access location ( proximal vs. distal ) , dialysis vintage and heart failure , and incorporation of country differences , the use of a 16-g needle was associated with a significantly higher risk of access failure ( hazard ratio ( hr ) 1.21 ) compared with the use of a 15-g needle . very few ( 1.4% ) patients were treated with the even smaller 17-g needles , but the direction of the results is the same , that is , increased hr for smaller needle size . using a blood flow of 300350 ml / min as a reference , the hr tended to decrease as the blood flow increased . with regard to cannulation technique , both rope - ladder and buttonhole techniques performed significantly better than the area technique . considering antegrade with the bevel up as reference , the retrograde direction of the arterial needle with bevel down is associated with a significant increase of access failure risk of 18% . all other options , that is , antegrade direction with bevel down or retrograde direction with bevel up , were not associated with a hr significantly different from 1.00 . with regard to venous pressure , using as reference the range between 100 and 150 mm hg , the hrs increased proportionally to 1.4 , 1.87 , and 2.09 with the increase of venous pressure from 150 to 200 mm hg , 200 to 300 mm hg , and > 300 mm hg , respectively ( all p0.008 ) . of note , venous pressures of > 300 mm hg are extreme cases and were only recorded in 0.6% of the patients . in addition , a venous pressure of < 100 mm hg was associated with a significantly higher hr of 1.51 . to investigate this further , we also looked for interaction effects between blood flow and venous pressure , as well as between arterial and venous pressures ; no significant associations were found . finally , the use of a tourniquet and not applying any pressure at the time of cannulation were associated with hrs of 1.30 and 1.25 ( p<0.008 and < 0.02 ) , respectively , compared with exertion of arm compression by the patient at the time of cannulation ( labeled patient assistance ' in table 1 ) . in summary , this study revealed that area cannulation technique , albeit being identified as the most commonly used technique in this population of over 7000 patients , was inferior to rope - ladder and to buttonhole for maintenance of va functionality . with regard to the effect of needle and bevel direction , the combination of antegrade positioning of the arterial needle with bevel - up orientation was significantly associated with better access survival than retrograde positioning with bevel down . the use of larger needles tended to favor access patency , with 15 g being superior to 16 or 17 g. the application of arm pressure by the patient at the time of cannulation had a favorable effect on access longevity compared with not applying pressure or using a tourniquet . results pertaining to the type and location of the access and the technical parameters ( i.e. , blood flow and venous pressure ) were as follows : there was an increased risk for access failure for grafts vs. fistulas , proximal location vs. distal , right arm vs. left arm , blood flows below 300 ml / min vs. those in the range of 300350 ml / min , and for the presence of a venous pressure > 150 mm hg vs. pressures between 100 and 150 mm hg . tissue reparative processes triggered by cannulation procedures may cause enlargement of the fistula and the formation of aneurisms and scars that , in turn , can favor the development of stenotic lesions and ultimately impact fistula survival . repetitive punctures at the va site cause vessel wall defects that are initially filled by thrombi before finally healing . of the three cannulation techniques , the buttonhole approach has the theoretical advantage of limiting the process of dilatation and fibrosis because the thrombus is displaced while being formed , favoring the formation of a cylindrical scar from the subcutaneous and vessel wall tissues . the rope - ladder technique may have the initial advantage of favoring progressive maturation along the entire length of the fistula , but it requires fistula with sufficiently long segments suitable for cannulation . the area puncture technique weakens the fistula wall and is associated with the least favorable consequences , that is , localized dilation , disruption of the vessel wall , and subsequent development of ( pseudo)aneurysms and strictures . despite this and the fact that area cannulation has been discouraged for over two decades , it was disheartening to observe that this was the predominant practice in almost two - thirds of patients . according to the ebpg and the clinical practice guidelines for va , the rope - ladder technique should be used for cannulation of grafts . specifically , according to the latter , this study showed a 22% lower risk for va failure in those patients whose va was cannulated with the buttonhole technique as opposed to area , confirming the results of a recently published randomized controlled clinical trial . although the buttonhole technique is associated with good results , one should also take into consideration that it is a practice performed in centers with highly trained personnel that work with strict protocols and that it may also be used for fistulas with only short segments available for cannulation . in our study , this practice is used in 22 centers , mainly in portugal , turkey , the united kingdom , and italy . research questions that arise from current guidelines address the effectiveness of structured cannulation training , increased remuneration for expert cannulators , and whether self - cannulation can lead to better outcomes . indeed , as buttonhole cannulation requires the designation of a reference nurse , especially for the initial 46 weeks , it is likely that this technique benefits from its association with centers offering the necessary training ( i.e. , centers capable of stemming the increased organizational effort and assigning the right cannulator to the right patient ) . in addition , once the tunnel is created , cannulation can be performed directly by patients . however , irrespective of the influence of cannulator training and center organizational issues , the underlying question to be addressed , optimally in a well - designed clinical study , is which cannulation techniques can be recommended to ensure long - term va functionality . this study showed that retrograde direction of arterial needle with bevel down is associated with the least favorable outcome . this is consistent with the findings of woodson and shapiro who reported that retrograde puncturing may be associated for an increased risk of hematoma formation , possibly owing to the related venous return of the blood ( i.e. , retrograde filling ) . antegrade puncturing , on the other hand , may be considered fistula - protective by the same reasoning , that is , tract closure through flow force . therefore , retrograde direction of the arterial needle is more likely to be associated with a higher risk for aneurism . despite recommendations by kdoqi to rotate the needle during insertion , the univariate analysis performed here found no evidence of any benefit of this practice . on the contrary , the authors share the opinion of many cannulators that the 180 rotation of the needle is unnecessary and may constitute an additional trauma to the va . further studies are needed to clarify whether rotation of the va needle during cannulation should be recommended or not . there are a number of possible reasons for the association of the higher failure risk with smaller needle sizes . while increased trauma and prolonged bleeding time are generally associated with the use of large needles , the use of small needles at the same blood flow results in a higher speed of the blood returning to the vasculature , possibly damaging the intima of the avfs . for example , at an operative blood flow of 350 ml / min , the maximum speed of the injected blood will be 8.79 m / s with a 17-g needle and 5.80 m / s with a 15-g needle ( presented by ralf jungmann at vascular access coursestockholm , 1112 october 2012 , stockholm , sweden ) . furthermore , the shear forces created by returning blood can have a role in inflammation and stenosis formation . stenotic fistula and graft lesions are associated with the induction of the expression of profibrotic cytokines , local inflammation , and neointimal proliferation . however , we can not exclude that this association may be a consequence of bias by indication . needles of smaller inner dimension are generally prescribed not only for a new va but also for problematic avfs , that is , those likely to fail in the following months . therefore , it is difficult to derive a conclusion from this association , but on the basis of figure 3 , 17-g needles are clearly linked to blood flow levels below 300 ml / min and , on the contrary , 14-g needles are mainly prescribed to patients with 350400 ml / min or greater blood flows . it is also of interest to underline that higher venous pressure is mainly associated with the 16-g needles , which have a wider distribution of different blood flows . measurement of venous pressure during dialysis is currently used as a surveillance tool within the dialysis session , and not as a standard monitoring strategy . this study showed a significant and proportionally increasing risk of va failure with venous pressures higher than 150 mm hg . an increased hr was also detected for venous pressure below 100 mm hg . as shown in figure 3 , an association between needle size , blood flow , and venous pressure is indicated in that for needle gauges 15 , 16 , and 17 low venous pressures appear to be associated with low blood flows . such an association could be an indication of stenosis in the artery . venous pressure is crucially dependent on the characteristics of the needle ( e.g. , the needle gauge , the length of the metallic portion , and the length and the thickness of the needle shaft ) , which vary among manufacturers . in this network , at the time of the study , the vast majority of the needles ( 85% ) were from a single producer and the length of the needle was 25 mm . the unexpectedly high hr associated with a venous pressure of under 100 mm hg compared with 150200 mm hg should motivate reflection on the currently accepted limits . one could consider integration of venous pressure monitoring into an algorithm for the detection of increased risk of access failure . this study has certain limitations over and beyond those inherent to observational studies , for example , that residual confounding can not be completely ruled out . being a retrospective study , patient data for those patients on dialysis before admission to the nephrocare clinic were not collectable , and thus robust information on the number of prior vaes , on their respective lengths , and on first cannulation was not available . particularly , the missing information on the length of the va , its depth , and the access flow constitute a major weakness because a particular cannulation technique could have been chosen on the basis of what is possible with the given access characteristics . in addition , the length of the access can influence the way in which the needles are placed . despite these missing data , we feel that this study has its merits , as it shows that traditional local practices have a significant influence on procedures exercised . a further limitation is that the va practice was surveyed in april 2009 and was assumed not to have been changed during the follow - up ( 31 march 2012 ) . however , as nursing practices in this field are strongly related to the clinic culture and experience , we have reason to believe that it is should not constitute a significant bias . of course , some cannulation particulars , such as needle size and arterial blood flow , may vary over time , in that smaller needle sizes and low blood flow rate are used for initial access use and that large needles are taken for mature accesses . however , we feel that the model selected here is also justified because it is an explanatory model , based on the association of baseline characteristics with access survival . other limitations are that we had follow - up of 65% of the patients and that most countries were in europe ( owing to deployment of the electronic reporting system ) . as reported , an association between clinical practice patterns and country has been detected , and consequently not all different practices were covered by our model . however , according to the results of this analysis , each country has a combination of practices that positively and negatively influence the va survival . for example , in romania , positive influences were the puncture direction being antegrade ( 82% ) , bevel orientation being predominantly upward ( 95% ) , and needle not rotated ( 84% ) ; negative associations were the use of area technique ( 77% ) , preferred needle size ( 75% with 16 g ) , and the use of blood flows < 300 ml / min ( 47% ) . for this reason , intracountry correlations were considered using a sandwich estimator in the multivariate model . to assess the influence of individual center practices , we also performed a sensitivity analysis by applying the sandwich estimator at the center level . there were only negligible differences to the results obtained with the original model at the country level , raising our confidence that there is no severe confounding of the model by center practice effects . given the relevant impact of the investigated variables on the survival of the va , itself a key driver of hemodialysis patient survival , we believe it is time to organize a large - scale randomized clinical trial to facilitate the formulation of practical and comprehensive cannulation practice guidelines . as the associations between practice patterns and va survival reported here are mainly related to national procedures and only partially related to actual patient limitations , they offer some promising indications for improving clinical practice . in april 2009 , a cross - sectional survey was conducted in 171 dialysis units located in europe , the middle east , and africa to collect details on va cannulation practices on a clinic by clinic level . all patients who were on double - needle hemodialysis or online hemodiafiltration during the week of the survey were selected for analysis , as long as a fistula or graft was used for va , survey data were complete , and follow - up data were available in our clinical database . primary outcome was time until the first surgical access intervention resulting in the generation of a new access ( i.e. , as opposed to any surgical intervention done just for revision , thrombectomy , etc . , or any endovascular intervention ) . patients were censored for transplantation , death , loss of follow - up , or end of the follow - up period ( 31 march 2012 ) . information on cannulation retrieved from the survey comprised fistula type and location , cannulation technique , needle size , needle and bevel direction , needle rotation , blood flow , arterial and venous pressure , use of disinfectants , use of local anesthesia , and application of arm compression at the time of cannulation . to adjust for individual patient characteristics , the following information was extracted from the clinical database : patient age , gender and body mass index , prevalence of diabetes , and the use of ace inhibitors , platelet antiaggregants , and anticoagulants . in addition , the median blood flow prescription was documented at a center level at the time of the survey . for univariate analysis , kaplan meier curves were calculated and comparisons were performed using the log - rank test . by combining univariate results with medical and statistical experience , a set of variables for multivariable analysis was determined . in particular , specific interaction terms ( e.g. , bevel vs. arterial needle direction ) were defined for statistical examination , and decisions were made regarding their inclusion or omission in the cox model depending on their significance or collinearity , respectively . a final cox model based on these variables was calculated , using the sandwich estimator to account for within - country correlation . step by step , the final model was reduced , setting a p - value of 0.1 for variable inclusion . all analyses were performed with sas v9.2 ( sas institute , cary , nc ) .

the initial step of atrioventricular ( av ) valve development involves the deposition of extracellular matrix ( ecm ) components of the endocardial cushion and the endocardialmesenchymal transition . while the appropriately regulated expression of the major ecm components , versican and hyaluronan , that form the endocardial cushion is important for heart valve development , the underlying mechanism that regulates ecm gene expression remains unclear . we found that zebrafish crip2 expression is restricted to a subset of cells in the av canal ( avc ) endocardium at 55 hours post - fertilization ( hpf ) . knockdown of crip2 induced a heart - looping defect in zebrafish embryos , although the development of cardiac chambers appeared to be normal . in the avc of crip2-deficient embryos , the expression of both versican a and hyaluronan synthase 2 ( has2 ) was highly upregulated , but the expression of bone morphogenetic protein 4 ( bmp4 ) and t - box 2b ( tbx2b ) in the myocardium and of notch1b in the endocardium in the avc did not change . taken together , these results indicate that crip2 plays an important role in av valve development by downregulating the expression of ecm components in the endocardial cushion .

the developing vertebrate heart tube contains an outer layer of myocardium and an inner layer of endocardium separated by an extracellular matrix ( ecm ) referred to as the cardiac jelly . during heart valve formation , a subset of endocardial cells in the atrioventricular ( av ) canal ( avc ) region is specified to delaminate , differentiate , and migrate into the cardiac jelly in a process called endocardial - mesenchymal transition ( emt ) ( armstrong and bischoff , 2004 ; combs and yutzey , 2009 ) . for av valve formation , locally swollen cardiac jelly together with mesenchymal cells derived from avc endocardial cells form the endocardial cushion ; however the extensive remodeling process that occurs subsequently is not clearly understood ( armstrong and bischoff , 2004 ; combs and yutzey , 2009 ) . signal molecules produced from both the myocardium and endocardium in the avc region are necessary for proper endocardial cushion formation and emt of endocardial cells ( krug et al . , 1985 ) . in the initial step of endocardial cushion formation , a bone morphogenetic protein ( bmp ) signal from avc myocardium inhibits the expression of chamber - specific genes in the avc region , while the endocardial cushion is enlarged by increased deposition of ecm or cardiac jelly between the myocardium and endocardium layers in the avc . upon receiving a myocardially derived bmp signal , a subset of avc endocardium cells undergo the emt process by producing multiple signals such as transforming growth factor , notch , and wnt/-catenin , and then migrate into the cardiac jelly where they transition into mesenchymal cells that form valve precursors during later development ( armstrong and bischoff , 2004 ; combs and yutzey , 2009 ; dlot , 2003 ; nakajima et al . , 2000 ) . therefore , deficiencies in bmp or notch signal - related genes inhibit or disrupt normal endocardial cushion formation and/or failure of avc endocardium emt in the mouse , chick , and zebrafish ( jiao et al . , 2003 ; ma et al . , 2005 ; in addition , fgfs , vegfs , nfat , and hdac are also prerequisites for normal heart valve development ( combs and yutzey , 2009 ; kim et al . , 2012b ) . glycosaminoglycan hyaluronan exists as a hydrated gel that expands the extracellular space , regulates ligand availability , and interacts with numerous ecm components including versican , a major constituent of cardiac jelly ( armstrong and bischoff , 2004 ) . disruption of hyaluronan synthase 2 ( has2 ) or versican in mice and zebrafish is reported to abrogate avc endocardial cushion formation and emt - associated mesenchymal cell migration , suggesting that an appropriate ecm environment is important for both emt of avc endocardium development and formation of the endocardial cushion ( camenisch et al . , 2000 ; although there is some debate on whether emt is a prerequisite for the migration of avc endocardial cells into the cardiac jelly during endocardial cushion formation in zebrafish , the mature av valve of the 2-chambered zebrafish adult heart and 4-chambered mammalian valves are structurally similar . in addition , cellular and molecular signaling events occurring during heart valve development are largely conserved between zebrafish and mammals ( beis et al . cysteine - rich protein 2 ( crip2 ) is a crip - type subgroup of the lim domain protein family and has two conserved cysteine - rich motifs that contain cysteine and histidine residues with zinc - binding properties ( karim et al . , 1996 ; tsui et al . mouse crip2 is reported to be highly expressed in the heart , brain , testis , kidney , and lung , and is involved in the dynamics of the cortical actin cytoskeleton by binding ptp - bl , a protein tyrosine phosphatase ( van ham et al . , 2003 ) . in zebrafish , crip2 is reported to be a target of wnt3a signaling and regulates smooth muscle cell differentiation ( kihara et al . , 2011 ) and the development of cardiac neural crest cells during early embryogenesis ( sun et al . , 2008 ) . however , the direct role of crip2 in the av valve formation has not yet been elucidated . zebrafish is a useful animal model for studying development of the heart and other circulation systems , including blood and lymphatic vessels ( kim and kim , 2014 , kim et al . , 2012a ; 2013a ; 2013b ; ) because the embryos are transparent and a multitude of transgenic lines that express fluorescent proteins in tissue- and cell - specific manners are readily available . in zebra - fish , the heart can be observed under a dissecting microscope soon after formation of the primitive heart tube . the heart starts beating at 24 h post - fertilization ( hpf ) , heart looping begins at 36 hpf , functional valves are formed by 48 hpf , and heart valve development is complete by approximately 55 hpf ( stainier et al . , 2002 ) . in this study , we found that crip2 expression in the avc endocardial cells , but not in the avc myocardial cells , in late heart development is critical for heart valve development in zebrafish embryos . furthermore , crip2-deficient embryos showed markedly increased expression of the has2 and versican a genes , which are responsible for the synthesis of versican a and hyaluronan . we provide data that crip2 expressed in the avc endothelial cells plays an important role in cardiac valve formation by suppressing genes involved in the synthesis of versican a and hyaluronan , which are essential ecm components in the cardiac jelly and endocardial cushion . zebrafish ( danio rerio ) were raised and maintained at 28.5c , and embryos were staged as described in a previous study ( kimmel et al . , 1995 ) . embryos were obtained from spontaneous spawnings and the embryos were manually dechorionated at appropriate stages by using watchmaker s forceps and then fixed using 4% paraformaldehyde in phosphate - buffered saline . tg ( cmlc2:egfp ) ( huang et al . , 2003 ) and tg ( flk1:egfp ) ( jin et al . , 2005 ) embryos were used for phenotypic analyses and immunostaining . antisense digoxigenin - labeled rna probes for crip2 , vmhc , cmlc2 , cdh5 , bmp4 , tbx2b , notch1b , versican a , and has2 were generated using in vitro transcription with t3 , t7 , or sp6 rna polymerase , according to the manufacturer s instructions ( roche , germany ) . whole - mount in situ hybridization was performed at the desired embryonic stages , as described by jowett and lettice ( 1994 ) . for fluorescent immunostaining after in situ hybridization , transgenic embryos were immunostained by anti - gfp antibody ( santa cruz biotechnology , inc . processed embryos were photographed using a microscope ( axioskop , zeiss , germany ) outfitted with a nikon coolpix 4500 digital camera system and a laser - scanning confocal microscope ( lsm 5 pascal ) . crip2 antisense morpholino oligonucleotide ( crip2 mo ) was designed and ontained from gene tools llc . ( usa ) to block the splicing of crip2 exon 2 and intron 2 and had the following sequence : 5-catgccgaggtgagtaaaacagaga-3. to generate crip2 sense mrna , the full open reading frame of crip2 ( nm_213497 ) was cloned into the pcs2 + expression vector . the construct was linearized using noti digestion and capped rna was synthesized using mg ( 5 ) ppp ( 5 ) g ( roche ) and sp6 rna polymerase ( roche ) . the rna was diluted to 100 ng/l and phenol red was added to a final concentration of 0.2% immediately before injection . the synthesized crip2 mo and sense mrna were injected into one- or two - cell stage embryos by using a gas driven microinjector ( world precision instruments , usa ) . zebrafish ( danio rerio ) were raised and maintained at 28.5c , and embryos were staged as described in a previous study ( kimmel et al . , 1995 ) . embryos were obtained from spontaneous spawnings and the embryos were manually dechorionated at appropriate stages by using watchmaker s forceps and then fixed using 4% paraformaldehyde in phosphate - buffered saline . tg ( cmlc2:egfp ) ( huang et al . , 2003 ) and tg ( flk1:egfp ) ( jin et al . , 2005 ) embryos were used for phenotypic analyses and immunostaining . antisense digoxigenin - labeled rna probes for crip2 , vmhc , cmlc2 , cdh5 , bmp4 , tbx2b , notch1b , versican a , and has2 were generated using in vitro transcription with t3 , t7 , or sp6 rna polymerase , according to the manufacturer s instructions ( roche , germany ) . whole - mount in situ hybridization was performed at the desired embryonic stages , as described by jowett and lettice ( 1994 ) . for fluorescent immunostaining after in situ hybridization , transgenic embryos were immunostained by anti - gfp antibody ( santa cruz biotechnology , inc . processed embryos were photographed using a microscope ( axioskop , zeiss , germany ) outfitted with a nikon coolpix 4500 digital camera system and a laser - scanning confocal microscope ( lsm 5 pascal ) . crip2 antisense morpholino oligonucleotide ( crip2 mo ) was designed and ontained from gene tools llc . ( usa ) to block the splicing of crip2 exon 2 and intron 2 and had the following sequence : 5-catgccgaggtgagtaaaacagaga-3. to generate crip2 sense mrna , the full open reading frame of crip2 ( nm_213497 ) was cloned into the pcs2 + expression vector . the construct was linearized using noti digestion and capped rna was synthesized using mg ( 5 ) ppp ( 5 ) g ( roche ) and sp6 rna polymerase ( roche ) . the rna was diluted to 100 ng/l and phenol red was added to a final concentration of 0.2% immediately before injection . the synthesized crip2 mo and sense mrna were injected into one- or two - cell stage embryos by using a gas driven microinjector ( world precision instruments , usa ) . in an in situ hybridization - based screen for candidate genes that regulate heart valve development , we found that crip2 expression was restricted to the avc region in 48-hpf zebrafish embryos ( fig . 1 and data not shown ) . detailed analysis of the gene expression profile showed that crip2 mrna was detected in zebrafish embryos at the one - cell stage , indicating that crip2 was maternally expressed ( fig . at the shield stage , crip2 was ubiquitously expressed in the entire embryonic cell ( fig . 1b ) , but its expression became restricted to the mesodermal region along the anterior - posterior axis at the 10 somite - stage ( ss ) ( fig . crip2 expression became further restricted to the head and trunk mesoderm , including the somites , lateral plate mesoderm , and notochord posterior edge ( fig . transverse sections of the posterior trunk region further confirmed its expression in the somitic muscle ( fig . 1h ) , and new expression appeared in the pharyngeal arch blood vessels ( fig . these gene expression profiling data suggest a function of zygotic crip2 in mesoderm - derived tissue formation during early embryogenesis . during heart development , crip2 expression was observed in the linear heart tube in 24 hpf embryos ( fig . 1k ) and its strong expression persisted in the ventricle and av canal regions at 36 hpf , but was barely detected in the atrium ( fig . crip2 expression was decreased in the ventricle and remained only at the avc and outflow tract ( figs . when heart valve development was completed at 55 hpf , crip2 expression at the avc and outflow tract had started to decrease ( fig . 1o ) . in tg(flk1:egfp ) transgenic zebrafish embryos , green fluorescent protein ( gfp ) is expressed in all endothelial cells ( jin et al . , 2005 ) . to verify which cells express crip2 , 48 hpf tg ( flk1:egfp ) embryos were immunostained with gfp antibodies and then whole - mount in situ hybridization was performed using a fluorescent crip2 riboprobe . endocardial cells in the ventricle , atrium , and avc region were visualized using the gfp antibody ( fig . 1p ) , and expression of crip2 was detected in cells at the avc and outflow tract ( fig . 1p and 1q , we confirmed that crip2 is expressed specifically in the endocardium of the avc and outflow tract and not in the heart chamber myocardium or endocardium during the late stage of heart development ( figs . to evaluate the contribution of crip2 to late heart development , we designed a crip2 splice morpholino ( crip2 mo ) ( fig . the crip2 mo effectively blocked splicing between exon 2 and intron 2 and resulted in a decrease of crip2 mrna transcript ( fig . 2b , * * ) and an increase in the aberrant transcript containing intron 2 ( fig . 2b , * ) in 24 hpf embryos . similarly , the abundance of the crip2 mrna transcripts was clearly reduced by the crip2 mo ( figs . 2d and 2d ) in 24 hpf embryos when compared to non - injection control embryos ( nics ; figs . 2c and 2c ) examined using whole - mount in situ hybridization . to analyze the effect of crip2 knockdown on heart tube formation , we used tg ( cmlc2:egfp ) transgenic embryos that express gfp in myocardial cells in both ventricular and atrial portions of the heart tube ( huang et al . , 2003 ) . depending on the amount of crip2 mo injected into one - cell stage embryos , three variable phenotypes were induced in 48 hpf embryos : normal ( fig . 2g ) and typical ring structures in the avc region ( arrow ) were observed ( fig . 2 g ) . however , typical ring structures were not formed in the type 1 and type 2 embryos because heart tubes failed to undergo looping ( figs . 2 h , 2h , 2j , and 2j ) . because type 2 morphant embryos ( fig . 2i ) showed more severe developmental defects than type 1 morphants , type 1 embryos were selected for further analysis of crip2 function in heart development . the ratios of the dose - dependent phenotypes were calculated and these data are summarized in fig . 2k . collectively , our data demonstrating that morphological heart valve defects occur in crip2 morphant embryos and that crip2 expression becomes spatiotemporally restricted to the endocardium of the avc and outflow tract strongly suggest an important role for crip2 in heart valve development . to further assess the function of crip2 in late heart development , including heart valve formation cardiac myosin light chain 2 ( cmlc2 ) is expressed in myocardia throughout both ventricular and atrial portions of the heart tube , whereas ventricular myosin heavy chain ( vmhc ) is restricted to the myocardia in the ventricular portion ( yelon et al . , 1999 ) . ve - cadherin ( cdh5 ) , on the other hand , is specifically expressed in the endocardium during heart development ( larson et al . , 2004 ) . in zebrafish embryos , the total abundance of cdh5 mrna was also not changed by crip2 mo ( figs . 3e and 3f ) , suggesting that crip2 is not involved in the development of myocardium and endocardium comprising the outer layers of heart chambers . next , we examined the expression of genes required for the development of the heart valve . bmp4 is known to be highly expressed specifically in the avc myocardium in zebrafish embryos ( larson et al . , 2004 ) . in the crip2 morphant embryos , bmp4 was expressed in the avc region ( fig . 3h , arrows ) , in a manner similar to that of the control embryos ( fig . 3 g ) . in agreement with this , expression of tbx2b , a marker of the avc myocardium ( walsh and stainier , 2001 ) , was also not altered ( figs . 3i and 3j , arrows ) . in mice , notch promotes emt during heart valve development and inhibition of notch activity prevents valve formation in mice and zebrafish ( timmerman et al . , 2004 ) . notch1b , a mouse notch1 ortholog , was expressed in the avc endocardium of 48 hpf zebrafish embryos ( hurlstone et al . , 2003 ) , thus we tested the expression of notch1b in crip2 morphant embryos . we found that the expression level of notch1b detected in the avc region of morphant embryos was comparable to that of control embryos at 48 hpf ( figs . 3k and 3l ) . this observation indicated that crip2 is not involved in regulating the emt process in the avc endocardium . an appropriate ecm environment in the endocardial cushion is reported to be required for normal heart valve formation ( camenisch et al . in zebrafish , it is well established that versican a and has2 synthesize versican a and hyaluronan , respectively , and that these genes have restricted expression in the avc region in 48 hpf embryos ( hurlstone et al . , 2003 ) . to further investigate whether crip2 regulates synthesis of ecm components , we analyzed the expression of versican a and has2 in crip2 morphant embryos . the expression of versican a in the avc region was significantly increased in the morphant embryos ( fig . 4a ) . similar to versican a , the expression of has2 in the avc region was markedly upregulated by the knockdown of crip2 ( figs . 4c and 4d ) . these results imply that crip2 functions as a negative regulator of ecm - related gene expression during av valve development . we also tested whether overexpression of crip2 could down - regulate versican a and has2 expression . in wild - type embryos , overexpression of crip2 did not decrease the expression of versican a and has 2 , although severe developmental defects , including curled body and cardiac edema , were observed ( supplementary fig . these results suggested that the expression level of crip2 in wild - type embryos is sufficient to suppress the expression of versican a and has 2 ; thus , further suppression of these ecm genes could not be achieved by the overexpression of crip2 . alternatively , crip2 might require a cofactor to suppress versican a and has 2 expression . therefore , overexpression of crip2 alone can not further suppress the expression of versican a and has 2 . adenomatous polyposis coli ( apc ) , a well - known tumor suppressor protein , negatively regulates wnt/-catenin signaling ( hurlstone et al . , 2003 ) . moreover , heart valve defects in apc null mice are reported to be caused by increased versican a and has2 expression in the avc region , indicating that versican a and has2 are transcriptional targets of the wnt/-catenin pathway ( hurlstone et al . according to our crip2 knockdown data , expression of versican a and has2 at the avc was markedly upregulated , suggesting an antagonistic role of crip2 towards wnt/-catenin signaling that activates versican a and has2 expression . our results , together with that of previous reports , indicate that either deficiency or over - production of versican and has2 causes heart valve defects ( hurlstone et al . , 2003 ; schroeder et al . , , we report a novel function of crip2 in suppressing the expression of versican a and has2 in the avc region , which is essential for normal heart valve development . through its spatiotemporal regulation of gene expression , crip2 likely contributes to the formation of an appropriate ecm environment in the endocardial cushion region during multiple stages of heart valve development .

a 38-year - old man without any symptoms was admitted to our institution because of an abnormal shadow found incidentally on a chest x - ray . chest computed tomography showed a round mass in the lingular segment of the left upper lobe . lingular segmentectomy was performed , and the histopathological diagnosis was intrapulmonary schwannoma . immunohistochemical staining revealed a positive result for s-100 protein and negative results for cd34 and desmin . we report this case of intrapulmonary schwannoma , which is extremely rare .

schwannoma may arise from any peripheral nerve and is often found in the chest wall and posterior mediastinum . however , case reports of intrapulmonary schwannoma are extremely rare . a 38-year - old man was referred to our institution because of an abnormal shadow found incidentally on the chest x - ray . the plain chest x - ray showed a mass lesion in the left middle zone . chest computed tomography ( ct ) showed a round and homogeneous mass 25 18 mm in size with a well - defined margin in the lingular segment of the left upper lobe ( fig . 18-fluorodeoxy - glucose ( fdg ) positron - emission tomography ( pet ) showed no accumulation in the tumor ( fig . the ct and fdg - pet findings suggested the possibility of a benign tumor or a low - grade malignancy . as a malignant tumor could not be definitively ruled out , we performed lingular segmentectomy.fig . 1 a computed tomography shows a mass with well - defined margins in the lingular segment of the left upper lobe . b 18-fluorodeoxy - glucose ( fdg ) positron - emission tomography ( pet ) showed no accumulation in the tumor a computed tomography shows a mass with well - defined margins in the lingular segment of the left upper lobe . b 18-fluorodeoxy - glucose ( fdg ) positron - emission tomography ( pet ) showed no accumulation in the tumor macroscopic examination of the resected specimen showed a well - demarcated round tumor in the lung , without any evidence of invasion of the surrounding tissues . microscopic examination revealed proliferation of elongated tumor cells having spindle - shaped nuclei , with cellular palisading ( fig . immunohistochemical staining demonstrated positive staining of the tumor cells for s-100 protein and bcl2 , but negative staining for cd34 and desmin . the histopathological diagnosis was intrapulmonary schwannoma.fig . 2 a macroscopic examination of the specimen showed a round , well - demarcated tumor in the lung , without invasion of the surrounding tissues . b microscopic examination revealed elongated tumor cells with spindle - shaped nuclei a macroscopic examination of the specimen showed a round , well - demarcated tumor in the lung , without invasion of the surrounding tissues . schwannoma is one of the few truly encapsulated neoplasms of the human body , and is almost always solitary . it most commonly occurs on the flexor surfaces of the extremities , neck , mediastinum , retroperitoneum , posterior spinal roots , and cerebellopontine angle . however , schwannomas are extremely rare in the lung , regardless of the patient age . ohtsuka and colleagues reported 62 patients ( 28 men , 34 women ) aged 583 years , with intrapulmonary or bronchial schwannomas , and stated that this neoplasm accounted for about 0.2 % of all pulmonary neoplasms . in 55 % of these patients , the tumor originated within the tissues in proximity to the terminal segmental bronchus . on radiographic images , peripheral intrapulmonary schwannoma appears as a round mass with well - defined margins . in patients with the tumor located proximal to a lobar bronchus , atelectasis or pneumonia about half of the patients have some symptoms ; however , most patients with peripheral intrapulmonary schwannomas , as in the present case , have no symptoms . today , fdg - pet is a useful approach to differentiate malignant solitary pulmonary nodules from benign nodules . beaulieu et al . reported the maximum standard uptake values ( suvmax ) of the 10 schwannomas ranged from 1.9 to 7.2 , and the variation in the suvmax could be explained by the variation in cellularity . however , the reason why high fdg accumulation is found in benign tumors such as schwannoma remains unclear , and a diagnostic value of fdg - pet with intrapulmonary schwannoma is not established . it is reported that in rare instances , schwannomas may undergo malignant transformation . the expression level of ki67 , a tumor cell proliferation marker , has been reported to be useful for determining the malignant potential of these tumors . kindblom and colleagues compared the ki67 expression levels in 26 malignant peripheral nerve sheath tumors and 24 benign nerve sheath tumors ( schwannomas ) , and reported that significantly high density of nuclear staining was found in malignant peripheral nerve sheath tumors . for the treatment of primary intrapulmonary schwannoma , surgical resection , intrabronchial resection with endoscopy , and yttrium aluminium garnet ( yag ) laser resection have been employed [ 57 ] . because of the low malignant potential of these tumors , tumor enucleation or partial lung resection is thought to be adequate , with lobectomy not being necessary . in the present case , we performed segmentectomy to accomplish complete resection , because we did not obtain the pathological diagnosis before the operation and the tumor was located in the central part of the superior lingular segment . in conclusion ,

the application of a 32p - postlabeling assay for 7-methylguanines in dna was studied either by labeling the imidazole ring - opened dinucleotide derivatives or by using strong - anion - exchange column chromatography for the adduct enrichment from normal nucleotides . data showed that 7-methylguanines can be efficiently labeled as dinucleotides when in vitro methylated dna was first imidazole ring - opened and then digested to the dinucleotide level with deoxyribonuclease i , snake venom phosphodiesterase , and prostatic acid phosphatase . when using ion exchange chromatography for the adduct enrichment , dna was digested with micrococcal nuclease and spleen phosphodiesterase . anion exchange chromatography was applied for 7-methylguanine measurements in white blood cell dna of healthy nonsmokers ( n = 17 ) and patients ( n = 4 ) treated with the methylating drugs procarbazine and decarbazine . we found that the mean level of 7-methylguanine residues in nonsmokers was 2.5 per 10(7 ) nucleotides . the corresponding level in the patient samples immediately after the drug treatment was 57 per 10(7 ) nucleotides.imagesfigure 2 .

abstractthis article describes the aetiology , epidemiology and clinical significance of incidental non - solid pulmonary nodules . non - solid nodules are more likely malignant . if malignant , they are mostly due to atypical adenomatous hyperplasia and bronchioloalveolar carcinoma . as these may be negative on positron emission tomography and slow growing , the diagnostic algorithms that are used for solid nodules have to be modified for non - solid nodules .

a pulmonary nodule is defined as a spherical well - circumscribed radiographic opacity that is surrounded completely by aerated lung . lesions that are larger than 3 cm are described as lung masses . as size has been shown to be important for classification of nodules ( see below ) the term micronodules is usually applied to very small nodules ( < 7 mm , < 5 mm ) which are almost always multiple and diffuse . if only one pulmonary nodule is detected , it is described as a solitary pulmonary nodule ( spn ) . if a nodule is detected in an examination performed for other reasons than a search for pulmonary nodules , it is called an incidental nodule , . most of the information on the aetiology and the natural course of incidentally found small pulmonary nodules is derived from studies on lung cancer screening with unenhanced low - radiation dose computed tomography ( low - dose ct ) in asymptomatic smokers and to a lesser extent non - smokers and workers exposed to asbestos . in these studies , at least one non - calcified pulmonary nodule was found in up to 66% of subjects , depending on the imaging technique ( e.g. multidetector - computed tomography ( ct ) versus single - detector ct ) . it was also shown that > 95% of these nodules were 10 mm in diameter and of these > 95% were benign . therefore , in order to avoid a high rate of invasive procedures for benign lesions in small nodules , algorithms were proposed that are based on mostly non - invasive strategies and , in particular , follow - up with low - dose ct . during follow - up changes in size ( or even better volume ) are recorded . nodules that decrease in size or resolve are obviously regarded as benign and no further follow - up is required . nodules that increase in size are likely malignant , particularly , if the growth rate is typical of malignant growth . most malignant tumors have been shown to double their volume ( i.e. increase their diameter in spherical lesions by 26% ) between 30 and 400 days . if malignancy is suspected , invasive procedures are usually performed to obtain a histological diagnosis ( bronchoscopic , percutaneous or thoracoscopic biopsy ) . in addition , positron emission tomography ( pet)-ct may be useful for small nodules of intermediate size ( 610 mm ) , as many malignant nodules show increased uptake of [ f]fluorodeoxyglucose ( fdg ) , whereas most benign nodules do not . pet - ct , however , is often negative in well - differentiated adenocarcinoma and bronchioloalveolar carcinoma ( bac ) and may be positive in inflammatory nodules ( e.g. sarcoid , tuberculosis ) . figure 1three non - solid nodules ( ground glass opacities ) in the posterior segment of the right upper lobe . three non - solid nodules ( ground glass opacities ) in the posterior segment of the right upper lobe . if the density of a nodule is lower than soft tissue attenuation , it does not obscure adjacent or transgressing vessels . part - solid nodules are characterised by a mixture of ground glass attenuation and solid components . benign non - solid nodules are due to inflammation ( eosinophilic pneumonia , cryptogenic organizing pneumonia ) , focal haemorrhage ( pulmonary endometriosis , pulmonary trauma , post biopsy ) , and focal interstitial fibrosis . persistent non - solid lesions , in contrast , mostly represent neoplasms , predominantly malignant ones . in a study by nakata et al . 54% of persistent ggos represented bronchioloalveolar carcinoma , 26% adenocarcinoma with mixed bronchioloalveolar carcinoma components and 21% atypical adenomatous hyperplasia . the proportion of malignant lesions differs between solid , part - solid and non - solid lesions . in a study by henschke et al . 7% of solid , 63% of part - solid and 18% of non - solid lesions were malignant . the majority of part - solid and non - solid malignant lesions were bronchioloalveolar carcinomas and adenocarcinomas with bronchioloalveolar features . noguchi et al . have described a classification of adenocarcinomas based on their histology which is reflected by their prognosis and this is also reflected by their thin - section ct morphology . types a and b represent bronchioloalveolar carcinomas , are characterised by a 100% 5-year survival rate and appear as non - solid lesions at thin - section ct , whereas types c , d , e and f represent more aggressive bacs or adenocarcinomas , exhibit part - solid or solid appearances with a less favourable prognosis . peripheral ill - defined lesion with mixed density ; the more peripheral component exhibits ground glass attenuation , whereas the more central part exhibits soft tissue ( solid ) attenuation . peripheral ill - defined lesion with mixed density ; the more peripheral component exhibits ground glass attenuation , whereas the more central part exhibits soft tissue ( solid ) attenuation . another non - solid lesion which was just recently defined is atypical adenomatous hyperplasia ( aah ) . this lesion is recognized as a separate entity which in itself is not malignant but may be a precursor of adenocarcinoma . it is characterised by proliferation of atypical cuboidal or columnar epithelial cells along the alveoli and respiratory bronchioles without invasion of the stroma or adjacent structures . at ct most of the recommendations for the management of solid nodules , also apply to non - solid and part - solid lesions . for example , the risk of malignancy should be estimated , previous imaging should be reviewed , and if growth is detected biopsy should be performed . there are , however , some recommendations unique to non - solid or part - solid nodules : if such a nodule is malignant the underlying lesion is likely to be bronchioloalveolar carcinoma or adenocarcinoma with bronchioloalveolar features. these may grow more slowly than other malignant tumors . therefore , the recommendation of follow - up after 3 , 6 , 12 and 24 months and no further follow - up when no growth is demonstrated in 2 years should not be applied to non - solid and part - solid lesions . these require longer follow - up to exclude growth for several years . because of the cumulative radiation exposure , dose levels during follow - up should be as low as possible. as bronchioloalveolar carcinoma and adenocarcinoma with bronchioloalveolar features may be negative at pet , examination with pet - ct should not be performed routinely as a negative result does not exclude malignancy . if such a nodule is malignant the underlying lesion is likely to be bronchioloalveolar carcinoma or adenocarcinoma with bronchioloalveolar features . therefore , the recommendation of follow - up after 3 , 6 , 12 and 24 months and no further follow - up when no growth is demonstrated in 2 years should not be applied to non - solid and part - solid lesions . these require longer follow - up to exclude growth for several years . because of the cumulative radiation exposure , dose levels during follow - up should be as low as possible . as bronchioloalveolar carcinoma and adenocarcinoma with bronchioloalveolar features may be negative at pet , examination with pet - ct should not be performed routinely as a negative result does not exclude malignancy . non - solid pulmonary nodules are more likely malignant than solid nodules . if malignant they are usually due to atypical adenomatous hyperplasia and bronchioloalveolar carcinoma . these lesions tend to grow more slowly than other malignant pulmonary lesions and are often not hypermetabolic at fdg - pet . therefore , follow - up to exclude growth in non - solid nodules needs to be longer than in solid lesions and pet - ct is not appropriate to rule out malignancy in non - solid nodules .

the relationship between stress challenges and adverse health outcomes , particularly for the development of affective disorders , is now well established . the highly conserved neuroimmune mechanisms through which responses to stressors are transcribed into effects on males and females have recently garnered much attention from researchers and clinicians alike . the use of animal models , from mice to guinea pigs to primates , has greatly increased our understanding of these mechanisms on the molecular , cellular , and behavioral levels , and research in humans has identified particular brain regions and connections of interest , as well as associations between stress - induced inflammation and psychiatric disorders . this review brings together findings from multiple species in order to better understand how the mechanisms of the neuroimmune response to stress contribute to stress - related psychopathologies , such as major depressive disorder , schizophrenia , and bipolar disorder .

stressful life experiences have the amazing ability to synchronize organism - wide physiological responses toward the common goal of surviving threat . in this way , the major stress - responsive systems are often regarded by evolutionary biologists as essential adaptive mechanisms that ultimately promote survival . however , as stress challenges become greater in magnitude or more protracted in length , the toll on the host organism can be quite severe , and the adaptive value of mobilizing physiological processes toward survival exacts a long - term cost . this essential framework for understanding the relationship between stress challenges and their impact on organism health was articulated by hans selye , who noted that nearly all stress challenges were followed by a syndrome of being sick . unbeknownst to selye , this prescient view of the relationship between stressful experiences and their sickness - like outcomes , in today 's vernacular , implies that immune processes are probably essential mediators of the link between stress and adverse health outcomes . with this in mind , the present review takes the position that neuroimmune mechanisms of stress are highly conserved across species and serve as a likely mechanistic bridge between classic stress - responsive systems and stress - related pathologies ( figure 1 ) . to support this position , we ( i ) provide a succinct overview of neuroimmune mechanisms of stress that have been largely established in preclinical ( mouse and rat ) models ; ( ii ) extend these findings to other mammalian systems ( guinea pigs and nonhuman primates ) ; and ( iii ) translate these findings to the human condition . the intent here is not to provide a comprehensive summary , but rather to succinctly link together commonalities across models / species that argue in favor of viewing neuroimmune consequences of stress as highly conserved across taxonomic orders . at the same time ( and perhaps ironically ) , we argue that key features of the stress challenge ( nature , intensity , and duration of threat ) , as well as individual subject characteristics ( sex , age , stress history ) , will be critical for delineating individualized therapeutic approaches for the future . in doing so , we hope to provide guidance on significant gaps in our knowledge that remain to be filled and some possible pathways for the future . although we focus largely on the link between stress and negative affective states , such as depression and anxiety , the same processes are probably involved in risk for other forms of psychopathology as well . the first step in understanding neuroimmune mechanisms of stress and their eventual role in stress - related health outcomes is to define what is meant by , most studies have focused on intercellular signaling factors , such as cytokines , chemokines , and the many species of prostaglandins , whose primary roles were initially defined within an immunologically relevant context . demonstration that such intercellular mediators are often invoked by stress challenges in which there is no apparent tissue damage or other immunological insult suggests a natural role for these agents in mediating stress outcomes . however , the identification and interpretation of such findings can be quite difficult because there are literally dozens of inflammatory signaling factors that can be simultaneously regulated ; cytokines often have redundant biological action with one another ; and nearly all cytokines have pleiotropic functions . even more troublesome is the fact that actions of cytokines can manifest as generally pro- or anti - inflammatory , depending upon the presence of other signaling molecules and the context in which they are induced . in addition to these conceptual issues surrounding the identification and interpretation of cytokine action , there are also technical considerations surrounding the assessments of cytokines and other immune mediators . because immune - related factors are expressed at appreciably low quantities in the normal central nervous system ( cns ) , the techniques to detect and measure such cytokines , particularly in early studies , have often been inadequate to yield precise results . for instance , many studies have utilized gross tissue dissections ( or micropunches ) that aggregate cytokine measures over large anatomical areas and across cell types within the dissected tissue , which can significantly influence outcomes and interpretation . a second major technical consideration lies in the use of quantification procedures that allow for signal amplification ( reverse transcription polymerase chain reaction ) versus those that do not ( in situ hybridization ) , and the more general juxtaposition of studies that measure protein versus messenger rna ( mrna ) . the availability of well - validated antibodies and the use of appropriate immunohistochemical controls for target specificity and cell type have also been a historical problem . finally , deploying standard neuroscientific approaches ( cannulation , lesions , etc ) for manipulation of inflammatory signaling in the cns can significantly alter sensitivity to later stress challenges . thus , one must carefully consider the technical approach employed by studies as a key constraint in interpreting experimental outcomes . with that said , several inflammatory mediators have emerged as highly stress - responsive , and their physiological impact has been delineated clearly . for instance , numerous studies have indicated that interleukin 1 ip is rapidly increased in key limbic structures ( paraventricular nucleus [ pvn ] ; amygdala ) in response to stress challenges that involve application of an aversive / noxious stimulus such as footshock , but not in response to social stress challenges . in contrast , social stressors appear to increase release of another proinflammatory cytokine , il-6 , in both plasma and brain of mice , thereby contributing to stress phenotypes . importantly , recent evidence suggests that early exposure to maternal separation in male rats may change cytokine reactivity to later social stress challenges incurred during adulthood . moving beyond some of the classic cytokines ( such as il-1 , il-6 , tumor necrosis factor a [ tnf- ] ) , recent evidence suggests that chemokines are also dynamically altered by stress challenges . as a separate class of immune - related molecules , these stress - induced chemokines probably play a key role in structural changes to the cytoskeleton of microglia that allows for the motility and retraction of processes , in recruitment of other intrinsic microglia to the site of release , and in potentially inducing passage of monocytes across the blood - brain barrier . indeed , genetic ablation of the chemokine cx3cl1 ( fractalkine ) was recently shown to prevent microglial activation associated with chronic unpredictable stress . in addition , prostaglandins have emerged as rapid , stress - sensitive inflammatory mediators , particularly within the cortex . given the role that prostaglandins play as final common mediators of the febrile response , prostaglandins are also likely mediators of stress - induced fever responses . thus , a multitude of inflammatory signaling families are mobilized by stressful experiences and significantly impact cns function . a growing number of studies have also examined cellular manifestations of neuroinflammation , with microglia emerging as highly reactive to stress challenges . early studies examining microglial activation established that administration of minocycline , a putative microglial inhibitor , blocked the induction of central , but not peripheral , il-1 by footshock . other studies have shown that chronic stress exposure drives proliferation of microglia and increases both the density and activational state of microglia within certain brain structures . stress challenges also alter the expression of receptors expressed on the cell surface of microglia that are both indicative of microglial activation state and positively coupled to cytokine expression within microglia . indeed , dynamic alterations in cell surface receptors on microglia probably accounts , at least in part , for certain priming / sensitization effects incurred by stress , including more rapid cytokine responses produced by later injection of lipopolysaccharide ( lps ) . together , these findings provide multiple avenues by which stress challenges impact microglia and strongly support the notion that microglial ( re)activity may be a key culprit in mediating stress - dependent changes in behavior , cognition , and potentially multiple forms of psychopathology . the importance of inflammatory phenotype to behavioral and mood - related changes incurred by stress has been strengthened in recent years through the use of adoptive transfer studies . these clever studies show that repeated exposure to various forms of repeated social stress in mice increases microglial activation and expression / release of cytokines . intriguingly , when circulating monocytes are then extracted from previously stressed mice and transferred to other mice in which the existing monocytes / lymphocytes have been depleted , the recipient mice display behavioral and mood tendencies that reflect those of the original stressed host . these findings , combined with recent evidence showing that circulating monocytes may transit into the cns as a result of stressor exposure and actively influence mood state , add a new dimension to our understanding of bidirectional communication between the brain and the peripheral immune system . though this provocative area of research is still in its infancy , the emerging body of evidence provides , for the first time , causal evidence for the notion that non - neuronal cells ( microglia ) may be responsible for encoding stress - dependent changes in mood regulation , particularly for negative affective states like depression and anxiety . a key issue that must be considered in understanding the relation between stress challenges and their neuroimmune consequences is timing . in laboratory models , acute stress challenges or the individual bouts of daily , intermittent chronic stress procedures are typically elaborated across a 30-min to 3-hour window of stress exposure ( figure 2 ) . the central questions requiring consideration here are ( i ) how rapidly are neuroimmune signaling agents induced ? ( ii ) how long do such changes persist ? ( iii ) are there downstream neuroimmune effects that cascade or coalesce into subsequent neuroimmune alterations ? and ( iv ) what are the functional outcomes that can be tied to individual components of such neuroimmune cascades ? although the discussion above did not address these issues by indicating the time point at which individual changes were observed , recent studies examining the time course for neuroimmune responses provide some guidance . for instance , induction of il-1 gene expression is prevalent and significant within 30 min of stress onset , with il-1 protein responses peaking shortly thereafter ( 60 to 120 minutes after stress onset ) . in contrast , most studies examining microglial morphology , proliferation , or activational state tend to show changes that are prevalent 24 hours or more after stress termination , by which point many cytokine changes have largely resolved . though perhaps correlational at this stage , one interpretation of the timing of these events is that the expression and release of intercellular mediators ( cytokines , chemokines , prostaglandins ) have a nearly immediate impact upon behavioral changes observed in the post - stress recuperative period , while at the same time eliciting parallel changes in other aspects of neuroimmune function that persist for days to weeks after cessation of stress . thus , it will be critical for future studies to conceptually discriminate between those neuroimmune changes that represent acute , activational responses to an individual bout of stress , versus those neuroimmune changes that reflect the aggregate influence of a repeated history of stress across a more protracted period of time . the multitude of inflammatory signaling pathways that appear to be activated by stress challenges raises questions regarding the mechanisms that control such neuroimmune changes . here , we suggest that multiple upstream signaling pathways converge upon regulatory elements associated with neuroimmune signaling , thereby propagating various features of the neuroimmune response to stress . for ease of description , we will describe these pathways in three functional clusters ( figure 3 ) , with full recognition that these pathways are not independent of one another . for instance , norepinephrine release during stress - associated activation of the sympathetic nervous system has long been regarded as a primary driver of neuroimmune consequences of stress . a wide range of pharmacological studies lend support to this notion , showing that administration of -adrenergic blockers ( eg , propranolol ) block the induction of il-1 incurred by stress , whereas -adrenergic agonists ( eg , isoproterenol ) potently induce il-1 expression . consistent with this , previous treatment with desipramine ( a norepinephrine - reuptake inhibitor ) potentiated both basal and stress - induced il-1 expression . gross neurotoxic lesions of norepinephrine - containing cells via intracerebroventricular injection of the selective neurotoxin n-(2-chloroethyl)-n - ethyl-2 bromobenzylamine ( dsp-4 ) blocked the induction of il-1 in the hippocampus , whereas more focal lesions of the ventral noradrenergic bundle produced modest effects on il-1 induction by stress in the pvn . together , these studies underscore the importance of norepinephrine signaling as a key driver of neuroimmune consequences of stress . however , other studies have clearly tied microglial proliferative responses incurred by chronic stress to glutamate signaling , since chronic administration of mk801 effectively blocked this proliferative response , and n - methyl - d - aspartate ( nmda)-receptor activation is positively coupled to inflammatory signaling pathways . interestingly , considering that adenosine triphosphate ( atp ) is co - packaged and released with many classic neurotransmitters ( including norepinephrine , in particular ) , other groups have recently posited purinergic signaling as a putative mechanism controlling functional release of il-1 ( and potentially other neuroimmune factors ) in response to stress . another emerging class of neuroimmune intermediaries falls into the category of what we describe as danger , damage , and disease signals ( figure 3 ) . the basic premise is that our innate immune defense network is constructed to respond to general molecular motifs associated with pathogens or self - injury . damage - associated molecular patterns are host - derived molecules that are released in response to tissue trauma or other insults and are thus capable of activating inflammatory signaling pathways . a variety of studies have convincingly demonstrated several families of damps that are activated by exposure to intense stress challenges , including heat shock proteins ( hsp72 , in particular ) and high - mobility group box 1 ( hmgb1 ) . when released , these damps interact with cognate receptors that are positively coupled to inflammatory signaling pathways . this family of receptors is often referred to as pathogen recognition receptors ( prrs ) because they recognize and bind to general molecular motifs associated with classes of bacteria and viruses , as well as to certain damps that may be activated by stress and/or damage . yet another intriguing twist is the recent notion that bacteria from the gut or other areas of the microbiome may be stress - sensitive , and perhaps even mobile in response to stress challenges , thereby incurring a mild form of endogenous infection . thus , the damp->prr pathways have emerged as important alternative pathways by which neuroimmune consequences of stress are primed and/or activated . the propagation of neuroimmune signaling cascades in response to stress is also constrained by other features of physiology . perhaps the most logical of these counter - regulatory elements is the release of glucocorticoid ( gc ) hormone ( corticosterone in rat and mouse ; cortisol in guinea pigs and primates ) as a result of hypothalamic - pituitary - adrenal - axis ( hpa ) activation that accompanies stress exposure . the anti - inflammatory action of gcs has been established for decades , with great progress being made in recent years in the cis- and trans regulatory elements that give rise to anti - inflammatory effects of gcs . within stressful contexts , this was particularly evident in early studies where induction of central il-1 was massively potentiated in adrenalectomized subjects , and normalized by replacement of corticosterone . similar effects have been observed after pretreatment with metyrapone ( a gc synthesis inhibitor ) . however , there appear to be certain instances in which gcs prime or modestly stimulate inflammation , a notion which is now challenging some of the central dogma on the relation between gc hormone signaling and inflammation . these latter , proinfiammatory - like effects of gcs probably represent a small proportion of the overall gc effects on inflammation and relate to very specific parameters of low gc dose , the timing of gc hormone exposure , and the specific tissue in which the gcs act . gene expression profiling studies predict such effects , and so it is quite interesting to see these effects bearing out on functional responsiveness within the neuroimmune system . furthermore , a recent study found that microglial activation was more profound after several weeks of repeated restraint , but not after an equivalent exposure to chronic variable stress . although the reason for this difference has yet to be determined , the authors propose that hpa - axis habituation , and thus the lack of circulating gc hormone that accompanies repeated restraint , led to a disinhibition of stress - dependent microglial activation . additional studies showed that pharmacological activation of the hpa axis ( in the absence of an overt stress challenge ) via corticotropin - releasing hormone or corticotropin injection led to an expected inhibition of cytokine expression in the pvn , though paradoxically , both peptides stimulated inflammatory signaling at the level of the adrenal gland . yet another added complexity is that gcs are not the only hormone system engaged by stress exposure . recent studies demonstrated that progesterone is also rapidly released in response to stress , even in male rats . this makes sense as progesterone is a precursor molecule in the biosynthesis of corticosterone , yet the possibility that progesterone may contribute to the anti - inflammatory influence of gcs has not been systematically tested . one major area of neglect in the literature has been the elaboration of sex differences in neuroimmune responses to stress . in a recent set of studies from our lab , we examined fluctuations in the il-1 response to stress as a function of estrous stage in female rats . these studies reported relatively uniform il-1 induction at all phases of the cycle except during metestrus where basal il-1 was moderately increased , and the il-1 response to stress appeared to be blunted in the pvn . however , this effect requires further study to generalize the effects from mrna to protein , to expand the analysis from the pvn to other stress - sensitive sites , to incorporate additional neuroimmune measures , and to replicate what was originally a slightly underpowered effect . nevertheless , subsequent studies demonstrated that stress - induced il-1 was potentiated in ovariectomized rats and restored by estradiol - replacement injections . however , because endogenous progesterone evinced a stress - induced surge , we can not at this time attribute the rescue effect of estradiol replacement exclusively- to estradiol ( versus an estradiol plus progesterone effect ) . what we do know , however , is that progesterone replacement alone did not recapitulate the effect of estrogen replacement . nevertheless , few studies , if any , have examined how sex - specific gonadal steroids differentially affect sex differences in neuroimmune responses to stress in rodents . thus , future studies need to give serious consideration to potential additive , cooperative , and synergistic effects of steroid hormones on stress - dependent changes in neuroimmune function . although the vast majority of studies on stress and inflammation have focused on rats and mice ( as indicated in the above discussion ) , investigators have sometimes turned to alternative rodent species . guinea pigs in particular offer a number of advantages , especially in developmental studies . unlike rats and mice , brain regions are well defined , and the skull is calcified even in the first few days , which facilitates procedures such as implanting indwelling cannula and immunohistochemistry . thermoregulation matures rapidly , so lps- or stress - induced fever can be investigated early in preweaning pups . but for studies of stress - induced inflammatory responses during infancy , perhaps the greatest advantage stems indirectly from the fact that the pups are precocial . guinea pigs are capable of coordinated behavior , including locomotion and ingestion , from shortly after birth . licking of the pups , the primary active maternal behavior , is infrequent , particularly after about a week of age . there is no retrieval , no nest , and mothers simply accommodate nursing attempts initiated by the pups . as a result , mother - young proximity is maintained almost exclusively by the strong attraction or attachment that pups display for the mother . indeed , pups exhibit evidence of the classic markers of attachment commonly used in primate studies namely , approach , recognition , and preference for the attachment object ; use of the attachment object as a secure base for exploration ; and distressful responses to separation . maternal separation in guinea pigs thus affords a compelling translational model for the effects of attachment disruption , a class of early stressors frequently linked to later psychopathology in humans as well as altered inflammatory activity . there are many similarities in the responses to maternal separation shown by young guinea pigs and infant monkeys . these include , for instance , increased hpa and sympathetic activity and central catecholamine turnover associated with stress . as is seen in some species of macaques , as well as in human infants , guinea pig pups also display a two - stage , active / passive behavioral response to separation . when placed alone in a novel cage , pups initially show protest by vocalizing in an apparent attempt to re - establish contact with the mother . but after about an hour of separation , the pups typically quiet and adopt a characteristic crouched stance , with closed eyes , extensive piloerection , and apparent disinterest in their surroundings ( figure 1 ) . neither the active nor passive stages are observed if the mother is placed in the novel cage with the pup , establishing that the mother 's absence , and not just the novelty of the environment , is responsible for these responses . the passive , second stage of separation in guinea pig infants is reminiscent of the despair shown by monkeys separated for much longer periods and even the although the nature of the passive stage of separated guinea pig pups can suggest depression to a comparative psychologist , it may just as readily suggest cytokine - induced sickness behavior to the psychoneuroimmunologist . each of the components of the response moreover , direct stimulation of sickness with lps results in the same behavioral constellation of these three behaviors . it appears then that the stressor of separation in a novel environment initiates an inflammatory reaction that mediates the behavioral response . this claim is bolstered by findings that the separation procedure elicits a transitory fever and increased expression of the proinflammatory cytokine tnf- in spleen , two physiological indicators of a sickness response . behaviors of the second stage of separation can also be reduced by a variety of anti - inflammatory agents , including -melanocyte - stimulating hormone ( -msh ) , indomethacin , il-10 , and naproxen . although other systems are also probably involved , it is clear that inflammatory mechanisms play a fundamental role in the depressive - like response of separated guinea pig pups . in the adult human literature , there is now overwhelming evidence for involvement of inflammation in depressive illness ( as described below ) . the guinea pig results suggest that the particular form of depressive response shown by separated nonhuman and perhaps human primate infants may also be mediated , at least in part , by inflammatory factors . most current research on attachment disruption and depression in humans focuses not on immediate effects , but rather on long - term vulnerability for developing depression engendered by early abuse , neglect , or prolonged separation . whereas increased risk for later depressive illness has long been suspected for infants exposed to such forms of maltreatment , recent research has solidified this link and begun to provide glimpses of potential neurobiological mechanisms . that is , the early stress of attachment disruption is seen as sensitizing underlying stress - responsive machinery so that , in later life , exposure to stressors with which other individuals would be able to effectively cope elicit disproportional , protracted , and inadequately regulated stress responses that lead to , or constitute the underlying basis of , the depressive episode . these diathesis - stress or two - hit models have most often emphasized effects on elements of the hpa axis and its control , including increases in central corticotropin - releasing factor secretion , amygdala activity , and gc resistance , as well as a reduction in hpa inhibition by the frontal cortex . however , recently , there also has been a proliferation of findings implicating inflammatory mechanisms in the sensitization process . attachment disruption and other forms of early stress have repeatedly been found to be associated with increased markers of inflammation at later ages . from an evolutionary perspective , the increased inflammation may represent a remnant of an ontogenetic adaptation originating from a time when persistent early - life stress was predictive of a hazardous adult environment , in which injury was common and a robust innate immune system was adaptive . the process by which early stress enhances later inflammation remains unclear but may involve sensitization of resident microglia or increased transport of peripheral monocytes to the cns , resulting in heightened central release of inflammatory mediators . the augmentation of inflammatory processes , in turn , may be driven by activation of , and alterations in , other stress - responsive systems , such as increased resistance to the suppressive action of gcs and elevated sympathetic activity . results of guinea pig studies implicate inflammatory factors in long - term effects of early stress . repeated separation increases , ie , sensitizes , both the depressive - like behavior and febrile response to later separations during both the preweaning period and beyond . moreover , administration of the cyclooxygenase ( cox)-inhibitor naproxen before the initial separation suppressed the sensitization response not only to the initial separation experience but also to separations that followed 1 and 10 days later . the sensitization that occurs appears to be related to some broader , depressive - like state rather than just the separation response , as previously separated guinea pigs also showed more immobility in the forced swim test , a measure and paradigm that is selectively sensitive to antidepressant medications . in these studies , as is the case in the field more generally , sex differences have yet to be a major focal point . although males and females have typically both been included in the guinea pig work , it has been in numbers too small to sufficiently power examination of male - female differences . however , as rodent models for the role of inflammation in stress - related disease are nowbecoming established , it is imperative that male - female differences be taken into account . this is particularly important for disorders in which sex differences are profound , such as major depression , for which women are about twice as likely as males to be afflicted . the above results argue that the guinea pig model has strong internal validity in that there is good evidence that attachment disruption in the form of maternal separation results in depressive - like behavior that sensitizes with repeated separation . the evidence also indicates that the sensitization involves inflammatory processes and reflects changes in an underlying state that is manifested in more than one depressive - like response . the external validity , ie , the generality of the results to humans , would , however , be bolstered if we could demonstrate that a similar experimental procedure produced similar results in a primate . indeed , the guinea pig work was always intended as a complement to primate research ; that is , as a way of doing the investigative work necessary to ultimately generate hypotheses that might then be tested in primate models . the challenge has been how this might be accomplished without proposing to repeat the prolonged separation of monkey infants from their mothers that was common in experiments of the 1950s and 1960s . a possible solution arose from observations of the behavioral management unit and others at the california national primate research center . it was noted that when adult monkeys were brought from large outdoor social groups ( figure 1 ) to restricted indoor housing , as is commonplace for the beginning of experiments or veterinary care , a small proportion exhibited a hunched posture with apparent disinterest in their surroundings , a response that both is widely regarded as indicating a depressive - like reaction in macaques and which mimics the reaction observed in separated monkey infants . furthermore , when recordings of adult male monkeys introduced to the restricted indoor housing with no human observer in the room were analyzed , an even larger percentage of animals were found to exhibit the depressive - like hunched posture . these findings suggest that presence of a human may evoke a defensive reaction incompatible with the depressive - like response . these findings are compatible with interpretation of these depressive - like consequences as a manifestation of sickness , since expression of sickness behaviors are easily perturbed in the face of threat . from a modeling standpoint , however , the observations suggested that the relatively simple procedure of transferring adult monkeys from outdoor social groups to indoor housing may provide a means of evaluating the general relevance of the guinea pig results for nonhuman primates . therefore a new model was developed in which adult male rhesus macaques were brought from half - acre outdoor social groups to standard indoor housing , either alone or together with an affiliative juvenile partner for 8 days on two occasions at an approximate 2-week interval . behavior was filmed without a human observer present and blood samples were taken for cytokine analysis at the end of each 8-day period of indoor housing , as well as when the males were residing in the outdoor field cages . during the first separation , all monkeys of both groups exhibited the hunched posture , with an average of about a third of all observation time spent in this posture . during the second separation , time spent in the hunched posture increased ( ie , sensitized ) , but the increase in time spent in the hunched posture was accompanied by a decline in activity and environmental exploration . the monkeys housed alone showed a relative decline from the first to the second period of indoor housing in lps - induced expression of the anti - inflammatory cytokine il-10 , whereas those monkeys housed with a partner showed a relative increase . furthermore , regardless of whether monkeys were alone or with a partner , indoor housing reduced sensitivity- of the proinflammatory cytokines il-1 and tnf- to the suppressive action of gcs . it should be noted that both of these measures response to lps stimulation and gc resistance were chosen because they appear responsive to early - life stress in humans . finally , evidence for a coupling of the behavioral and cytokine findings was also observed . for monkeys tested alone , a large and significant positive correlation was observed between the number of seconds spent in the hunched posture during each period indoors and circulating levels of each of the three cytokines measured . although many particulars of the experimental design and measures in the guinea pig and monkey studies differed , the broad similarity of results suggests some cross - species commonality or conservation of basic relations between social separation , inflammatory activity , and depressive - like behavior and its sensitization . the findings also suggest relatively simple rodent and monkey models that might be used to continue to disentangle the way in which earlylife stress and inflammation contribute to depressive illness and other negative affective states . the above results argue that the guinea pig model has strong internal validity in that there is good evidence that attachment disruption in the form of maternal separation results in depressive - like behavior that sensitizes with repeated separation . the evidence also indicates that the sensitization involves inflammatory processes and reflects changes in an underlying state that is manifested in more than one depressive - like response . the external validity , ie , the generality of the results to humans , would , however , be bolstered if we could demonstrate that a similar experimental procedure produced similar results in a primate . indeed , the guinea pig work was always intended as a complement to primate research ; that is , as a way of doing the investigative work necessary to ultimately generate hypotheses that might then be tested in primate models . the challenge has been how this might be accomplished without proposing to repeat the prolonged separation of monkey infants from their mothers that was common in experiments of the 1950s and 1960s . a possible solution arose from observations of the behavioral management unit and others at the california national primate research center . it was noted that when adult monkeys were brought from large outdoor social groups ( figure 1 ) to restricted indoor housing , as is commonplace for the beginning of experiments or veterinary care , a small proportion exhibited a hunched posture with apparent disinterest in their surroundings , a response that both is widely regarded as indicating a depressive - like reaction in macaques and which mimics the reaction observed in separated monkey infants . furthermore , when recordings of adult male monkeys introduced to the restricted indoor housing with no human observer in the room were analyzed , an even larger percentage of animals were found to exhibit the depressive - like hunched posture . these findings suggest that presence of a human may evoke a defensive reaction incompatible with the depressive - like response . these findings are compatible with interpretation of these depressive - like consequences as a manifestation of sickness , since expression of sickness behaviors are easily perturbed in the face of threat . from a modeling standpoint , however , the observations suggested that the relatively simple procedure of transferring adult monkeys from outdoor social groups to indoor housing may provide a means of evaluating the general relevance of the guinea pig results for nonhuman primates . therefore a new model was developed in which adult male rhesus macaques were brought from half - acre outdoor social groups to standard indoor housing , either alone or together with an affiliative juvenile partner for 8 days on two occasions at an approximate 2-week interval . behavior was filmed without a human observer present and blood samples were taken for cytokine analysis at the end of each 8-day period of indoor housing , as well as when the males were residing in the outdoor field cages . during the first separation , all monkeys of both groups exhibited the hunched posture , with an average of about a third of all observation time spent in this posture . during the second separation , time spent in the hunched posture increased ( ie , sensitized ) , but the increase in time spent in the hunched posture was accompanied by a decline in activity and environmental exploration . the monkeys housed alone showed a relative decline from the first to the second period of indoor housing in lps - induced expression of the anti - inflammatory cytokine il-10 , whereas those monkeys housed with a partner showed a relative increase . furthermore , regardless of whether monkeys were alone or with a partner , indoor housing reduced sensitivity- of the proinflammatory cytokines il-1 and tnf- to the suppressive action of gcs . it should be noted that both of these measures response to lps stimulation and gc resistance were chosen because they appear responsive to early - life stress in humans . finally , evidence for a coupling of the behavioral and cytokine findings was also observed . for monkeys tested alone , a large and significant positive correlation was observed between the number of seconds spent in the hunched posture during each period indoors and circulating levels of each of the three cytokines measured . although many particulars of the experimental design and measures in the guinea pig and monkey studies differed , the broad similarity of results suggests some cross - species commonality or conservation of basic relations between social separation , inflammatory activity , and depressive - like behavior and its sensitization . the findings also suggest relatively simple rodent and monkey models that might be used to continue to disentangle the way in which earlylife stress and inflammation contribute to depressive illness and other negative affective states . the key question , of course , is how well these animal models translate to humans and help us better understand the human stress response and risk for psychopathology . psychosocial stress is a well - established risk factor for the development of various forms of psycho - pathology in humans , including major depressive disorder ( mdd ) , posttraumatic stress disorder ( ptsd ) , and other anxiety disorders . similar to findings from animal models of stress , evidence from human studies shows that exposure to stressors provokes neurochemical changes , including changes in levels of inflammation , that are frequently associated with psychopathology , and which may help to explain the mechanisms by which stress increases risk for psychiatric disorders , including mdd . neuroimmune effects of exposure to stressful events can persist beyond the immediate impact and potentiate an individual 's response to future stressors , thus increasing risk for future psychopathology . indeed , among individuals with mdd , those who experienced childhood adversity had higher circulating levels of c - reactive protein ( crp ) than individuals with no history of childhood trauma . additionally , adults who experienced childhood adversity exhibited higher levels of circulating il-6 levels in response to an acute stressor . finally , there is preliminary evidence that a haplotype in il-33 moderated the link between women 's history of childhood abuse and their risk for depression in adulthood . across a variety of stressors , ranging from low socioeconomic status to traumatic events , mammalian immune cells show an immediate conserved transcriptional response to adversity , which involves increased expression of inflammatory genes and decreases in the genes underlying antiviral responses . interestingly the inflammatory component of the leukocyte conserved transcriptional response to adversity may be driven by stress - induced upregulation of myelopoiesis and could contribute to development of negative health outcomes associated with adverse social conditions , including psychiatric disorders . overall , one of the pathways for the effects of exposure to stressors on psychopathology risk seems to be an increase in immediate and long - term expression of immune -related genes , outcomes that appear to recapitulate effects observed in preclinical ( animal ) models . immune - related transcripts involved in the cellular stress response have been shown to be upregulated in the prefrontal cortex ( pfc ) of individuals with schizophrenia , indicating that these genes may play an important role in chronic psychopathology . importantly , these observed transcriptome changes were not a result of acute immune system activation , as there were no differences in markers of acute inflammation or responses to infections between individuals with schizophrenia and controls . similarly , in the hippocampus , general upregulation of inflammatory transcripts was found in patients with mdd , bipolar disorder , and schizophrenia , though the authors reported no overlap in specific genes across disorders . this suggests that there are common immune - related gene changes in psychiatric disorders , but that there are also changes in expression that are differentially regulated between diseases . for example , circulating cytokines and upregulation of immune - related genes have been found to occur in patients undergoing a first episode of psychosis , whereas those with comorbid depression displayed a unique expression profile , suggesting separable transcriptional phenotypes . additionally , increased mrna levels of chemokine ( c - c motif ) ligand 24 ( ccl24 ) were found in circulating leukocytes among participants with mdd compared with individuals with bipolar personality disorder and healthy controls . in contrast , c - c chemokine receptor type 6 ( ccr6 ) was expressed consistently less among mdd patients than in controls . another example can be found in common variants in long - range enhancer elements that modulate transcriptional response to activation of gc receptors in human blood cells , thereby increasing the risk for later psychopathology , including mdd and schizophrenia . moreover , those functional genetic variants were associated with overgeneralized amygdala reactivity , suggesting that individual differences in the leukocyte 's immediate transcriptional reactivity to stress may influence an individual 's neurophysiological response to stress . epigenetic alterations in genes related to immune function are one of the plausible mechanisms underlying the lasting neuroimmune effects of stress exposure and increased risk for psychopathology . one of the first epigenome - wide association studies ( ewas ) that examined dna methylation among participants with a history of mdd identified tryptophan metabolism - related genes as one of the top three functional clusters in individuals with no history of mdd , one of which was a cytokine - induced reduction in tryptophan , the primary serotonin precursor often implicated in mdd . there is also evidence that methylation levels of the il-6 and crp genes are inversely related to circulating levels of il-6 and crp among individuals with a history of mdd . similarly , immune - related genes were shown to be over - represented among unmethylated genes among individuals with ptsd . furthermore , hypomethylation of immune - related genes among ptsd - affected individuals was linked to increased peripheral levels of inflammatory cytokines , which were , in turn , related to history of childhood abuse and life stress . overall , these findings suggest that exposure to stressors , perhaps particularly early - life stress , could result in immune - associated epigenetic changes that increase an individual 's susceptibility to psychiatric disorders . complementing these findings , researchers have recently begun exploring neural mechanisms underlying immune activation after stress exposure in humans via structural and functional imaging . for example , a functional magnetic resonance imaging study that examined changes in mood and neural activity after an in vivo immune challenge found that immune - induced mood decline was associated with increased activity in the subgenual anterior cingulate cortex ( sacc ) during emotional face processing . inflammation - induced mood deterioration was also associated with decreased functional connectivity of the sacc with the amygdala , medial pfc , nucleus accumbens , and superior temporal sulcus . additionally , exposure to an acute social stressor has been associated with increased circulating levels of il-6 and tnf- , along with increased activity in the dorsal acc ( dacc ) during a social rejection task . these findings suggest that stress - induced effects on inflammation may increase activity in brain regions associated with emotion processing , while decreasing connectivity with regions involved in emotion regulation . this increased sensitivity to social stressors and decreased emotion regulation may , in turn , increase the risk for various psychiatric disorders , including mdd . there is growing evidence that inflammation may also impact corticostriatal reward circuitry , which underlies symptoms of anhedonia that are common in various psychiatric disorders . specifically , there is evidence that increased circulating levels of crp , a common inflammatory marker , were associated with decreased connectivity between ventral striatum and ventromedial pfc and decreased dorsal striatal to ventromedial pfc connectivity among participants with mdd . notably , the association between the differences in connectivity and symptoms of anhedonia and motor slowing were significantly predicted by participants peripheral crp levels . similarly , depressive symptoms after exposure to an in vivo immune challenge was contingent , at least in part , on a reduction in ventral striatum activity in response to anticipated rewards . these findings parallel the results of neuroimaging studies showing that administration of interferon- , a potent proinflammatory cytokine , led to reduced activity in the ventral striatum during a hedonic reward task . moreover , positron emission tomography was utilized to show an interferon--induced increase in f - dopa ( radiolabeled dopamine precursor ) uptake , but decreased f - dopa turnover , in the basal ganglia , which correlated with increased depressive symptoms . together , these findings suggest that inflammation may adversely impact motivation and goal - directed behavior by decreasing activation and connectivity of brain regions involved in processing of rewarding stimuli and psychomotor speed , plausibly though the modulation of dopamine function . the recent understanding of the above interactions between stress and inflammation has given rise to research that applied these findings to both new and longstanding treatment approaches . for instance , there is a growing body of research that suggests that cognitive behavior therapy ( cbt ) reduces inflammation in the context of improving disturbed sleep and depressed mood . other treatment approaches , such as mindfulness meditation and yoga , are also associated with decreased stress - induced inflammation . finally , there is evidence in human clinical populations that targeting inflammation directly may help to alleviate symptoms of psychopathology , including mdd , ptsd , and schizophrenia , but only in a subgroup of patients who exhibit increased initial levels of inflammatory markers . specifically , meta - analyses have supported the beneficial use of anti - inflammatory medication in schizophrenia - affected individuals , particularly those who are in the early stages of this disorder . although preliminary results of the anti - inflammatory therapy in the treatment of psychiatric disorders are promising , it is important to identify specific subgroups that would benefit the most from such treatment . for example , one study examined the potential antidepressant effect of the tnf- inhibitor infliximab in patients with treatment - resistant depression who were otherwise healthy . infliximab is a monoclonal antibodydirected against tnf- that prevents this cytokine from binding to its receptor via immunoneutralization . interestingly , the anti - inflammatory therapy outperformed placebo , but only in patients with high peripheral levels of crp before treatment ( > 5 mg / l ) . baseline levels of inflammation could , therefore , serve as a biomarker of an individual 's likelihood of responding to anti - inflammatory therapies . intriguingly , among participants with a baseline crp value greater than 5 mg / l , anti - inflammatory therapy led to a reduction in a variety of symptoms , including sad mood , psychomotor retardation , anhedonia , anxiety , and suicidal ideation , all of which are linked to the neurocircuits typically targeted by inflammatory cytokines . overall , therefore , anti - inflammatory therapy may be a promising treatment for specific subgroups of patients with a variety of psychiatric disorders , such as those with elevated circulating inflammatory markers . the identification of , and ability to detect , specific biomarkers that can identify individuals who would benefit from anti - inflammatory therapy is a critical step in delivering individualized therapy . despite recent advances made in understanding the role of inflammatory processes in human psychopathology , direct evidence examining potential sex differences historically , men and women have been known to display pronounced biological and psychological differences in responses to stress , with females typically displaying nearly double the gc response relative to males . stress reactivity is also not constant across developmental epoch or across hormonal cycles ; women who are between the ages of puberty and menopause typically exhibit lower hpa - axis and autonomic nervous system responses to acute psychosocial stressors than older women . what is striking is that women have higher rates of stress - related psychiatric disorders that have been strongly linked to inflammation , including mdd and anxiety disorders . the sex - specific epidemiological pattern of psychiatric disorders highlights the role of sex hormones in stress reactivity , since many of these sex differences emerge in puberty . interestingly , there is also evidence of sexual dimorphism in the susceptibility of women and men to immune - related disorders . for instance , the prevalence of many inflammatory conditions , including autoimmune diseases is significantly higher in women , whereas men are more likely to suffer from infectious diseases . notably , young women were reported to exhibit higher peripheral levels of il-6 than men after mental or physical stress . clearly , more research is needed in this area , as it may be that sex differences in stress reactivity , including inflammatory and , potentially , neuroimmune responses to stress , could provide much needed insight into the vast sex differences in the rate of stress - related psychiatric disorders . the goal of this review was to highlight the neuroimmune mechanisms underlying the response to stress with an emphasis on extending findings from animal models toward the human experience . research on rodents has served as a starting point for understanding the molecular and cellular responses to stress , allowing for a greater understanding of how inflammatory factors , such as cytokines , chemokines , and prostaglandins , ultimately influence brain function . importantly , microglia have emerged as a key interface between stress - related signals and neuroimmune consequences of stress . the guinea pig in particular serves as a useful model of early - life stress with excellent early - life translatability that recapitulates findings from nonhuman primates and humans . further validation of the guinea pig model , and expansion of genetic tools and antibodies directed toward guinea pig - specific proteins , will allow for bridging the findings in rats and mice with a complementary , tractable model system . the use of nonhuman primates confers significant advantages not only in being able to bridge findings in rodent models to humans , but also in that they lead highly social lives allowing for examination of stress and social interactions ( eg , buffering effect of a cage partner on social separation discussed earlier in the review . ) . through studying conserved transcriptional responses to stress that have been examined in circulating leukocytes in humans , researchers have made connections between clinically relevant psychological disorders and expression levels of immune and inflammatory factors . the finding that epigenetic modulation of cytokines differs in individuals with stress - related disorders , such as mdd and ptsd , provides an intriguing avenue for animal model research in examining how stress can affect future gene expression . thus , viewing neuroimmune consequences of stress through a multispecies lens provides a compelling argument for the highly conserved nature of the relationship between cytokines , stress , and multiple forms of psychopathology . our challenge for the future , therefore , will be to dive deeper into individual differences in stress reactivity , using a combination of highly tractable animal models from different species to better discriminate between those neuroimmune consequences of stress that are constant within and across species , relative to those that differ as a function of the individual ( sex , age , recent stress history ) or that are species - specific . in doing so , we can exploit the strengths of individual model systems while at the same time circumventing their limitations , with the hope of defining novel therapeutic strategies to ameliorate adverse health consequences of stress .

backgroundpelvic bleeding from trauma and postpartum hemorrhage is often difficult to treat successfully by emergency providers particularly in low resource environments , when hospital presentation is delayed or there is a lack of immediate surgical , anesthesia , and transfusion capabilities . pneumatic anti - shock garments ( pasg ) decrease pelvic blood flow and hemorrhage . a tightly fitted neoprene non - pneumatic anti - shock garment ( nasg ) has been shown to decrease blood loss and improve survival rates from postpartum hemorrhage.aimsthe objective of this study was to determine whether blood flow to the pelvis is decreased by use of the nasg or by an improvised pasg.methodsa pasg was made using three bicycle tubes , placing one tube on each leg and one on the lower abdomen / pelvis , wrapping firmly with sheets and inflating the tubes to approximately 3.5 bar ( 45 psi ) . a doppler ultrasound was used to measure distal aortic blood flow in 12 healthy adults at baseline and in both devices . data were analyzed with one sample and paired t tests.resultsmean flow was 1.99 l / min at baseline . mean flow decrease was 1.11 [ 95% confidence interval ( ci ) : 0.641.57 , p = 0.0003 for the difference ] for the pasg and 0.65 ( 95% ci : 0.031.26 , p = 0.04 ) for the nasg . the pasg decreased blood flow more than the nasg ( mean difference : 0.46 , 95% ci : 0.020.90 , p = 0.04).conclusionsboth devices decreased distal aortic blood flow , but the improvised pasg device decreased it by a larger margin .

hemorrhage from pelvic blood vessels after trauma or obstetric delivery are major health care problems . trauma is a leading cause of death worldwide and a major cause of lost quality - adjusted life years ( qalys ) . treatment of pelvic trauma is difficult since hemorrhage can be massive and it is difficult to apply direct pressure to the bleeding vessels . postpartum hemorrhage is the leading cause of obstetric deaths and causes an estimated 150,000 maternal deaths per year . definitive control of severe pelvic bleeding from any cause often requires aggressive measures such as arterial embolization , laparotomy , internal fixation , or pelvic packing . these procedures are difficult to perform because they require immediate access to sophisticated medical and surgical expertise and equipment . blood volume must be maintained by transfusion of red blood cells , platelets , and clotting factors . this could provide time to mobilize more sophisticated resources and might decrease transfusion requirements and the need for aggressive therapy . pneumatic anti - shock garments ( pasg ) , also called medical or military anti - shock trousers ( mast ) , have been shown to greatly decrease pelvic blood flow [ 2 , 3 ] , and case reports imply that they are effective in patients with pelvic injuries and postpartum hemorrhage . however , most of the patients enrolled in clinical trials had thoracic or abdominal trauma and had ready access to definitive surgery . they only autotransfuse about 250 cc of blood from the lower body into the central circulation and have little or no effect on cardiac output . these findings would explain differing outcomes for upper body versus pelvic hemorrhage . in their favor pasg unfortunately pasg are complicated , expensive , and rarely available in the low resource areas where they would seem most useful . a simple and less expensive alternative to pasg this non - pneumatic anti - shock garment ( nasg ) is made of neoprene and is tightly applied to the patient using velcro straps . it has been commercialized as the zoex non - inflatable anti - shock garment ( zoex , ashland , or , usa ) . the nasg has been shown to decrease postpartum blood loss by 50% [ 8 , 9 ] and improve survival rates from postpartum hemorrhage . we measured pelvic blood flow in volunteers before and after being placed in the nasg and pasg . subjects ranged in age from 21 to 60 years of age and weighed 5282 kg . an acuson sequoia 512 ultrasound and 4vi probe operating at 23 mhz ( siemens , mountain view , ca , usa ) was used to measure heart rate , apparent artery diameter , doppler - to - vessel angle , and blood flow velocity at the abdominal aorta below the superior mesenteric artery ( sma ) . measurements were obtained from each subject at baseline and immediately after being placed in each device . the pasg was made by wrapping individual folded hospital sheets around each leg and the abdomen / pelvis . after the first wrap a bicycle tube was placed on each sheet and the sheet was further wrapped around the patient and tube . a second set of three sheets was then wrapped firmly around the first set being careful to allow the tube stem but no other part to protrude . the tubes were then inflated with a bicycle pump to 2.83.5 bar ( 4050 psi ) . the ultrasonographer has 20 years experience and is certified by the american registry for diagnostic medical sonography ( rockville , md , usa ) as both a registered diagnostic medical sonographer ( rdms ) and a registered vascular technician ( rvt ) . data were analyzed with one sample and paired t tests using two - tailed p values . mean blood flow was 1.99 l / min at baseline with a range of 0.393.05 l / min . mean flow decrease was 1.11 l / min or 56% [ 95% confidence interval ( ci ) : 0.641.57 , p = 0.0003 for the difference ] for the pasg and 0.65 l / min or 33% ( 95% ci : 0.031.26 , p = 0.04 ) for the nasg . the pasg decreased blood flow more than the nasg ( mean difference : 0.46 , 95% ci : 0.020.90 , p = 0.04 ) . our results are compatible with the medical literature regarding the regional hemodynamic effects of circumferential abdominal pelvic pressure using pasg [ 2 , 3 ] . commercial pasg are quite rigid and their air bladders are considerably larger than bicycle tubes . a previous study showed that when inflated to two thirds of their maximum , commercial pasg decrease blood flow by approximately one half , which is similar to the magnitude of decrease we demonstrated with our improvised pasg . fully inflated commercial pasg decrease distal aortic flow by about three quarters [ 2 , 3 ] . pelvic hemorrhage and particularly postpartum hemorrhage bleeding can be arterial from branches of the internal iliac , pelvic , ovarian , and middle sacral arteries . bleeding will stop when externally applied pressure exceeds the perfusion pressure of the injured vessel . low pressure venous bleeding theoretically requires only a few millibars , but this must be at the actual bleeding sites . this is impossible to apply externally and would in addition completely eliminate tissue perfusion , causing undesirable side effects . it provides indirect pressure on the pelvic organs that exceeds venous and capillary filling pressure . this directly decreases arterial bleeding and hence should give normal hemostatic mechanisms more opportunity to act . it is unfortunate that the term anti - shock became incorporated into the names of these devices because they have essentially no utility in reversing shock in general or in preventing it from injuries that are above the pelvis . however , emergency care and childbirth providers need a means of slowing or stopping pelvic hemorrhage . this is particularly true in low resource environments or when patients present late or to facilities that lack interventional radiology , immediate surgical capability , and safe blood . delays in gaining access to definitive care are even more serious if patients suffer from preexisting anemia or have little reserve . although we did not do any formal testing of device acceptability , we did ask all of the subjects if they wanted the trial terminated because the devices were too uncomfortable and none did . one subject had participated in a previous pasg study and spontaneously volunteered that all three devices felt about the same which is reassuring given the long clinical experience with commercial pasg hypovolemia ordinarily causes peripheral vasoconstriction which would lead to less blood flow at the distal aorta it would be unethical and impractical to perform an initial trial on ill and bleeding subjects . our within - subject experimental design specifically compensates for differing amounts of pelvic blood flow between subjects . although the amount of blood flow in the distal aorta might be different , the effect of mechanical pressure and hence the comparison between devices would be the same . although the improvised device can be made locally , the nasg is available in a manufactured version which may make it more suitable in places that can afford the initial investment . the pasg decreased blood flow by a larger margin than the nasg , but this does not prove that it is clinically either equivalent or superior . it should be noted that the optimal pressure to treat pelvic and postpartum bleeding is not known but that the pressure in each device can be changed by adding or removing air from the tubes or adjusting the tension on the straps of the nasg . currently available reports of nasg use in obstetric hemorrhage have not noted problems with compartment syndrome , but the number of subjects studied is small .

we compare the ability of shrna and crispr / cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line k562 . we find that the precision of the two libraries in detecting essential genes is similar and that combining data from both screens improves performance . notably , results from the two screens show little correlation , which can be partially explained by identification of distinct essential biological processes with each technology .

cell culture performed as previously described . briefly , k562 cells ( atcc ) were cultured in rpmi 1640 ( gibco ) media and supplemented with 10% fbs ( hyclone ) , penicillin ( 10,000 i.u./ml ) , streptomycin ( 10,000 ug / ml ) , and l - glutamine ( 2 mm ) . cells were grown in log phase during all biological assays by returning the population to 500,000 cells / ml each day . k562 cells were maintained in a controlled humidified incubator at 37 c , with 5% co2 . briefly , to generate sufficient lentivirus to infect the genome - wide shrna library into k562 cells , we plated 293 t cells on 15 cm tissue culture plates . 293 t cells were transfected with third generation packaging plasmids and shrna encoding vectors . after 48 hrs and 72 hrs of incubation we filtered the pooled lentivirus through a 0.45 m pvdf filter ( millipore ) to remove any cellular debris . approximately 560 million k562 cells were infected with our next - generation genome - wide lentiviral shrna library to maintain roughly 1,000-fold coverage of the shrna library after selection . infected cells grew for 3 days before selecting the cells with puromycin ( 0.7 g / ml , sigma ) . after three days of selection , infection efficiency was monitored using flow cytometry ( bd accuri c6 ) . once the cells reached 90100% mcherry positive cells , they were spun out of selection and allowed to recover in normal rpmi 1640 media . at t0 , 500 million cells were pelleted by centrifugation ( 300 g for 5 min ) . cells were then split into two populations and maintained at logarithmic growth ( 500,000 cells / ml ) each day for 14 days . after 14 days of growth genomic dna was extracted for all 3 time points separately following qiagen s blood maxi kit protocol . a previously designed 4 sgrna / gene crispr - cas9 library was used targeting 5 ends of conserved exons with sgrnas varying in length between 19 and 25 base - pairs . the library was generated first by infecting k562 cells with a sffv - cas9-bfp vector to create a stably expressed cas9 cell line . we then infected the lentiviral genome - wide sgrna library into approximately 120 million cells following the same protocol as the genome - wide shrna library to maintain at least 1,000-fold representation in cells . infected cells were selected with puromycin ( 0.7 g / ml , sigma ) for 3 days . percentage of mcherry positive cells was measured by flow cytometry ( bd accuri c6 ) . selected cells were spun out of selection and into normal rpmi 1640 media . at t0 cells were then split into two populations and grown for 14 days , maintaining logarithmic growth ( 500,000 cells / ml ) each day . after 14 days of growth , cells were pelleted by centrifugation , and genomic dna was extracted for all three time samples separately following qiagen s blood maxi kit instructions . data from previous shrna screen for modifiers of ricin toxicity were obtained as pre- computed hairpin - level enrichments averaged from two replicates , pooled data was analyzed with castle , and compared to signed , log - transformed published results ( supplementary data 5 ) . count data for two replicates from a previous crispr / cas9 cutting screen for lps - induced tnf expression were obtained from the authors , analyzed with castle , and compared to signed , log - transformed published deseq results ( supplementary data 6 ) . data from previous crispri and crispra screens were obtained as pre - computed guide - level enrichment averaged from two replicates , analyzed with castle , and compared to published results ( supplementary data 7 ) . where available , positive and negative results from published low - throughput validations are also presented . trimmed sequences were aligned to the library using bowtie with zero mismatches tolerated . all alignment from multi - mapped reads were used . enrichment of individual hairpins was calculated as a median - normalized log - ratio of fraction of counts , as previously described . raw count files are available ( supplementary data 2 ) , and raw fastq files have been deposited at the sequence read archive ( accession srp072806 ) and bioproject ( accession prjna317269 ; http://www.ncbi.nlm.nih.gov/bioproject/prjna317269 ) . briefly , we built cas9 high throughput maximum likelihood estimator ( castle ) that uses an empirical bayesian framework to account for multiple sources of variability , including reagent efficacy and off - target effects ( for more complete description , see supplementary methods ) . for each gene , we have the phenotypes of multiple targeting reagents . from this , and the phenotypes of negative controls , we obtain an effect size estimate for each gene and an associated log - likelihood ratio . in the figures we present this as the castle score , which is twice the log - likelihood ratio , signed to match the effect size . all screens are analyzed with the same parameters , and no optimization was performed using the gold standard or related sets . these , along with a complete screen analysis pipeline , are available at https://bitbucket.org/dmorgens/castle . for comparison , riger was implemented with default settings on precomputed element enrichments with gene - e ( http://www.broadinstitute.org/cancer/software/gene-e/ ) . rsa was implemented with python scripts available from http://carrier.gnf.org / publications / rsa/. mageck was implemented with default settings with software available from https://sourceforge.net / projects / mageck/. hitselect was implemented with default settings with software available from https://sourceforge.net / projects / hitselect/. median and highest effect heuristics as well as the mw test were implemented with custom python scripts . go terms were generated using gorilla available at http://cbl-gorilla.cs.technion.ac.il/ by ranking genes from highest confidence negative growth phenotypes to lowest confidence genes to highest confidence positive growth phenotypes . for direct comparison to the 4 sgrna per gene crispr / cas9 library , the 25 hairpin per gene library rnai library was down - sampled to 4 shrnas per gene . hairpins were ranked according to their original computational design and the top four unique shrnas were used as well as all negative control shrnas . note that this is independent of the data set used here and represents the 4 shrna library that would have been designed . essential gene predictions were then repeated using this reduced shrna library as above ( supplementary fig . gene sets were defined by 10% fpr cutoff for cas9 , shrna , and combination screens . cas9 and shrna unique gene sets were defined as genes under the 10% fpr cutoff in the cas9 or shrna screen but neither the other nor the combination screen . an overlap set was defined as genes under the 10% fpr cutoff for both cas9 and shrna screens . public rna - seq data ( accession encff934ybo ) for the k562 cell line was obtained from encode experiment encsr000aem . genes were filtered for fpkm greater than one and successful mapping to genes present in our libraries , leaving expression data for ~7,000 genes . genes were then ranked from highest to lowest expressed and binned in increments of 500 . the number of genes in each bin were counted for each gene set and normalized by the total number of genes in each gene set present in the rna - seq data . was then graphed versus the mean log(fpkm ) value for each bin ( supplementary figure 11a , b ; supplementary data 10 ) . yeast essential genes annotated as viable or inviable as well as their human homologs were obtained from the saccharomyces genome database . the number of homologs in each gene set were counted for both inviable and viable annotations , and the fraction inviable is presented ( supplementary figure 11c , d ; supplementary data 11 ) . p - values are calculated using fisher s exact test . the total fraction of all homologs mapped from inviable yeast genes is also presented . cell culture performed as previously described . briefly , k562 cells ( atcc ) were cultured in rpmi 1640 ( gibco ) media and supplemented with 10% fbs ( hyclone ) , penicillin ( 10,000 i.u./ml ) , streptomycin ( 10,000 ug / ml ) , and l - glutamine ( 2 mm ) . cells were grown in log phase during all biological assays by returning the population to 500,000 cells / ml each day . k562 cells were maintained in a controlled humidified incubator at 37 c , with 5% co2 . briefly , to generate sufficient lentivirus to infect the genome - wide shrna library into k562 cells , we plated 293 t cells on 15 cm tissue culture plates . 293 t cells were transfected with third generation packaging plasmids and shrna encoding vectors . after 48 hrs and 72 hrs of incubation , lentivirus was harvested . we filtered the pooled lentivirus through a 0.45 m pvdf filter ( millipore ) to remove any cellular debris . approximately 560 million k562 cells were infected with our next - generation genome - wide lentiviral shrna library to maintain roughly 1,000-fold coverage of the shrna library after selection . infected cells grew for 3 days before selecting the cells with puromycin ( 0.7 g / ml , sigma ) . after three days of selection , infection efficiency was monitored using flow cytometry ( bd accuri c6 ) . once the cells reached 90100% mcherry positive cells , they were spun out of selection and allowed to recover in normal rpmi 1640 media . at t0 , 500 million cells were pelleted by centrifugation ( 300 g for 5 min ) . cells were then split into two populations and maintained at logarithmic growth ( 500,000 cells / ml ) each day for 14 days . after 14 days of growth genomic dna was extracted for all 3 time points separately following qiagen s blood maxi kit protocol . a previously designed 4 sgrna / gene crispr - cas9 library was used targeting 5 ends of conserved exons with sgrnas varying in length between 19 and 25 base - pairs . the library was generated first by infecting k562 cells with a sffv - cas9-bfp vector to create a stably expressed cas9 cell line . we then infected the lentiviral genome - wide sgrna library into approximately 120 million cells following the same protocol as the genome - wide shrna library to maintain at least 1,000-fold representation in cells . infected cells were selected with puromycin ( 0.7 g / ml , sigma ) for 3 days . percentage of mcherry positive cells was measured by flow cytometry ( bd accuri c6 ) . selected cells were spun out of selection and into normal rpmi 1640 media . at t0 cells were then split into two populations and grown for 14 days , maintaining logarithmic growth ( 500,000 cells / ml ) each day . after 14 days of growth , cells were pelleted by centrifugation , and genomic dna was extracted for all three time samples separately following qiagen s blood maxi kit instructions . data from previous shrna screen for modifiers of ricin toxicity were obtained as pre- computed hairpin - level enrichments averaged from two replicates , pooled data was analyzed with castle , and compared to signed , log - transformed published results ( supplementary data 5 ) . count data for two replicates from a previous crispr / cas9 cutting screen for lps - induced tnf expression were obtained from the authors , analyzed with castle , and compared to signed , log - transformed published deseq results ( supplementary data 6 ) . data from previous crispri and crispra screens were obtained as pre - computed guide - level enrichment averaged from two replicates , analyzed with castle , and compared to published results ( supplementary data 7 ) . where available , positive and negative results from published low - throughput validations are also presented . trimmed sequences were aligned to the library using bowtie with zero mismatches tolerated . all alignment from multi - mapped reads were used . enrichment of individual hairpins was calculated as a median - normalized log - ratio of fraction of counts , as previously described . raw count files are available ( supplementary data 2 ) , and raw fastq files have been deposited at the sequence read archive ( accession srp072806 ) and bioproject ( accession prjna317269 ; http://www.ncbi.nlm.nih.gov/bioproject/prjna317269 ) . briefly , we built cas9 high throughput maximum likelihood estimator ( castle ) that uses an empirical bayesian framework to account for multiple sources of variability , including reagent efficacy and off - target effects ( for more complete description , see supplementary methods ) . for each gene , we have the phenotypes of multiple targeting reagents . from this , and the phenotypes of negative controls , we obtain an effect size estimate for each gene and an associated log - likelihood ratio . in the figures we present this as the castle score , which is twice the log - likelihood ratio , signed to match the effect size . all screens are analyzed with the same parameters , and no optimization was performed using the gold standard or related sets . these , along with a complete screen analysis pipeline , are available at https://bitbucket.org/dmorgens/castle . for comparison , riger was implemented with default settings on precomputed element enrichments with gene - e ( http://www.broadinstitute.org/cancer/software/gene-e/ ) . rsa was implemented with python scripts available from http://carrier.gnf.org / publications / rsa/. mageck was implemented with default settings with software available from https://sourceforge.net / projects / mageck/. hitselect was implemented with default settings with software available from https://sourceforge.net / projects / hitselect/. median and highest effect heuristics as well as the mw test were implemented with custom python scripts . go terms were generated using gorilla available at http://cbl-gorilla.cs.technion.ac.il/ by ranking genes from highest confidence negative growth phenotypes to lowest confidence genes to highest confidence positive growth phenotypes . for direct comparison to the 4 sgrna per gene crispr / cas9 library , the 25 hairpin per gene library rnai library was down - sampled to 4 shrnas per gene . hairpins were ranked according to their original computational design and the top four unique shrnas were used as well as all negative control shrnas . note that this is independent of the data set used here and represents the 4 shrna library that would have been designed . essential gene predictions were then repeated using this reduced shrna library as above ( supplementary fig . gene sets were defined by 10% fpr cutoff for cas9 , shrna , and combination screens . cas9 and shrna unique gene sets were defined as genes under the 10% fpr cutoff in the cas9 or shrna screen but neither the other nor the combination screen . an overlap set was defined as genes under the 10% fpr cutoff for both cas9 and shrna screens . public rna - seq data ( accession encff934ybo ) for the k562 cell line was obtained from encode experiment encsr000aem . genes were filtered for fpkm greater than one and successful mapping to genes present in our libraries , leaving expression data for ~7,000 genes . genes were then ranked from highest to lowest expressed and binned in increments of 500 . the number of genes in each bin were counted for each gene set and normalized by the total number of genes in each gene set present in the rna - seq data . this fraction of essential genes for each gene set was then graphed versus the mean log(fpkm ) value for each bin ( supplementary figure 11a , b ; supplementary data 10 ) . yeast essential genes annotated as viable or inviable as well as their human homologs were obtained from the saccharomyces genome database . the number of homologs in each gene set were counted for both inviable and viable annotations , and the fraction inviable is presented ( supplementary figure 11c , d ; supplementary data 11 ) . p - values are calculated using fisher s exact test . the total fraction of all homologs mapped from inviable yeast genes is also presented . distribution of targeting and control elements . ( a ) distribution of negative controls for a single replicate of cas9 and shrna screens . ( b , c ) distribution of targeting elements is shown in meta - gene plots for the top 50 ( b ) enriched and ( c ) disenriched genes found in a single replicate of the cas9 and shrna screens as identified by castle . to normalize , the enrichment of each individual element was divided by the effect size estimate for the gene generated by castle . ( a d ) enrichment of targeting elements and estimated effect size is shown for the top four disenriched genes from cas9 data from a single replicate . ( a d ) enrichment of targeting elements and estimated effect size is shown for the top four disenriched genes from shrna data from a single replicate . the unknown relationship between gene dosage and measured phenotype as well as the unknown distribution of shrna and cas9 efficacies restricts the predicted effect size of reagents to a bounded region , marked as the blue shaded region , between 0 and the maximum effect i , marked by the dotted line . some fraction ( 1 ) of the reagents the phenotype observed is thus the true effect obscured by noise , which is estimated using the distribution of non - targeting controls . the likelihood of models for different values of i and are calculated and by marginalizing the most likely effect size is selected . a likelihood ratio is then calculated by comparing to a null model where i is zero . ( a ) results are shown for a previously published shrna screen for ricin sensitivity reanalyzed with castle and compared to published results based on a mw test . ( b ) previous crispr / cas9 deletion screen for lps - induced tnf expression in primary mouse bone - marrow derived dendritic cells , analyzed with castle and the published deseq results . ( c ) previous crispri screen for sensitivity to the fusion toxin ctx - dta , analyzed with castle versus the average of the top three sgrna effects . ( d ) previous crispra screen for sensitivity to the fusion toxin ctx - dta , analyzed with castle versus the average of the top three sgrna effects . ( a d ) roc curves indicate screen performance in identifying essential genes from changing composition between the plasmid library and two weeks growth . true positive rates and false positive rates are calculated using a previously established gold standard set of essential and nonessential genes . genes are ranked by likelihood to be essential using the indicated methods , including castle . highest effect heuristic was calculated by ranking the genes according to their most disenriched element . data is shown from single replicates of the ( a , c ) cas9 and ( b , d ) shrna screens for ( a , b ) replicate 1 and ( c , d ) replicate 2 . supplementary figure 7 . ( a ) roc curves from combination of different replicates of cas9 and shrna using castle . roc curves indicate screen performance in identifying essential genes from changing composition between the plasmid library and two weeks growth . true positive rates and false positive rates are calculated using a previously established gold standard set of essential and nonessential genes . a large positive castle score indicates a high confidence increase in growth rate , while a highly negative castle indicates a high confidence decrease in growth rate , i.e. gene essentiality . the graphs compare replicate measurements of likelihood ratio between plasmid and t14 of the combination score based on replicates 1 for cas9 and shrna and replicates 2 for cas9 and shrna . ( a ) roc curves indicate screen performance in identifying essential genes by comparing the library composition between the plasmid library and cells after two weeks growth . roc curves for cas9 ( red ) and shrna ( blue ) screens based on duplicate data combined using castle . alternatively , data from single replicates of both cas9 and shrna screens were combined using castle ( purple ) . ( b ) the number of essential genes at 10% false positive rate and their overlap based on the duplicate data from cas9 and shrna screens , as well as combination of a single replicate from both screens . ( c ) precision recall curve for cas9 , shrna , and combination data using castle . results from the 25 shrna library were downsampled by only including four hairpins per gene , selected by previous computational ranking . ( a ) roc curves indicate screen performance in identifying essential genes by comparing the library composition between the plasmid library and cells after two weeks growth . ( b ) the number of essential genes at 10% false positive rate and their overlap based on the duplicate data from cas9 and shrna screens , as well as combination of a single replicate from both screens . ( c ) comparison of castle scores derived from castle between single replicates of cas9 and shrna data . ( d ) adjusted p - values for select go terms for shrna and cas9 screens as well as for data from both screens combined with castle . supplementary figure 10 . a large positive castle score indicates a high confidence increase in growth rate , while a highly negative castle score indicates a high confidence decrease in growth rate , i.e. gene essentiality . the graphs compare replicate measurements of castle scores between plasmid and t14 for ( a ) cas9 and ( b ) shrna screens . castle scores in different time - frames for ( c ) cas9 and ( d ) shrna screens . genesets are defined for cas9 , shrna , and combination by a 10% fpr cutoff . genesets are defined for cas9-combo and shrna - combo by the genes present in cas9 or shrna set and not in the combination set . overlap set is defined as genes present in both the cas9 and shrna set ( see supplementary fig . ( a , b ) ~7,000 genes with detectable expression in k562 were binned by expression . the fraction of genes identified as essential in each bin is reported versus the average expression level of the bin . ( c , d ) fraction of genes that are homologs of essential yeast genes versus genes that are homologs of nonessential yeast genes .

myocarditis often occurs due to viral infections and postviral immune - mediated responses . hypersensitivity myocarditis is a rare form of myocarditis . numerous drugs can induce myocarditis , which is typically reversible after withdrawal of the causative agent . here , we report a case of hypersensitivity myocarditis that was probably triggered by amoxicillin and that resolved completely with heart failure management as well as discontinuation of the drug . a 68-year - old woman presented with acute chest pain mimicking acute coronary syndromes , but the coronary angiography was normal . a recent history of taking medications , skin rash , and peripheral eosinophilia suggested a diagnosis of hypersensitivity myocarditis , which was confirmed by cardiac magnetic resonance imaging and endomyocardial biopsy .

clinical presentations vary from nonspecific systemic symptoms ( e.g. , fever , myalgia , palpitation , chest pain , dyspnea ) to fulminant hemodynamic collapse and sudden death . infections ( viral , bacterial , fungal , protozoal , parasitic ) , toxins , immunological syndromes , and hypersensitivity can cause myocarditis . among these lewin et al . reported that seven explanted hearts from 288 heart transplants ( 2.4% ) had histological evidence of hypersensitivity myocarditis . unlike other forms of myocarditis , the prognosis of hypersensitivity myocarditis is excellent , usually improving after administration of the causative drug is ceased . , we report a case of hypersensitivity myocarditis , as confirmed by cardiac magnetic resonance imaging ( cmr ) and endomyocardial biopsy ( emb ) , which subsequently improved after cessation of the causative drug and administration of prednisolone . she had a history of hypertension , transient cerebral ischemic attack , and osteoarthritis in both knees . her blood pressure was 91/53 mmhg , pulse rate was 63 beats per minute , respiration rate was 20 breaths per minute , and body temperature was 37. upon physical examination , heart sounds were normal without murmur . multiple itchy erythematous lesions were present on the hands , buttocks , abdomen , and trunk ( fig . the results of laboratory examinations were as follows : white blood cell count , 9,140/mm ; eosinophil count , 1,380/mm ; hemoglobin , 10.3 g / dl ; platelet count , 123,000/mm ; c - reactive protein level , 1.7 mg / dl ; and erythrocyte sedimentation rate , 72 mm / sec . the levels of brain natriuretic peptide , creatine kinase - mb ( ck - mb ) , and troponin - i were 944 pg / ml , 56.73 ng / ml , and 38.85 ng / ml , respectively . initial electrocardiography showed st segment elevation 1 mm in leads ii , iii , avf , v5 , and v6 ( fig . emergency coronary angiography was performed under the assumption of acute st segment elevation myocardial infarction at the inferolateral wall , but coronary atherosclerosis was not evident . echocardiography showed the " sparkling " appearance of the left ventricular ( lv ) myocardium , concentric lv hypertrophy , and mild hypokinesia on the anterior , anterolateral , and posterior walls from the mid - lv to the apex with 50% lv ejection fraction . peripheral blood eosinophilia , skin rash , and a history of taking multiple medications suggested hypersensitivity myocarditis . 3 . management of heart failure using aldosterone and furosemide was initiated promptly . on the second day of hospital admission , however , dyspnea worsened , and newly developed bilateral pulmonary congestion was noted on chest radiography . therefore , cmr and emb were carried out to confirm the diagnosis of hypersensitivity myocarditis . contrast - enhanced cmr ( ce - cmr ) showed high signal intensities in circular and nodular patterns mainly in the epicardium and septum of the left ventricle ( fig . these patterns suggested infiltrative myocarditis , such as amyloidosis , sarcoidosis , or eosinophilic myocarditis . emb obtained from the septum of the right ventricle revealed lymphocytic and eosinophilic infiltration in the interstitium and perivascular area , findings that were consistent with a diagnosis of hypersensitivity myocarditis ( fig . 5 ) . levels of ck - mb and troponin - i were normalized on days 6 and 9 , respectively . the peripheral eosinophil count decreased from 1,380/mm to 620/mm on day 2 and was normalized ( 50/mm ) on day 4 . the skin rash disappeared almost completely after use of 5 mg prednisolone , and the patient was discharged from the hospital on day 12 . echocardiography at the 6-month follow - up showed normalized regional wall motion abnormalities and improved lv systolic function from 50% to 70% of the ejection fraction . at the 6-month follow - up , ce - cmr showed that previously high signal intensities in circular patterns in the epicardium of the left ventricle had disappeared and the oval - shaped area of high signal intensity in the lv septum had decreased in size ( fig . eosinophilic myocarditis can be classified according to cause and includes those types associated with systemic disease ( e.g. , hypereosinophilic syndrome , churg - strauss syndrome , malignancies ) , parasitic infections ( e.g. , toxocara canis ) , and drugs or vaccines ( hypersensitivity myocarditis ) . eosinophilic myocarditis associated with hypereosinophilic syndrome is usually underpinned by a history of peripheral eosinophilia ( > 1.5 10/l ) lasting > 6 months and is related to systemic involvement ( e.g. , central nervous system , gastrointestinal tract , heart , skin , or lung ) . in the present case , hypereosinophilic syndrome and parasitic infection were excluded because the patient did not have a history of prolonged eosinophilia , other organ involvement , or ingestion of raw meat . instead , she had a history of taking multiple medications , urticaria , and peripheral eosinophilia drug - related myocarditis can be classified into five types : 1 ) hypersensitivity myocarditis ; 2 ) toxic myocarditis ; 3 ) endocardial fibrosis ( e.g. , ergotamine tartrate , methysergide , phentermine , fenfluramine ) ; 4 ) drug - induced cardiomyopathy ( e.g. , anthracycline , chloroquine ) ; and 5 ) giant cell myocarditis . numerous medications , including clozapine , sulfonamide , penicillin antibiotics , methyldopa , and some antiseizure drugs , have been reported to be associated with hypersensitivity myocarditis . delayed hypersensitivity is the main pathogenesis and occurs from several days to months after administration of the causative agent . hypersensitivity myocarditis is not easy to recognize because the clinical features suggestive of drug hypersensitivity reactions ( e.g. , urticaria , angioedema , malaise , fever , eosinophilia ) do not occur uniformly . however , if tissue necrosis or deterioration of clinical status is observed , corticosteroid and cytotoxic agents can be used . emb is the gold standard tool in the diagnosis of myocarditis . according to the dallas criteria , eosinophils may be a minor component of postviral and giant cell myocarditis but are predominant in hypersensitivity and parasitic myocarditis as well as hypereosinophilic syndrome . lymphocytic and eosinophilic infiltrates with slight myocyte necrosis were observed in our case , which coincided with hypersensitivity myocarditis . myocarditis is often a focal process , so sampling error may occur and increase the false - negative rate . a previous study of endomyocardial samples obtained from the hearts of patients who died of myocarditis performed at the mayo clinic reported a false - negative rate of 37% for the right ventricle . other limitations of emb include the aggressiveness of the procedure as well as the delay until confirmation of the result . the lake louise criteria were found to have a sensitivity of 67% , specificity of 91% , accuracy of 78% , positive predictive value of 91% , and negative predictive value of 69% , when the criteria were compared with clinical or histopathological data . mahrholdt and sechtem reported recently that cmr - guided biopsy improved the sensitivity of emb . thus , the combined application of cmr and emb may yield diagnostic synergy and overcome some of the limitations of cmr or emb when applied individually . in our case , we performed cmr - guided emb on the right ventricular septum , in which we detected regional high signal intensity on late gadolinium enhancement . in conclusion , careful history taking ( especially with regard to drugs ) , thorough physical examination , and clinical suspicion can help in recognizing hypersensitivity myocarditis both accurately and promptly . cmr - guided emb is a valuable tool for the early and appropriate diagnosis for hypersensitivity myocarditis . our patient recovered completely after cessation of administration of the causative drug and short - term administration of prednisolone for rapidly worsening hypersensitivity myocarditis .

hepatocellular carcinoma ( hcc ) is the fastest rising cause of cancer - related death in the united states and carries a very poor prognosis , with a median survival time of < 50% at 1 year for advanced disease . to date , sorafenib is the only therapy approved by the food and drug administration for the treatment of advanced hcc . tivantinib ( arq-197 ) , a non - atp competitive inhibitor of cellular mesenchymal epithelial transcription factor ( c - met ) , has shown a survival benefit in patients with advanced hcc who have failed or are intolerant to sorafenib in phase i and ii trials . those patients who have tumors with high concentrations of met ( met - high ) appear to derive the greatest benefit from tivantinib therapy . currently , two large randomized double - blind placebo - controlled phase iii trials ( metiv - hcc [ nct01755767 ] and jet - hcc [ nct02029157 ] ) are evaluating tivantinib in patients with met - high advanced hcc , with the primary end points of overall survival and progression - free survival , respectively . this study reviews the evidence for the use of tivantinib in advanced hcc . specific topics addressed include the pharmacology , dosing , toxicity , and biomarkers associated with tivantinib use .

liver cancer is the second most common cause of cancer - related death worldwide , the fifth most common cancer among men and the ninth most common cancer among women.1 hepatocellular carcinoma ( hcc ) is responsible for ~90% of primary liver cancers.2 in the united states , hcc is the fastest rising cause of cancer - related death , and its incidence has nearly tripled since the 1980s.3,4 despite increased focus and research , the overall prognosis for hcc remains poor . curative modalities , such as liver transplant , resection and radiofrequency ablation , do exist when hcc is diagnosed early ; however , only ~15% of patients are eligible for such treatments ( table 1).3,5,6 the majority of patients are diagnosed with advanced - stage hcc , for which the median survival time is < 50% at 1 year and only 12% at 5 years.7,8 chemotherapy is rarely used for the treatment of hcc due to its minimal success rate of 1020% and high levels of toxicity.2 the only therapy currently approved by the food and drug administration ( fda ) for the treatment of advanced hcc is sorafenib , a tyrosine kinase inhibitor that has been shown to decrease cell proliferation and tumor angiogenesis.9,10 while sorafenib represents a major advancement in the treatment of hcc , its effects are modest , with the sharp trial demonstrating an increase in overall survival from 7.9 months to 10.7 months and an increase from 2.8 months to 5.5 months in the median time to radiological progression.10 it demonstrated even less robust benefits in the asia - pacific trial , with overall survival in the treatment group of 6.5 months and median time to radiological progression of 2.8 months.9 furthermore , after experiencing an initial response to treatment , most hcc patients develop a decrease in efficacy with sorafenib.11 many novel therapies are currently under investigation in clinical trials . in a recently published phase iii trial , regorafenib , an oral multikinase inhibitor , has demonstrated an increase in median overall survival ( 10.6 months vs. 7.8 months ) and an increase in median progression - free survival ( pfs ; 3.1 months vs. 1.5 months ) compared to placebo in patients with advanced hcc who have progressed on sorafenib.12 to date , however , there are no fda - approved second - line or salvage therapies for those patients who progress with or are intolerant to sorafenib.13 one of the most promising new therapies under investigation is tivantinib ( arq 197 ) , which has demonstrated positive results in early - phase clinical trials as a second - line agent for multiple solid tumors . tivantinib is an oral , highly selective , non - atp competitive inhibitor of c - met receptor tyrosine kinase.14 the met proto - oncogene was initially discovered in human osteosarcoma , and it encodes for cellular mesenchymal epithelial transition factor ( c - met).15 c - met and hepatocyte growth factor ( hgf ) , its only known ligand , are necessary for normal embryonic development and tissue repair ; however , their expression is normally very low and only found in cells of epithelial or mesenchymal origin.1618 when hgf binds to c - met , there is activation of multiple metabolic pathways , including the mitogen - activated protein kinase , protein kinase b , mammalian target of rapamycin , phophatidylinositol 3-kinase and focal adhesion kinase , which can lead to tumor growth , proliferation , cell invasion and distant metastasis ( figure 1).1921 upregulation of the c - met pathway is found in multiple cancers , including hcc , where it is present 2040% of the time and is associated with increased risk for metastases and poor prognosis.22,23 hgf is also over - expressed in hcc , which results in increased hepatic regeneration and suppression of hepatocyte apoptosis.24,25 similarly , livers with hcc have been shown to have high levels of mitogen - activated protein kinase , which can go on to activate the raf / mek / erk signaling pathway , leading to unopposed cellular proliferation , migration and survival.25,26 the inhibition of c - met by tivantinib disrupts the deregulated signaling cascade , promotes apoptosis and inhibits cellular growth in those cells that express c - met . munshi et al14 demonstrated the selectivity of tivantinib by showing that tivantinib inhibited only four of 229 human kinases with a ki for c - met of 355 nmol / l . nmol / l is less than that of other available c - met inhibitors , cellular assays demonstrated similar biochemical potency . tivantinib targets the inactive , unphosphorylated form of c - met , locking it in the inactive configuration and preventing downstream signaling.27 controversy does exist regarding the mechanism of action of tivantinib and whether c - met inhibition is the major anti - tumor effect of the drug . several studies have shown that tivantinib possesses cytotoxic activity , even in cells that lack c - met.2832 other proposed mechanisms include microtubule , glycogen synthase kinase 3 , cyclin b1 and proteasome inhibition . microtubule inhibition is the most widely theorized alternative mechanism of action , with three independent in vitro studies corroborating this hypothesis.2931 significant challenges to this alternative hypothesis have been raised . notably , in vivo studies have not documented evidence of neurotoxicity , which is known to be a major adverse effect of microtubule inhibitors.23 similarly , tivantinib has demonstrated only minimal , if any , response in patients who have tumors with low levels of met expression , while those tumors with high levels of met have had the most profound responses to the drug.33,34 from a pharmacokinetic standpoint , tivantinib has an oral bioavailability of > 20% , with a half life of 29 minutes . it is metabolized by the cyp2c19 and cyp3a4 pathways , with 19% elimination via the kidneys and 68% via the stool.35 possible drug interactions could occur with known cyp2c19 inhibitors , such as clopidogrel , esomeprazole , fluconazole , fluoxetine and omeprazole , or with known cyp2c19 inducers , such as carbamazepine , phenytoin and rifampin . maximum inhibition of c - met is obtained after just 24 hours of treatment , and this effect is sustained for 12 hours after the drug is withdrawn.36 three phase i trials have evaluated tivantinib monotherapy in samples without hcc and found tivantinib to be well tolerated with mild clinical benefit.3739 the first of these was conducted as an open - label , sequential 3 + 3 dose escalation design with an initial dose of 100 mg bid at a single center in the united kingdom.37 fifty - one patients with advanced solid organ tumors including prostate , melanoma , gastric , colorectal , sarcoma and breast were included in the trial . the primary objective was to evaluate safety and tolerability and determine the maximum tolerated dose ( mtd ) , establish a recommended phase ii dose ( rp2d ) and define dose - limited toxicities . tivantinib was deemed safe and well tolerated , with an mtd / rp2d of 360 mg bid . stable disease was observed in 14 patients , as defined by response evaluation criteria in solid tumors.40 similarly , a multicenter open - label single - arm dose escalation trial with an initial dose of 10 mg bid was conducted in the united states.38 seventy - nine patients with advanced solid organ tumors , including colorectal , renal , ovarian , pancreatic , sarcoma and thyroid among others , were enrolled . the pfs for all 79 patients was 77% at 6 weeks , 52% at 12 weeks and 34% at 21 weeks . the same concept was evaluated in japan in a multicenter dose - escalating open - label trial with a 3 + 3 design at an initial dose of 70 mg bid.39 forty - seven japanese patients with solid organ tumors were enrolled , although no hcc patients were included in the cohort . it is of interest that these patients were enrolled based on their cyp2c19 polymorphism status . it has been shown that 3080% of asians have a genetic polymorphism of cyp2c19 , making them very poor metabolizers of tivantinib . this same polymorphism , in contrast , is found in only 1219% of whites or blacks.41 tivantinib was well tolerated by both normal and poor metabolizers of cyp2c19 , although a dose adjustment down to 240 mg bid was recommended for poor metabolizers . the first study of tivantinib monotherapy in patients with hcc was a phase ib study conducted by santoro et al.42 three hundred and sixty milligrams of tivantinib was administered twice per day to 21 adult patients with child - pugh a and b cirrhosis and hcc in 28-day treatment cycles . all 21 patients eventually discontinued the treatment , 17 due to disease progression and four due to adverse events . only 16 patients were able to be evaluated for tumor response , and while none of them demonstrated any objective response to therapy , nine did show stable disease ( table 2 ) . the overall median time to disease progression was 3.3 months ( range 1.475.3 months ) among those who were able to be evaluated and 1.8 months ( range 1.65.3 months ) in the intention - to - treat analysis . pharmacokinetic analysis found significant tivantinib accumulation in the plasma of hcc patients compared to the accumulation in other solid tumors , but no correlation was found between tivantinib exposure and adverse events . several years later , a phase i trial using tivantinib monotherapy was performed in a japanese sample with advanced hcc.43 only 28 patients were included in the trial , and though the authors found no complete or partial responses , they did see stable disease in 20 of the 28 patients . multiple phase i studies have been performed using tivantinib as part of a combination therapy with sorafenib , sunitinib , erlotinib and gemcitabine.4447 a preclinical study showed that tivantinib and sorafenib had an additive effect in three hcc cell lines ( jhh-4 , plc / prf/5 and sk - hep-1 ) but did not find any degree of synergy between tivantinib and sunitinib in any of the 87 cancer cell lines tested.48 one of the clinical trials , a phase i trial , assessed the combination of tivantinib and sorafenib in 87 adults with advanced solid organ tumors.44 twenty patients had hcc with child - pugh a ( n=14 ) and b ( n=6 ) cirrhosis . an overall response rate of 11.5% and a 10% response in the hcc population were observed . among the hcc patients , there was one complete response , one partial response and 12 cases of stable disease . interestingly , both the complete response and the partial response were noted in patients who had a high concentration of tumor met , and overall three of four patients with high concentrations of tumor met achieved disease control . the median pfs was 3.5 months ( 95% confidence interval [ 95% ci ] : 311.1 ) , and the disease control rate was 70% . patients with hcc who had received prior treatment with vascular endothelial growth factor ( vegf ) therapy , sorafenib and/or sunitinib had a longer pfs than those who did not receive prior vegf therapy ( 15.9 months [ 95% ci : 1.715.9 ] vs. 3.5 months [ 95% ci : 37.4 ] ) . a pooled analysis was conducted by chai et al46 on all phase i studies with tivantinib in hcc and biliary tract cancer patients . overall , 53 patients were treated with tivantinib : 42 were diagnosed with hcc , ten with cholangiocarcinoma and one with gallbladder adenoma . twenty - three patients received tivantinib monotherapy , and 30 received a combination of tivantinib and either sorafenib ( n=20 ) , gemcitabine ( n=8 ) or erlotinib ( n=2 ) . the overall response rate was 6% , but the disease control rate was 62% , with one complete response , two partial responses and 30 cases of stable disease . based on positive results from phase i trials , a phase ii international multicenter double - blind randomized placebo - controlled trial was conducted.34 one hundred and seven adult patients with advanced - stage hcc who had previously progressed or had demonstrated intolerance to one prior systemic therapy ( sorafenib or sunitinib ) were randomized in a 2:1 ratio to receive tivantinib or placebo . the initial dose of tivantinib was 360 mg bid ; however , this was later decreased to 240 mg bid due to a higher than expected incidence of grade 3 or higher neutropenia . in total , 38 patients received tivantinib 360 mg bid , 33 patients received tivantinib 240 mg bid and 36 patients received placebo . the median time to progression ( ttp ) was significantly longer in the tivantinib group ( 1.6 months [ 95% ci : 1.42.8 ] ) compared to that in the placebo group ( 1.4 months [ 95% ci : 1.41.5 ] ) , with a hazard ratio ( hr ) of 0.64 , 90% ci 0.430.94 , p=0.04 . the median pfs in the tivantinib group ( 1.5 months [ 95% ci : 1.42.7 ] ) was longer than that in the placebo group ( 1.4 months [ 95% ci : 1.41.5 ] ) , with this trend approaching significance , hr 0.67 , 95% ci : 0.441.04 , p=0.06 . there was no significant difference in overall survival between the two groups . according to the statistical analysis plan , the study participants were also subdivided based on their tumor met status into high - expression ( met - high ) and low - expression ( met - low ) subgroups . there were 37 patients with met - high tumors , 22 of whom received tivantinib ( ten patients at 360 mg bid and 12 patients at 240 mg bid ) , and 40 patients with met - low tumors , 27 of whom received tivantinib ( 13 patients at 360 mg bid and 14 patients at 240 mg bid ) . patients who had met - high tumors and received tivantinib had a longer median ttp ( 2.7 months vs. 1.4 months [ hr 0.43 , 95% ci : 0.190.97 , p=0.03 ] ) , a longer median pfs ( 2.2 months vs. 1.4 months [ hr 0.45 , 95% ci : 0.210.95 , p=0.02 ] ) and a longer median overall survival ( 7.2 months vs. 3.8 months [ hr 0.38 , 95% ci : 0.180.81 , p=0.01 ] ) compared to placebo . there was no difference in those with met - low tumors who received tivantinib and those who received placebo . based on the significant benefits seen in the met - high group in the above - mentioned phase ii study , two phase iii trials have been initiated to further evaluate the benefit of tivantinib in patients with met - high hcc tumors . the metiv - hcc trial ( nct01755767 ) is a large international randomized double - blind , placebo - controlled trial comparing tivantinib 120 mg bid to placebo in patients with met - high hcc tumors who have failed or progressed with sorafenib.49 the primary end point is overall survival , and secondary end points are pfs and safety . a planned interim analysis was recently completed , and the study will continue to the final analysis as recommended by the data monitoring committee.50 the jet - hcc trial ( nct02029157 ) is a randomized , double - blind , placebo - controlled trial being conducted in japan to evaluate the effect of tivantinib at 120 mg bid on met - high hcc in patients previously treated with one prior course of sorafenib therapy . the primary end point of this study is pfs , and the secondary end point is overall survival . tivantinib dosing for hcc has continued to evolve as more studies have been performed in this patient population . the two initial phase i dosing trials did not include any patients with hcc , and both recommended a tivantinib dose of 360 mg bid.37,38 the three most common adverse events in both trials were fatigue , nausea and vomiting . dose - limiting toxicities observed by yap et al included one case of grade 3 fatigue at 200 mg bid , two cases of febrile neutropenia at 400 mg bid and one incidence of a combination of mucositis , palmar two cases of pancytopenia , vomiting and dehydration at 360 mg bid were seen in the rosen et al trial . the hematologic toxicity associated with tivantinib is expected , as hgf promotes hematopoiesis by stimulating production of cytokines by the stromal cells in the bone marrow.51,52 the santoro et al42 trial was the first to evaluate tivantinib dosing exclusively in hcc patients . based on the results of prior studies , a starting dose of 360 mg bid was selected . overall , 20 of the 21 patients developed a drug - related event , with the most frequent events being neutropenia ( 52% ) , anemia ( 48% ) , asthenia ( 48% ) and leukopenia ( 38% ) . eleven patients ( 52% ) developed adverse reactions that were grade 3 or grade 4 in severity , the most common of which were neutropenia ( 73% ) , anemia ( 45% ) and leukopenia ( 36% ) . in general , neutropenia was the main cause of dose reduction , treatment interruption and treatment discontinuation . these reactions included one case of grade 3 anemia , one case of grade 3 anemia and grade 4 neutropenia , one case of grade 4 leukopenia and grade 4 neutropenia , and one case of grade 4 neutropenia with grade 5 septic shock leading to death . due to interpatient variability , the authors did not find any correlations among adverse events with tivantinib dose , exposure or child - pugh status ; however , they did recommend that tivantinib dosing in hcc patients be reduced to 240 mg bid due to the high amount of hematologic toxicity . a pooled analysis from all phase i / ib and phase ii studies assessed the exposure response of tivantinib in 289 patients , 73 of whom had hcc.53 the pharmacokinetic analysis demonstrated that the clearance of tivantinib in hcc patients was reduced by 67% , which resulted in a threefold increase in exposure compared to other types of cancer . a significant relationship was also found between tivantinib exposure and incidence of grade 2/3 neutropenia . the authors suggested that a starting dose of 240 mg bid of tivantinib rather than 360 mg bid would result in a decrease in the incidence of grade 3 neutropenia from 28% to 16% in hcc patients . similar results were seen in a later phase ii trial by santoro et al,34 in which the treatment dose was reduced from 360 mg bid to 240 mg bid due to a high incidence of grade 3 neutropenia . overall , hematologic toxicities ( neutropenia , anemia and leukopenia ) occurred more frequently in the tivantinib group than in the placebo group , and the incidence of grade 3 neutropenia was higher in the tivantinib 360 mg bid group than either the tivantinib 240 mg bid or the placebo group . there was even a trend toward longer overall survival in the tivantinib 240 mg bid group ( 7.5 months , 95% ci 4.4not available ) compared to the 360 mg bid group ( 6.4 months , 95% ci 49 ) . both the currently active phase iii trials , metiv - hcc and jet - hcc , are evaluating tivantinib at 120 mg bid . the metiv - hcc trial initially started patients on 240 mg bid ; however , the dose was decreased to 120 mg bid in response to a higher than expected incidence of neutropenia.54 however , it should be noted that the metiv - hcc trial used a different formulation of tivantinib than the santoro et al phase ii trial.23 once the dose was decreased to 120 mg bid , a safety analysis showed that a dose of 120 mg bid of the new formulation resulted in an incidence of neutropenia similar to that seen at a dose of 240 mg in the phase ii trial.55 in addition , a pharmacokinetic analysis showed that the plasma exposure of the new formulation at 120 mg bid was comparable to that of the capsule formulation used in the santoro et al trial.55 the jet - hcc trial set its dosing at 120 mg bid based on dosing studies in japanese samples.43 patients with a cyp2c19 polymorphism may require even lower doses of tivantinib . yamamoto et al39 were the first to examine the effect of the cyp2c19 polymorphism in patients taking tivantinib . forty - seven patients were included in the study , 14 of whom were considered poor metabolizers . the study was unable to demonstrate a relationship between dose level and plasma exposure ; however , it was found that the mean exposure of tivantinib in poor metabolizers at 240 mg bid was equivalent to that of normal metabolizers at a dose of 360 mg bid . based on this finding , the authors recommended that those with the cyp2c19 polymorphism receive doses of 240 mg bid , while those with normal cyp2c19 enzymes receive 360 mg bid . a second trial was conducted by okusaka et al43 to specifically look at tivantinib dosing in those with the cyp2c19 polymorphism and hcc . twenty - eight patients were included in the study , seven of whom had the cyp2c19 polymorphism . all patients tolerated tivantinib at a dose of 120 mg bid , but once the dose was increased to 240 mg bid , five of nine normal metabolizers developed dose - limiting toxicities , either neutropenia or febrile neutropenia , which resolved with drug interruption and treatment with granulocyte colony - stimulating factor medications . because normal metabolizers were unable to tolerate a dose of 240 mg bid , those with the cyp2c19 polymorphism were never escalated to a dose higher than 120 mg bid . the authors concluded that japanese patients with hcc were unable to tolerate a dose of tivantinib > 120 mg bid , irrespective of cyp2c19 polymorphism status . this study served as the basis for the dosing regimen in the jet - hcc trial . the future of tivantinib , like that of many other drugs , rests on appropriate patient selection . the identification of biomarkers for tivantinib sensitivity is an active area of research with promising early results . it was published that patients with met - high tumors showed a significant survival benefit , with longer median ttp ( 2.7 months vs. 1.4 months ) , longer median pfs ( 2.2 months vs. 1.4 months ) and longer median overall survival ( 7.2 months vs. 3.8 months ) on tivantinib compared to placebo.34 no difference in efficacy was found between tivantinib and placebo in patients with met - low tumors . in a combination study of tivantinib with sorafenib , the combination therapy demonstrated a trend toward significance regarding increased disease control rate , defined as the combination of complete responses , partial responses and stable disease for at least 8 weeks.44 while only four cases of met - high hcc were observed , three of the four patients achieved disease control . based on these promising results , both of the active phase iii trials are evaluating tivantinib in hcc patients with met - high tumor status . a preclinical study evaluated whether hgf tumor concentration could be used as a marker for hcc , but no significant differences were found between hgf - high and hgf - low groups in clinicopathological factors or patient outcomes.56 a more recent study that specifically evaluated met , hgf and afp as circulating biomarkers ( by 75 percentile ) found that they were prognostic markers for overall survival in patients with hcc.57 circulating met was also a pharmacodynamic biomarker for tivantinib : patients on treatment with a 10% decrease in circulating met levels demonstrated increased overall survival compared to those with a < 10% decrease ( 13.3 months vs. 6.3 months ; hr : 0.46 [ 95% ci : 0.240.86 ] , p=0.01 ) . additionally , tumor met levels were correlated with response to tivantinib treatment , the only biomarker thus far to predict response to treatment . these results support the use of tivantinib exclusively in met - high tumor patients , although more definitive data will be forthcoming with the results of the metiv - hcc trial , which will analyze over 900 tumor samples and validate the use of select biomarkers in hcc . additional studies are needed to assess the use of tivantinib in those with more severe cirrhosis . the vast majority of trials have evaluated tivantinib in patients who are child - pugh class a , with only a few trials including patients who are child - pugh class b. although theoretically tivantinib could also be used for those who are child - pugh class c , this has not yet been studied . moreover , it may not be safe to attempt such a trial given the anticipated side effects . nevertheless , future development of more specific targeted biologic markers may enable treating physicians to administer agents such as tivantinib in a safer and more effective way . tivantinib , a potent inhibitor of c - met , appears to be a promising therapy for those with advanced - stage hcc that has progressed or is intolerant to sorafenib . tivantinib has demonstrated increased overall survival , pfs and ttp compared to placebo in patients with met - high tumors . serious hematologic side effects ( neutropenia , anemia and leukopenia ) are anticipated to be less prominent at lower dosing regimens ( 120 mg bid ) , at which dose good treatment efficacy is still maintained . two large phase iii trials , metiv - hcc and jet - hcc , are currently ongoing . to date , there is no fda - approved salvage or second - line therapy for patients with hcc who have failed or progressed with sorafenib .

members of the genus aspergillus are the most common fungi and all reproduce asexually by forming long chains of conidiospores ( or conidia ) . the impact of various aspergillus species on humans ranges from beneficial to harmful . for example , several species including aspergillus oryzae and aspergillus niger are used in industry for enzyme production and food processing . in contrast , aspergillus flavus produce the most potent naturally present carcinogen aflatoxins , which contaminate various plant- and animal - based foods . importantly , the opportunistic human pathogen aspergillus fumigatus has become the most prevalent airborne fungal pathogen in developed countries , causing invasive aspergillosis in immunocompromised patients with a high mortality rate . a. fumigatus produces a massive number of small hydrophobic conidia as the primary means of dispersal , survival , genome - protection , and infecting hosts . large - scale genome - wide expression studies can now be conducted due to completion of a. fumigatus genome sequencing . however , genomics becomes more powerful and informative when combined with genetics . we have been investigating the mechanisms underlying the regulation of asexual development ( conidiation ) and gliotoxin biosynthesis in a. fumigatus , primarily focusing on a characterization of key developmental regulators identified in the model fungus aspergillus nidulans . in this review , i will summarize our current understanding of how conidiation in two aspergilli is regulated .

conidiation in aspergillus involves many common developmental themes including spatial and temporal control of gene expression , specialized differentiation of cells , and intra and intercellular communications . conidiation is regulated by an asexual developmental signaling pathway that directs expression control elements and other genes required for conidiophore assembly . as developmental mechanisms in a. nidulans have been intensively studied and results have provided important clues for understanding conidiation in other aspergilli [ 2 , 4 , 7 ] , what is known in a. nidulans ( ani ) will be described first followed by what has been found in a. fumigatus ( afu ) . in ani , a key and essential step for conidiophore development is the activation of brla encoding a c2h2 zinc finger transcription factor ( tf ) , which induces expression of other genes required for conidiation . loss - of - function brla mutants form structures that resemble conidiophore stalks ( thus named " bristle " ) , except that they grow indeterminately and fail to produce vesicles , metuale , phialides , and conidia , indicating that brla controls the initiation of conidiophore development ( fig . , brla overexpression in vegetative cells causes termination of polar growth coupled with the commencement of abnormal sporulation leading to the formation of viable spores from hyphae . no environmental signals , including nutrient limitations or various ( osmotic and oxidative ) stresses , can bypass the brla requirement for conidiation , indicating that activation of brla expression early in conidiophore development represents a key and essential control point for commencing conidiation . the anibrla ( but not afubrla ) gene is a compound gene consisting of two overlapping transcriptional units designated brlaa and brlab . important information on the complex regulation of the two overlapping brla transcription units is well described elsewhere . afubrla , the afu brla homologue , shows 68% identity and 77% similarity to the ani brla . mah and yu tested whether brla - dependent conidiation is conserved in these two aspergilli using genetics and found that afubrla deletion completely eliminated asexual development in afu , resulting in elongated aerial hyphae and increased hyphal mass in the colonies . these studies clearly demonstrated that afubrla is essential for conidiophore development in afu , and that the role of the core downstream tf brla in conidiation is conserved in these aspergilli ( fig . brla is a putative tf ( activator ) with two c2h2 zinc finger motifs at the c - terminus . disruption of either one of the fingers by mutating the key cysteine to serine results in complete loss of brla activity . chang and timberlake further demonstrated that brla expression in the budding yeast saccharomyces cerevisiae results in brla dependent activation of aspergillus genes . furthermore , they proposed the consensus brla binding sites ( brla response elements [ bres ] ; 5'-(c / a)(g / a)aggg(g / a)-3 ' ) . although direct binding of brla to bres remains to be verified in vitro , a number of developmental genes including abaa , weta , roda , and ya have multiple bres in their promoter regions . importantly , tao and yu reported that multiple bres are present in the promoter regions of afu brla , abaa , weta , roda and the velvet regulators , implying a potentially conserved regulatory circuit in the two aspergilli . the abaa gene , activated by brla during the middle stages of conidiation when metulae and phialides are being formed , functions in the differentiation of phialides . abacus ( abaa ) mutants have nearly normal conidiophores bearing abacus - like structures with swellings at intervals instead of chains of conidia . morphological studies have revealed that the metulae of abaa mutants produce supernumerary tiers of cells with properties of metulae , not phialides , indicating that abaa is essential for the differentiation and functionality of phialides as conidiogenous cells . abaa expression is dependent on brla activity , and bres are found in the abaa promoter region , suggesting that brla directly activates abaa . the abaa gene encodes a developmental regulator , which begins to be expressed when phialides form at 15~18 hr after developmental induction ( fig . 3 ) , and loss of abaa function results in abnormal expression of a number of developmentally regulated genes . abaa overexpression in vegetative hyphae causes growth cessation and enhances cellular vacuolization but not spore formation in ani . abaa overexpression activates the expression of weta and brla , and it is thought that abaa induces the expression of brla at certain times of development . however , the genetic interaction between abaa and brla seems more complex , as brla mrna levels increase in abaa null mutants [ 15 , 20 ] , which might involve vosa ( fig . 3 ) . the abaa protein contains an atts / tea dna - binding motif [ 21 , 22 ] and a leucine zipper for potential dimerization . expression studies in both ani and s. cerevisiae have demonstrated that abaa binds to the cis - regulatory elements upstream of the ya gene . results of a gel mobility shift assay indicate that abaa binds to the consensus sequence 5'-cattcy-3 ' ( abaa response element [ are ] where y is pyrimidine ) . in fact , multiple ares are found in the promoter regions of developmentally regulated genes , including brla , weta , ya , roda , and abaa . importantly , park et al . demonstrated that abaa binds to the chitin synthase gene promoter , chsc , suggesting that abaa also regulates chitin biosynthesis during conidiophore development by controlling the expression of certain chitin synthetases . wet - white ( weta ) mutants produce colorless conidia that completely autolyze within a few days , leading to the formation of liquid droplets ( wet ) on the tops of conidial heads . sewall et al . have shown that the weta gene is required for the synthesis of crucial cell wall components late in the development of the inner c4 layer ( fig . the ani weta gene encodes two transcription units , and weta transcription is predicted to be complex and subjected to developmental control . abaa , but not brla , activates weta expression , as abaa overexpression activates weta in the absence of wild type brla . furthermore , weta is auto - activated , as it is not expressed in weta temperature - sensitive mutants ( fig . the ani weta gene is predicted to encode a 60 kda protein rich in serine ( 14% ) , threonine ( 7% ) , and proline ( 10% ) . while no known dna binding domains are present in weta , it has been proposed to function as a regulator of spore - specific gene expression . this is based on observations that weta alone is sufficient to activate many sporulation - specific genes , and that the weta mutants fails to accumulate many sporulation - specific mrnas . furthermore , weta overexpression in vegetative cells inhibits hyphal growth , resulting in excessive hyphal branching and the activation of spore - specific genes . however , weta overexpression does not result in brla or abaa activation or lead to precocious conidiation . taken together , weta is proposed to activate a set of genes required for spore formation and maturation , which may function to complete the final two conidial wall layers and/or direct their assembly ( fig . , the brlaabaaweta cascade has been proposed to define a central regulatory pathway that controls temporal and spatial expression of conidiation - specific genes during conidiophore development and spore maturation ( fig . vosa is a multifunctional regulator that acts in concert with the central regulatory genes , couples trehalose biogenesis and conidia maturation , and exerts negative feedback regulation of brla , thereby completing conidiation in ani ( fig . 3 ) . afuabaa ( eal88194 , 60% identity , 74% similarity to aniabaa ) and afuweta ( eal89470 , 58% identity , 68% similarity ) were previously identified through a genome search . the afuabaa open reading frame ( orf ) is composed of 2,518 bp with three exons and two introns and is predicted to encode a 797 aa - length protein containing an atts / tea dna - binding domain . the afuweta orf comprises 1,701 bp with no introns and is predicted to encode a 566 aa - length protein with a conserved c - terminal domain . tao and yu examined the levels of the afuabaa and afuweta transcripts throughout the lifecycle and found that afuabaa and afuweta are highly expressed during asexual development . afuabaa mrna starts to accumulate at 6 hr post - developmental induction , reaches its highest level at 12 hr , and disappears at 48 hr . afuweta mrna begins to accumulate at 12 hr post - developmental induction , reaches its highest level at 48 hr post induction , and is present in conidia . deletion of afuabaa results in the formation of aberrant conidiophores exhibiting reiterated cylinder - like terminal cells without conidia , indicating that , as in ani , afuabaa is essential for differentiation and functionalization of afu phialides as conidiogenous cells . importantly , tao and yu found that the most distal cells of the afuabaa conidiophores are capable of apical hyphal growth , whereas the fractions of cylinder - like elongated phialides do not undergo vegetative growth . they also examined hyphal dry weights and found that the absence of afuabaa causes delayed hyphal mass loss compared to that in the wild type . when cell viability was examined by determining the percent alamar blue reduction , which represents living cell mitochondrial activity , the afuabaa mutant clearly exhibited prolonged cell viability compared to the wild type . tao and yu then examined the effects of afuabaa overexpression and found that it caused accelerated hyphal fragmentation , disintegration , a dramatic reduction of mycelial mass , and precocious cell death . the afuweta gene is subsequently activated by afuabaa in the middle to late phases of conidiation . furthermore , tem analyses of the afuweta mutant revealed that weta conidiophores exhibit an interconnected conidia phenotype , i.e. , incomplete conidial separation and maturation . as afuweta plays an essential role in completion of the conidial wall , tao and yu hypothesized that the absence of afuweta may affect spore viability and/or integrity , and found that the afuweta mutant exhibits dramatically reduced viability starting from day 10 , whereas wild type conidia maintain high viability until day 20 . moreover , tem studies revealed that , even at day 2 , about 20% of the afuweta conidia appear to lack cytoplasm and exhibit sheared conidial walls . tao and yu then examined the effects of afuweta on spore tolerance to various stressors and found that the afuweta conidia were much more sensitive to heat , and oxidative and osmotic stresses . as trehalose is essential for long - term spore viability and stress resistance [ 15 , 27 ] , tao and yu determined that the afuweta conidia do not contain any trehalose , whereas wild type conidia harbor about 3.4 pg of trehalose per conidium . these results indicate that afuweta plays an essential role in trehalose biogenesis in conidia , which probably affects their viability and stress tolerance . this was the first report that weta is essential for trehalose biogenesis in fungal conidia . additionally , given that the afuweta gene is activated during the late stage of conidiation , and that its mrna accumulates preferentially in mature conidia , tao and yu further determined whether the absence of afuweta had an effect on spore germination and early vegetative growth . they found that after a 8-hr incubation in liquid submerged culture , the afuweta mutant conidia formed only one unidirectional germ tube without branching , which resulted in loosened mycelial aggregates , whereas conidia of wild type and complemented strains produced bi- or multi - directional germ tubes that formed branches . moreover , after a 14-hr incubation , the afuweta mutant clearly showed a five - fold reduction in hyphal branching . these results suggest that afuweta is associated with proper germ tube formation and vegetative growth . this is consistent with the previous finding that aniweta overexpression in hyphae causes excessive branching in ani . tao and yu further dissected the genetic interactions among the central regulatory genes in afu . the absence of afubrla eliminates the expression of all conidiation - specific genes tested , indicating that afubrla functions upstream of afuabaa , afuweta , afuvosa , and afuroda . as afuabaa is necessary for activating afuweta , afuweta mrna accumulation is not detected in the afuabaa mutant . moreover , levels of afuvosa mrna decrease considerably in both afuabaa and afuweta strains , indicating the role of these genes in proper afuvosa expression . however , as afuvosa is activated before afuweta in the wild type , it is proposed that afuabaa primarily activates afuvosa with the assistance of afuweta . importantly , afubrla is highly upregulated in the afuabaa and afuweta mutants , particularly in conidia ( aberrant conidiophores in afuabaa ) and early ( 6 hr ) and late ( 24 hr ) phases of vegetative growth , suggesting that afuabaa and afuweta are necessary for proper negative regulation of afubrla upon completion of conidiation and during certain phases of vegetative growth . afuroda is a small , moderately secreted hydrophobic polypeptide that forms the outermost rodlet layer of conidia . conidia of afuroda mutants lack the external rodlet layer and are hydrophilic . as no afuroda mrna is detected in the afubrla mutant and afuroda levels are low in the afuabaa mutant , tao and yu speculated that afuroda is primarily activated by afubrla with the assistance of afuabaa . a recent genetic study identified the novel regulator vosa , which functions in maturation of conidia and completion of ani development . importantly , vosa deletion results in the lack of trehalose in both types of spores , resulting in a rapid disappearance ( evaporation ) of the cytoplasm , disintegration of cellular organelles , and loss of long - term spore viability . moreover , the vosa mutant spores exhibit dramatically reduced tolerance to heat and oxidative stress . as vosa mainly localizes in the nucleus of mature conidia and contains a potential transcriptional activation domain at the c terminus , it may be a tf that primarily controls the late process of sporulation , including trehalose biogenesis . the vosa protein expressed in metulae and phialides and later localized to the nucleus of the conidia plays two chief roles : 1 ) activation of genes involved in spore maturation ; 2 ) negative feedback regulation of brla and developmental specific genes ( fig . 3 ) . vosa studies clearly divide conidiophore formation and conidia maturation into two distinct genetic phases . vosa has been identified with three other ani proteins sharing high levels of similarity ; vea , velb ( ef540815 ) , and velc ( ef540816 ) . these , together with vosa , define the velvet regulators , and they are highly conserved in many filamentous and dimorphic fungi , sharing at least one highly conserved domain [ 15 , 30 ] . importantly , recent studies have shown that vea , velb , and vosa bridge light - responding development and secondary metabolism by forming trimeric complexes with the nuclear master regulator of secondary metabolism , laea , in ani [ 30 , 31 ] . the afuvosa gene shows an mrna accumulation pattern almost identical to that of anivosa . to begin to understand the roles of the afu velvet proteins , we generated a fu mutants lacking afuvea , afuvelb , afuvosa , and afuvelc , and found that , while the deletion of afuvosa caused an approximate 50% reduction in the spore trehalose content and viability , it did not result in uncontrolled activation of conidiation in afu ( park and yu , unpublished ) . our recent unpublished data suggest that the feedback regulation of conidiation in afu is primarily exerted by velb and vea , whereas trehalose biogenesis is conferred by both afuvosa and afuvelb ( additive roles ) ( fig . identification and characterization of six upstream genes ( flug , flba , flbb , flbc , flbd , and flbe ) required for proper expression of brla in ani have illuminated genetic regulatory cascades for activating conidiation . among these , flbb , flbc , flbd , and flbe are defined by mutants exhibiting the fluffy delayed conidiation phenotypes . flbb , flbc , and flbd are putative tfs containing a basic leucine zipper ( b - zip ) , two c2h2 zinc fingers , and a cmyb - dna binding domain , respectively . thus , it has been thought that flbb , flbb , and flbd likely function as dna binding proteins that may control the transcriptional activation of other developmental regulators , such as brla , in response to sporulation signals . detailed functional studies of these potential tfs have demonstrated that flbb / c / d activate brla expression [ 33 - 38 ] . moreover , b - zip tf flbb is necessary for the activation of flbd expression , while flbb and flbd cooperatively activate brla . they found that flbc mrna is present throughout the lifecycle at relatively high levels during vegetative growth and during early asexual and late sexual developmental phases . deletion of flbc causes a delay / reduction in sporulation , brla and vosa expression , and conidial germination . while flbc overexpression does not cause conidiophore development , it inhibits hyphal growth and activates expression of brla , abaa , and vosa , but not weta . further reported that flbc contains two c2h2 zinc - fingers at the c - terminus and a putative activation domain at the n - terminus . they also found that flbc localizes to the nuclei of both hyphae and developmental cells . localization and expression of flbc is not affected by the absence of flbb or flbe and vice versa . importantly , as flbc overexpression inhibits growth and activates abaa and vosa in the absence of brla and abaa , respectively , kwon et al . proposed that flbc plays a direct activating role in the expression of these genes . consistent with this idea , an in vitro dna binding assay revealed that flbc binds to the promoter regions of brla , abaa , and vosa , but not to that of weta . the aniflbe gene is predicted to encode a 201 aa - length polypeptide with two conserved yet uncharacterized domains , and it has been demonstrated that flbe and flbb are functionally interdependent , that they physically interact in vivo , and co - localize to the hyphal tip in ani . our recent studies revealed that both deletion and overexpression of flbe in ani results in developmental defects , enhanced autolysis , precocious cell death , and delayed expression of brla / vosa , suggesting that balanced flbe activity is crucial for proper growth and development . the n - terminal portion of flbe exhibits a transactivation ability in budding yeast , whereas the c - terminal half negatively affects this activity . site - directed mutagenesis of certain conserved n - terminal amino acids abolishes transactivation ability , overexpression - induced autolysis , and complements the null mutation . these results suggest that the conserved n - terminal domain might be crucial for the functionality of flbe . finally , flbd overexpression , but not that of flbb or flbc , restores conidiation in ani dflbe , generally supporting the current genetic model of developmental regulation . in a series of recent studies , we characterized the functions of flbe and flbb in afu [ 39 , 40 ] . the predicted afu flbe protein is composed of 222 aa in length . while flbe is transiently expressed during the early growth phase in ani , deletion of afuflbe causes reduced conidiation and delayed expression of brla and vosa in both species . moreover , afuflbe is necessary for salt - induced development of afu in a liquid submerged culture . as the ani flbe null mutation can be fully complemented by afuflbe , the developmental function of flbe appears to be conserved in aspergilli . conducted a series of molecular analyses and found that afuflbb produces two transcripts predicted to encode two b - zip polypeptides , afuflbbb ( 420 aa ) and afuflbba ( 390 aa ) . afuflbb deletion results in delayed and reduced conidiation , precocious cell death , the absence of conidiophore development in liquid submerged culture , altered expression of afubrla and afuabaa , and the lack ( or a reduction ) in gliotoxin production . importantly , they found that while introducing the wild type afuflbb allele fully complemented these defects , disruption of the atg start codon to either one of the afuflbb polypeptides led to partial complementation , indicating that both polypeptides are needed for wild type level sporulation and gliotoxin biogenesis . consistent with these observations , introducing ani flbb encoding one polypeptide ( 426 aa ) into the afuflbb null mutant partially restores asexual and chemical development . . also found that the presence of 0.6 m kcl in a liquid submerged culture suppresses the defects caused by the lack of one , but not both , of the afuflbb polypeptides , suggesting a genetic prerequisite for afuflbb in afu development . northern blot analyses revealed that both afuflbb and afuflbe are necessary for afuflbd expression , suggesting that flbd functions downstream of flbb and flbe in both aspergilli . further suggested that afubrla might be necessary for gliotoxin biosynthesis , which was based on the observations that the afubrla mutant lacks gliotoxin production , and that multiple bres are present in the promoter regions of many gliotoxin biosynthetic and regulatory genes . regarding the upstream region of flbb / c / d / e , mah and as discovered in ani , levels of the afuflug transcript are relatively constant throughout the lifecycle . the afuflug deletion mutant conidiated normally , similar to wild type on solid medium , indicating that activation of afu conidiation in the presence of air does not require afuflug . however , the afuflug deletion mutant did not produce conidiophores in liquid submerged culture , whereas afu wild type strains sporulated abundantly at approximately 24 hr . moreover , the afuflug deletion mutant showed reduced conidiation levels and delayed afubrla expression upon induction of synchronized asexual development . these results led mah and yu to conclude that while the presence of air bypasses the need for afuflug in conidiophore development , afuflug plays a particular role in afu conidiation and afubrla expression . these findings led to the hypothesis that afu has multiple pathways to activate afubrla expression ( fig . in ani , a key and essential step for conidiophore development is the activation of brla encoding a c2h2 zinc finger transcription factor ( tf ) , which induces expression of other genes required for conidiation . loss - of - function brla mutants form structures that resemble conidiophore stalks ( thus named " bristle " ) , except that they grow indeterminately and fail to produce vesicles , metuale , phialides , and conidia , indicating that brla controls the initiation of conidiophore development ( fig . , brla overexpression in vegetative cells causes termination of polar growth coupled with the commencement of abnormal sporulation leading to the formation of viable spores from hyphae . no environmental signals , including nutrient limitations or various ( osmotic and oxidative ) stresses , can bypass the brla requirement for conidiation , indicating that activation of brla expression early in conidiophore development represents a key and essential control point for commencing conidiation . the anibrla ( but not afubrla ) gene is a compound gene consisting of two overlapping transcriptional units designated brlaa and brlab . important information on the complex regulation of the two overlapping brla transcription units is well described elsewhere . afubrla , the afu brla homologue , shows 68% identity and 77% similarity to the ani brla . mah and yu tested whether brla - dependent conidiation is conserved in these two aspergilli using genetics and found that afubrla deletion completely eliminated asexual development in afu , resulting in elongated aerial hyphae and increased hyphal mass in the colonies . these studies clearly demonstrated that afubrla is essential for conidiophore development in afu , and that the role of the core downstream tf brla in conidiation is conserved in these aspergilli ( fig . brla is a putative tf ( activator ) with two c2h2 zinc finger motifs at the c - terminus . disruption of either one of the fingers by mutating the key cysteine to serine results in complete loss of brla activity . chang and timberlake further demonstrated that brla expression in the budding yeast saccharomyces cerevisiae results in brla dependent activation of aspergillus genes . furthermore , they proposed the consensus brla binding sites ( brla response elements [ bres ] ; 5'-(c / a)(g / a)aggg(g / a)-3 ' ) . although direct binding of brla to bres remains to be verified in vitro , a number of developmental genes including abaa , weta , roda , and ya have multiple bres in their promoter regions . importantly , tao and yu reported that multiple bres are present in the promoter regions of afu brla , abaa , weta , roda and the velvet regulators , implying a potentially conserved regulatory circuit in the two aspergilli . the abaa gene , activated by brla during the middle stages of conidiation when metulae and phialides are being formed , functions in the differentiation of phialides . abacus ( abaa ) mutants have nearly normal conidiophores bearing abacus - like structures with swellings at intervals instead of chains of conidia . morphological studies have revealed that the metulae of abaa mutants produce supernumerary tiers of cells with properties of metulae , not phialides , indicating that abaa is essential for the differentiation and functionality of phialides as conidiogenous cells . abaa expression is dependent on brla activity , and bres are found in the abaa promoter region , suggesting that brla directly activates abaa . the abaa gene encodes a developmental regulator , which begins to be expressed when phialides form at 15~18 hr after developmental induction ( fig . 3 ) , and loss of abaa function results in abnormal expression of a number of developmentally regulated genes . abaa overexpression in vegetative hyphae causes growth cessation and enhances cellular vacuolization but not spore formation in ani . abaa overexpression activates the expression of weta and brla , and it is thought that abaa induces the expression of brla at certain times of development . however , the genetic interaction between abaa and brla seems more complex , as brla mrna levels increase in abaa null mutants [ 15 , 20 ] , which might involve vosa ( fig . 3 ) . the abaa protein contains an atts / tea dna - binding motif [ 21 , 22 ] and a leucine zipper for potential dimerization . expression studies in both ani and s. cerevisiae have demonstrated that abaa binds to the cis - regulatory elements upstream of the ya gene . results of a gel mobility shift assay indicate that abaa binds to the consensus sequence 5'-cattcy-3 ' ( abaa response element [ are ] where y is pyrimidine ) . in fact , multiple ares are found in the promoter regions of developmentally regulated genes , including brla , weta , ya , roda , and abaa . importantly , park et al . demonstrated that abaa binds to the chitin synthase gene promoter , chsc , suggesting that abaa also regulates chitin biosynthesis during conidiophore development by controlling the expression of certain chitin synthetases . wet - white ( weta ) mutants produce colorless conidia that completely autolyze within a few days , leading to the formation of liquid droplets ( wet ) on the tops of conidial heads . sewall et al . have shown that the weta gene is required for the synthesis of crucial cell wall components late in the development of the inner c4 layer ( fig . the ani weta gene encodes two transcription units , and weta transcription is predicted to be complex and subjected to developmental control . abaa , but not brla , activates weta expression , as abaa overexpression activates weta in the absence of wild type brla . furthermore , weta is auto - activated , as it is not expressed in weta temperature - sensitive mutants ( fig . the ani weta gene is predicted to encode a 60 kda protein rich in serine ( 14% ) , threonine ( 7% ) , and proline ( 10% ) . while no known dna binding domains are present in weta , it has been proposed to function as a regulator of spore - specific gene expression . this is based on observations that weta alone is sufficient to activate many sporulation - specific genes , and that the weta mutants fails to accumulate many sporulation - specific mrnas . furthermore , weta overexpression in vegetative cells inhibits hyphal growth , resulting in excessive hyphal branching and the activation of spore - specific genes . however , weta overexpression does not result in brla or abaa activation or lead to precocious conidiation . taken together , weta is proposed to activate a set of genes required for spore formation and maturation , which may function to complete the final two conidial wall layers and/or direct their assembly ( fig . together , the brlaabaaweta cascade has been proposed to define a central regulatory pathway that controls temporal and spatial expression of conidiation - specific genes during conidiophore development and spore maturation ( fig . vosa is a multifunctional regulator that acts in concert with the central regulatory genes , couples trehalose biogenesis and conidia maturation , and exerts negative feedback regulation of brla , thereby completing conidiation in ani ( fig . afuabaa ( eal88194 , 60% identity , 74% similarity to aniabaa ) and afuweta ( eal89470 , 58% identity , 68% similarity ) were previously identified through a genome search . the afuabaa open reading frame ( orf ) is composed of 2,518 bp with three exons and two introns and is predicted to encode a 797 aa - length protein containing an atts / tea dna - binding domain . the afuweta orf comprises 1,701 bp with no introns and is predicted to encode a 566 aa - length protein with a conserved c - terminal domain . tao and yu examined the levels of the afuabaa and afuweta transcripts throughout the lifecycle and found that afuabaa and afuweta are highly expressed during asexual development . afuabaa mrna starts to accumulate at 6 hr post - developmental induction , reaches its highest level at 12 hr , and disappears at 48 hr . afuweta mrna begins to accumulate at 12 hr post - developmental induction , reaches its highest level at 48 hr post induction , and is present in conidia . deletion of afuabaa results in the formation of aberrant conidiophores exhibiting reiterated cylinder - like terminal cells without conidia , indicating that , as in ani , afuabaa is essential for differentiation and functionalization of afu phialides as conidiogenous cells . importantly , tao and yu found that the most distal cells of the afuabaa conidiophores are capable of apical hyphal growth , whereas the fractions of cylinder - like elongated phialides do not undergo vegetative growth . they also examined hyphal dry weights and found that the absence of afuabaa causes delayed hyphal mass loss compared to that in the wild type . when cell viability was examined by determining the percent alamar blue reduction , which represents living cell mitochondrial activity , the afuabaa mutant clearly exhibited prolonged cell viability compared to the wild type . tao and yu then examined the effects of afuabaa overexpression and found that it caused accelerated hyphal fragmentation , disintegration , a dramatic reduction of mycelial mass , and precocious cell death . these results indicate that afuabaa functions in afu autolysis and cell death . the afuweta gene is subsequently activated by afuabaa in the middle to late phases of conidiation . furthermore , tem analyses of the afuweta mutant revealed that weta conidiophores exhibit an interconnected conidia phenotype , i.e. , incomplete conidial separation and maturation . as afuweta plays an essential role in completion of the conidial wall , tao and yu hypothesized that the absence of afuweta may affect spore viability and/or integrity , and found that the afuweta mutant exhibits dramatically reduced viability starting from day 10 , whereas wild type conidia maintain high viability until day 20 . moreover , tem studies revealed that , even at day 2 , about 20% of the afuweta conidia appear to lack cytoplasm and exhibit sheared conidial walls . tao and yu then examined the effects of afuweta on spore tolerance to various stressors and found that the afuweta conidia were much more sensitive to heat , and oxidative and osmotic stresses . as trehalose is essential for long - term spore viability and stress resistance [ 15 , 27 ] , tao and yu determined that the afuweta conidia do not contain any trehalose , whereas wild type conidia harbor about 3.4 pg of trehalose per conidium . these results indicate that afuweta plays an essential role in trehalose biogenesis in conidia , which probably affects their viability and stress tolerance . this was the first report that weta is essential for trehalose biogenesis in fungal conidia . additionally , given that the afuweta gene is activated during the late stage of conidiation , and that its mrna accumulates preferentially in mature conidia , tao and yu further determined whether the absence of afuweta had an effect on spore germination and early vegetative growth . they found that after a 8-hr incubation in liquid submerged culture , the afuweta mutant conidia formed only one unidirectional germ tube without branching , which resulted in loosened mycelial aggregates , whereas conidia of wild type and complemented strains produced bi- or multi - directional germ tubes that formed branches . moreover , after a 14-hr incubation , the afuweta mutant clearly showed a five - fold reduction in hyphal branching . these results suggest that afuweta is associated with proper germ tube formation and vegetative growth . this is consistent with the previous finding that aniweta overexpression in hyphae causes excessive branching in ani . tao and yu further dissected the genetic interactions among the central regulatory genes in afu . the absence of afubrla eliminates the expression of all conidiation - specific genes tested , indicating that afubrla functions upstream of afuabaa , afuweta , afuvosa , and afuroda . as afuabaa is necessary for activating afuweta , afuweta mrna accumulation is not detected in the afuabaa mutant . moreover , levels of afuvosa mrna decrease considerably in both afuabaa and afuweta strains , indicating the role of these genes in proper afuvosa expression . however , as afuvosa is activated before afuweta in the wild type , it is proposed that afuabaa primarily activates afuvosa with the assistance of afuweta . importantly , afubrla is highly upregulated in the afuabaa and afuweta mutants , particularly in conidia ( aberrant conidiophores in afuabaa ) and early ( 6 hr ) and late ( 24 hr ) phases of vegetative growth , suggesting that afuabaa and afuweta are necessary for proper negative regulation of afubrla upon completion of conidiation and during certain phases of vegetative growth . afuroda is a small , moderately secreted hydrophobic polypeptide that forms the outermost rodlet layer of conidia . conidia of afuroda mutants lack the external rodlet layer and are hydrophilic . as no afuroda mrna is detected in the afubrla mutant and afuroda levels are low in the afuabaa mutant , tao and yu speculated that afuroda is primarily activated by afubrla with the assistance of afuabaa . a recent genetic study identified the novel regulator vosa , which functions in maturation of conidia and completion of ani development . importantly , vosa deletion results in the lack of trehalose in both types of spores , resulting in a rapid disappearance ( evaporation ) of the cytoplasm , disintegration of cellular organelles , and loss of long - term spore viability . moreover , the vosa mutant spores exhibit dramatically reduced tolerance to heat and oxidative stress . as vosa mainly localizes in the nucleus of mature conidia and contains a potential transcriptional activation domain at the c terminus , it may be a tf that primarily controls the late process of sporulation , including trehalose biogenesis . the vosa protein expressed in metulae and phialides and later localized to the nucleus of the conidia plays two chief roles : 1 ) activation of genes involved in spore maturation ; 2 ) negative feedback regulation of brla and developmental specific genes ( fig . 3 ) . vosa studies clearly divide conidiophore formation and conidia maturation into two distinct genetic phases . vosa has been identified with three other ani proteins sharing high levels of similarity ; vea , velb ( ef540815 ) , and velc ( ef540816 ) . these , together with vosa , define the velvet regulators , and they are highly conserved in many filamentous and dimorphic fungi , sharing at least one highly conserved domain [ 15 , 30 ] . importantly , recent studies have shown that vea , velb , and vosa bridge light - responding development and secondary metabolism by forming trimeric complexes with the nuclear master regulator of secondary metabolism , laea , in ani [ 30 , 31 ] . the afuvosa gene shows an mrna accumulation pattern almost identical to that of anivosa . to begin to understand the roles of the afu velvet proteins , we generated a fu mutants lacking afuvea , afuvelb , afuvosa , and afuvelc , and found that , while the deletion of afuvosa caused an approximate 50% reduction in the spore trehalose content and viability , it did not result in uncontrolled activation of conidiation in afu ( park and yu , unpublished ) . our recent unpublished data suggest that the feedback regulation of conidiation in afu is primarily exerted by velb and vea , whereas trehalose biogenesis is conferred by both afuvosa and afuvelb ( additive roles ) ( fig . identification and characterization of six upstream genes ( flug , flba , flbb , flbc , flbd , and flbe ) required for proper expression of brla in ani have illuminated genetic regulatory cascades for activating conidiation . among these , flbb , flbc , flbd , and flbe are defined by mutants exhibiting the fluffy delayed conidiation phenotypes . flbb , flbc , and flbd are putative tfs containing a basic leucine zipper ( b - zip ) , two c2h2 zinc fingers , and a cmyb - dna binding domain , respectively . thus , it has been thought that flbb , flbb , and flbd likely function as dna binding proteins that may control the transcriptional activation of other developmental regulators , such as brla , in response to sporulation signals . detailed functional studies of these potential tfs have demonstrated that flbb / c / d activate brla expression [ 33 - 38 ] . moreover , b - zip tf flbb is necessary for the activation of flbd expression , while flbb and flbd cooperatively activate brla . they found that flbc mrna is present throughout the lifecycle at relatively high levels during vegetative growth and during early asexual and late sexual developmental phases . deletion of flbc causes a delay / reduction in sporulation , brla and vosa expression , and conidial germination . while flbc overexpression does not cause conidiophore development , it inhibits hyphal growth and activates expression of brla , abaa , and vosa , but not weta . further reported that flbc contains two c2h2 zinc - fingers at the c - terminus and a putative activation domain at the n - terminus . they also found that flbc localizes to the nuclei of both hyphae and developmental cells . localization and expression of flbc is not affected by the absence of flbb or flbe and vice versa . importantly , as flbc overexpression inhibits growth and activates abaa and vosa in the absence of brla and abaa , respectively , kwon et al . proposed that flbc plays a direct activating role in the expression of these genes . consistent with this idea , an in vitro dna binding assay revealed that flbc binds to the promoter regions of brla , abaa , and vosa , but not to that of weta . the aniflbe gene is predicted to encode a 201 aa - length polypeptide with two conserved yet uncharacterized domains , and it has been demonstrated that flbe and flbb are functionally interdependent , that they physically interact in vivo , and co - localize to the hyphal tip in ani . our recent studies revealed that both deletion and overexpression of flbe in ani results in developmental defects , enhanced autolysis , precocious cell death , and delayed expression of brla / vosa , suggesting that balanced flbe activity is crucial for proper growth and development . the n - terminal portion of flbe exhibits a transactivation ability in budding yeast , whereas the c - terminal half negatively affects this activity . site - directed mutagenesis of certain conserved n - terminal amino acids abolishes transactivation ability , overexpression - induced autolysis , and complements the null mutation . these results suggest that the conserved n - terminal domain might be crucial for the functionality of flbe . finally , flbd overexpression , but not that of flbb or flbc , restores conidiation in ani dflbe , generally supporting the current genetic model of developmental regulation . in a series of recent studies , we characterized the functions of flbe and flbb in afu [ 39 , 40 ] . the predicted afu flbe protein is composed of 222 aa in length . while flbe is transiently expressed during the early growth phase in ani , deletion of afuflbe causes reduced conidiation and delayed expression of brla and vosa in both species . moreover , afuflbe is necessary for salt - induced development of afu in a liquid submerged culture . as the ani flbe null mutation can be fully complemented by afuflbe , the developmental function of flbe appears to be conserved in aspergilli . xiao et al . conducted a series of molecular analyses and found that afuflbb produces two transcripts predicted to encode two b - zip polypeptides , afuflbbb ( 420 aa ) and afuflbba ( 390 aa ) . afuflbb deletion results in delayed and reduced conidiation , precocious cell death , the absence of conidiophore development in liquid submerged culture , altered expression of afubrla and afuabaa , and the lack ( or a reduction ) in gliotoxin production . importantly , they found that while introducing the wild type afuflbb allele fully complemented these defects , disruption of the atg start codon to either one of the afuflbb polypeptides led to partial complementation , indicating that both polypeptides are needed for wild type level sporulation and gliotoxin biogenesis . consistent with these observations , introducing ani flbb encoding one polypeptide ( 426 aa ) into the afuflbb null mutant partially restores asexual and chemical development . . also found that the presence of 0.6 m kcl in a liquid submerged culture suppresses the defects caused by the lack of one , but not both , of the afuflbb polypeptides , suggesting a genetic prerequisite for afuflbb in afu development . northern blot analyses revealed that both afuflbb and afuflbe are necessary for afuflbd expression , suggesting that flbd functions downstream of flbb and flbe in both aspergilli . xiao et al . further suggested that afubrla might be necessary for gliotoxin biosynthesis , which was based on the observations that the afubrla mutant lacks gliotoxin production , and that multiple bres are present in the promoter regions of many gliotoxin biosynthetic and regulatory genes . regarding the upstream region of flbb / c / d / e , mah and as discovered in ani , levels of the afuflug transcript are relatively constant throughout the lifecycle . the afuflug deletion mutant conidiated normally , similar to wild type on solid medium , indicating that activation of afu conidiation in the presence of air does not require afuflug . however , the afuflug deletion mutant did not produce conidiophores in liquid submerged culture , whereas afu wild type strains sporulated abundantly at approximately 24 hr . moreover , the afuflug deletion mutant showed reduced conidiation levels and delayed afubrla expression upon induction of synchronized asexual development . these results led mah and yu to conclude that while the presence of air bypasses the need for afuflug in conidiophore development , afuflug plays a particular role in afu conidiation and afubrla expression . these findings led to the hypothesis that afu has multiple pathways to activate afubrla expression ( fig . our further studies suggest that afuflbd is essential for proper asexual development in afu ( xiao and yu , unpublished data ) . to examine potential genetic interactions among upstream developmental regulators in afu , xiao et al . conducted a series of expression studies and proposed that afu has an upstream regulatory cascade slightly different from the one proposed for ani ( fig . afuflbe expression is independent , and both are required for proper afuflbd expression , xiao et al . proposed that afuflbb and afuflbe function upstream of afuflbd and cooperatively activate afuflbd , which subsequently activates afubrla . while the transcriptional activation of afuflbb and afuflbe is independent , it may be that the afuflbb and afuflbe proteins interact and form a functional complex as in ani . furthermore , as afuflbc expression is independent of afuflbb and afuflbe , afuflbc is predicted to function in a separate pathway . because the afubrla mutant is defective in gliotoxin production and multiple bres are present in the promoter regions of many gliotoxin biosynthetic and regulatory genes , xiao et al . further functional and molecular studies of afuflbc and the identification of additional developmental regulators , including afusfga and afuwetb , are in progress in my laboratory . in this review , i have briefly summarized our current understanding of the upstream and downstream regulation of aspergillus conidiation focusing on key tfs . in summary , these two aspergilli have common essential downstream activators of conidiophore development ; brla , abaa , and weta . moreover , velvet regulator functions are generally conserved in these aspergilli . however , as clear differences in the upstream regulation of conidiation between the two species exist , much remains to be learned . further genetic and genomics studies aimed at identifying and characterizing the unique regulators and control networks in afu will illuminate the complex mechanisms of development in this opportunistic pathogenic fungus .