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conditions such as multiple chemical sensitivity ( mcs ) , fibromyalgia ( fm ) , and chronic fatigue syndrome ( cfs ) are a significant problem to the health care system due to the lack of effective management strategies.14 mcs is defined as a chronic condition with reproducible symptoms involving multiple organ systems whose symptoms are produced by low levels of exposure to multiple , chemically unrelated substances and improve or resolve when the chemical agents are removed.5 the canadian clinical working case definition for fm is that the condition is categorized by a history of widespread pain and pain on palpation at 11 or more of the defined tender point sites . patients with this condition also experience significant fatigue and cognitive problems.6 a patient receives a diagnosis of cfs if they have severe chronic fatigue of six months or longer duration with other known medical conditions excluded by clinical diagnosis and they have concurrently four or more of the following symptoms : substantial impairment in short - term memory or concentration ; sore throat ; tender lymph nodes ; muscle pain ; multi - joint pain without swelling or redness ; headaches of a new type , pattern or severity ; unrefreshing sleep ; and post - exertional malaise lasting more than 24 hours.7 aaron and buchwald8 reviewed the shared features of mcs , cfs , and fm such as fatigue / pain , inconsistent demonstration of laboratory abnormalities , disability out of proportion to examination findings , and association with stress and psychosocial factors . they found significant overlap among definitions for the core symptoms of these conditions suggesting co - morbidity of these conditions for patients . mcs , fm , cfs , and in some cases chronic pain have been attributed to the environment . for these conditions there are no specific or standardized management strategies for treatment that have been shown to alleviate or reverse the condition.912 the challenges in developing standardized care lie in the number of overlapping conditions with a wide variety of symptoms which are poorly characterized , but appear to be linked to the environment . predominantly , patients require an individualized care plan that takes into account all aspects of their illness.9 however , since these conditions are poorly understood with limited information in the literature and a multitude of overlapping symptoms and diagnoses , they often leave health care professionals challenged when attempting to develop a management strategy . these problems may be recognized by the patients , and this may lead to requests for referral to multiple specialists and the ordering of various costly laboratory tests.13 patients also learn to move beyond the traditional medical approach and begin to manage their own treatment.14,15 the nova scotia environmental health centre ( nsehc ) is a medical facility with a mandate for treatment and research of medical conditions such as mcs , cfs , and fm.16,17 at the nsehc , a multidisciplinary approach to treatment is taken in which the various disciplines work together in generating information on all aspects of the patient s illness before developing an individualized care plan for every patient . research at the nsehc is targeted towards understanding the nature of these illnesses , type and extent of symptom occurrence and their triggers , and the study of effective management strategies . joffres and colleagues13 looked at the prevalence of major symptoms in 351 patients from the nsehc . general symptoms such as difficulty concentrating , fatigue , forgetfulness , and irritability dominated the overall prevalence of symptoms since the start of their illness . those related to irritation such as sneezing , itchy or burning eyes , and hoarseness or loss of voice were more common when exposed to environmental stressors . a review of 100 patients18 who fulfilled the consensus criteria for mcs5 has shown that major senses are frequently reported as being modified , with 88% of patients reporting increased sense of smell , 74% reporting light sensitivity and 74% reporting sound sensitivity . another study conducted at the nsehc,19 investigated the ability to adapt to environmental challenges in individuals with and without mcs . using a variety of physiological readings ( skin conductance , skin temperature , respiratory rate , surface electro - myography , and heart rate ) and from self - reported symptoms , we showed evidence that the majority of participants are in a heightened state of reactivity or central nervous system arousal and have altered sensitivity in the five major senses . subjects with chemical sensitivities took longer to adapt to baseline protocols such as variation in noise levels in the booth environment , than did controls . after adaptation ( stability in physiological measures to baseline protocols ) , despite small study numbers , individuals with mcs displayed statistically significant responses ( p < 0.02 ) in tonic skin conductance response to test substances compared with controls and compared with the control substance . reported symptoms were also higher in cases than in controls for the test substances , body wash solution ( p = 0.05 ) and dryer sheets ( p = 0.02 ) . clinical evaluations at the nsehc have revealed that the patient population may have to cope with a variety of biopsychosocial stressors in addition to the symptoms and stressors related to their illness . this additional stress may have a negative impact on their health and well being . despite the concise definition for mcs,5 patients who receive this diagnosis often report being subjected to a variety of biases and misinterpretations from within the medical community , employers , family , and friends . some of our patients report marginalization and negative labeling such as malingering or neurotic personality disorders.20 for patients who are desperate to feel well , and want to live as productive members of society in their work / professions and interpersonal relationships , this adds another layer of stress of being misunderstood and viewed through the lens of a psychiatric disorder . in extreme cases , this may lead individuals to become fearful and skeptical of doctors , employers , insurance caseworkers , the justice system , and other agencies that become involved in an attempt to provide answers , solutions , and support to their state of illness and disease . while this may not be the only cause for the stress experienced by individuals with this diagnosis , it could add to the overall perception of stress in the management of their illness . the goal of the care management at the nsehc is to address the whole person and offer interventions that can strengthen resiliency towards management of the illness and build coping skills to all types of stressors . the body mind awareness program , an approach based on the mindfulness - based stress reduction model,21,22 was initiated as a pilot project in 1997 and , given its initial success , was subsequently incorporated into the stream of interventions and treatment options available to individuals at the nsehc . the body mind awareness program ( bmap ) at the nsehc follows the guidelines of the original mbsr program developed by jon kabat - zinn.21 illness is a major stressor , and how one copes with stress has consequences for health outcomes . results from other trials using the mbsr approach have shown that it is effective in reducing anxiety , depression , pain , and psychological symptoms associated with illness.2229 evaluations based on anecdotal reports collected at the end of each bmap program have revealed the beneficial nature of this technique as applied to individuals seeking treatment at the nsehc . patients report feeling less anxious , less depressed , use less pain medication , experience better sleep , and achieve a better quality of life . a controlled study was thus undertaken to quantify these narratives and identify the type and extent of changes in patients undergoing intervention versus wait - list controls using the symptoms checklist inventory ( scl-90r).30 the objective of the study was to determine the impact and extent of changes in women diagnosed with mcs , cfs , and fm following the bmap intervention when compared to a group of individuals that were waiting to be seen at the nsehc . participants for the intervention group were selected from women who received a referral to bmap from a physician at the nsehc . the intervention group in the study continued to receive the usual care management that was essential to manage their illness . the control group consisted of the first 50 women from the wait list group who consented to participate in the study . the study compared the results of a 10-week intervention group that underwent training in the mbap to a control group that was comprised of women waitlisted to be seen as patients at the nsehc . the participants were screened for the following exclusionary criteria : subjects with major illnesses such as cancer ; and subjects with an untreated psychiatric disorder and that might be negatively affected by the program or that might affect the group process . there were no people excluded on the basis of the outlined criteria for this study . men who participated in the bmap intervention were excluded from the study sample since women were a fair representation of the patient population.13 following the receipt of informed consent , an intervention group undertook 10-week training in mindfulness - based stress reduction of 2.5 hours each week , in addition to committing to a daily home practice . the bmap typically consists of 10 weekly group meetings , each one lasting approximately 2.5 hours . the facilitator is trained in mbsr through the omega institute , rhinebeck , new york with 12 years experience in facilitating the bmap intervention . each session provides an opportunity for participants to share their experiences of the week of mindfulness practice of the body scan , sitting meditation , or mindfulness yoga . one session is reserved as a silent retreat of six hours at week 8 of the program . commitment to the homework exercises which involve the practice of mindfulness both formally ( sitting meditation , yoga , and body scan ) and informally ( eating , cooking , driving , and other day - to - day activities ) is an essential component of the program . with the guidance and support from the instructor , participants begin to generalize what they learn in class and in daily home practice towards the management of their daily lives and their illness challenges in particular . participants are provided with compact discs that guide them through the formal mindfulness practices : yoga , mindfulness meditation practices , and the body scan . in addition , as the practices develop , patients are introduced to the concept that thoughts are not facts and that one can develop the ability to respond to stressors , rather than react to them . the goal is to help participants become more aware of thoughts , feelings , and sensations and their inter - related connections . to ensure the consistent application of the principles learned , a workbook is provided for each person to follow throughout the intervention . with the commitment to repeated mindfulness practices , participants begin to develop the ability to step out of habitual reactive patterns or negative - thinking patterns that might otherwise escalate to a cycle of stress reactivity and a heightened arousal state during stressful life - moments . the intervention group answered the scl-90r pre- and post - intervention ( before commencement of bmap intervention and at the end of 10-week intervention ) and at a three - month follow - up . scl-90r was administered to the control group at the same time periods . in a clinical evaluation conducted at the nsehc using scl-90r , brief symptoms inventory , mcgill pain questionnaire and whoqol - bref ( world health organization quality of life ) , scl-90r was identified as the most compelling outcome for bmap based on the consistency and sensitivity in capturing the changes pre and post intervention . data analysis for this study utilized sas ( version 9.1 ; sas institute inc . , cary , nc , usa ) , and the usual procedures for continuous data ( t - test , analysis of variance , general linear model [ glm ] ) and categorical data ( sas processes : freq , logistic ) . the objective of the study was to determine the impact and extent of changes in women diagnosed with mcs , cfs , and fm following the bmap intervention when compared to a group of individuals that were waiting to be seen at the nsehc . participants for the intervention group were selected from women who received a referral to bmap from a physician at the nsehc . the intervention group in the study continued to receive the usual care management that was essential to manage their illness . the control group consisted of the first 50 women from the wait list group who consented to participate in the study . the study compared the results of a 10-week intervention group that underwent training in the mbap to a control group that was comprised of women waitlisted to be seen as patients at the nsehc . the participants were screened for the following exclusionary criteria : subjects with major illnesses such as cancer ; and subjects with an untreated psychiatric disorder and that might be negatively affected by the program or that might affect the group process . there were no people excluded on the basis of the outlined criteria for this study . men who participated in the bmap intervention were excluded from the study sample since women were a fair representation of the patient population.13 following the receipt of informed consent , an intervention group undertook 10-week training in mindfulness - based stress reduction of 2.5 hours each week , in addition to committing to a daily home practice . the bmap typically consists of 10 weekly group meetings , each one lasting approximately 2.5 hours . the facilitator is trained in mbsr through the omega institute , rhinebeck , new york with 12 years experience in facilitating the bmap intervention . each session provides an opportunity for participants to share their experiences of the week of mindfulness practice of the body scan , sitting meditation , or mindfulness yoga . one session is reserved as a silent retreat of six hours at week 8 of the program . commitment to the homework exercises which involve the practice of mindfulness both formally ( sitting meditation , yoga , and body scan ) and informally ( eating , cooking , driving , and other day - to - day activities ) is an essential component of the program . with the guidance and support from the instructor , participants begin to generalize what they learn in class and in daily home practice towards the management of their daily lives and their illness challenges in particular . participants are provided with compact discs that guide them through the formal mindfulness practices : yoga , mindfulness meditation practices , and the body scan . in addition , as the practices develop , patients are introduced to the concept that thoughts are not facts and that one can develop the ability to respond to stressors , rather than react to them . the goal is to help participants become more aware of thoughts , feelings , and sensations and their inter - related connections . to ensure the consistent application of the principles learned , a workbook is provided for each person to follow throughout the intervention . with the commitment to repeated mindfulness practices , participants begin to develop the ability to step out of habitual reactive patterns or negative - thinking patterns that might otherwise escalate to a cycle of stress reactivity and a heightened arousal state during stressful life - moments . the intervention group answered the scl-90r pre- and post - intervention ( before commencement of bmap intervention and at the end of 10-week intervention ) and at a three - month follow - up . scl-90r was administered to the control group at the same time periods . in a clinical evaluation conducted at the nsehc using scl-90r , brief symptoms inventory , mcgill pain questionnaire and whoqol - bref ( world health organization quality of life ) , scl-90r was identified as the most compelling outcome for bmap based on the consistency and sensitivity in capturing the changes pre and post intervention . data analysis for this study utilized sas ( version 9.1 ; sas institute inc . , cary , nc , usa ) , and the usual procedures for continuous data ( t - test , analysis of variance , general linear model [ glm ] ) and categorical data ( sas processes : freq , logistic ) . the study included 50 women in the intervention group and 26 women in the control group . mean age of participants in the intervention group was 46.5 ( standard deviation [ sd ] = 9.3 , range = 3564 ) and in the control group was 44.5 ( sd = 8.5 , range = 3260 ) . approximately 16% of the total recruited participants dropped out of the intervention group for reasons such as work commitments , deterioration in health , or family reasons . in the control group , 26 of the 50 wait - listed patients approached for participation completed the questionnaires at the set time periods ( pre- , post- , and 76% of the intervention group had a diagnosis of mcs in combination with fm and cfs , 15% had a diagnosis of fm , and the remaining participants had a diagnosis of cfs . the wait - list control group had not received a diagnosis from the nsehc s physician at the time of recruitment . the group comprising all women , was selected because they had a referral to be seen at the nsehc . independent sample t - tests were used to compare scl90-r global severity index ( gsi ) and subscale scores between the wait - list control and mbsr treatment group at baseline , post - treatment ( 10 weeks ) , and three - month follow - up . the two groups were equal on gsi scores at baseline , with the mbsr treatment group having significantly lower scores than the control group at post - treatment ( t = 2.35 , p < 0.05 ) , and at follow - up ( t = 3.86 , p < 0.001 ) . all subscale scores except for the somatization subscale were equal at baseline between the two groups . five of nine subscales were significantly lower for the mbsr group following treatment , and eight of nine subscales were significantly lower for the mbsr group at three - month follow - up as summarized in table 1 . paired samples tests were used to compare changes in scl90-r gsi and subscale scores from baseline to post - treatment and from post - treatment to three - month follow - up , within each group . the long - term effects of treatment were examined using a glm repeated - measures procedure for the gsi , as well as the somatization , anxiety , and depression subscales , with treatment group as the between - subject variable and time ( baseline , post - treatment , and follow - up ) as the within - subject variable . a significant group time interaction was found for gsi ( f = 6.83 , p < 0.01 ) , as well as the depression ( f = 7.88 , p < 0.01 ) and anxiety ( f = 3.15 , p < 0.05 ) subscales , but no significant interaction was found for the somatization subscale ( f = 0.773 , not significant ) . the broader objective of the study was to determine the efficacy of a mbsr approach ( bmap ) in individuals with conditions such as mcs , cfs , and fm . results from other trials using the mbsr approach have shown reductions in anxiety , depression , pain , and psychological symptoms associated with illness . clinical evaluations conducted at the nsehc have shown that bmap is able to alleviate some of the stressful symptoms of mcs , cfs , and fm , and promotes a sense of well being in our patient population . also , our research has shown individuals with mcs exist in a state of hypervigilance19 and bmap is targeted towards altering this state of arousal . this study was an attempt to quantify the positive changes and benefits reported by nsehc patients upon completion of the bmap as measured in our clinical evaluations , and compare the outcome with women that were waiting to receive intervention , wait - list controls . the type and the extent of changes measured in the intervention group were consistent with the reports measured in clinical evaluations between 1998 and 2003 conducted at the nsehc . the results show significant improvements in the intervention group in the gsi scores of the scl-90r at pre- to post- and pre - intervention to follow - up when compared to the control group . while the scores for the control group stayed the same at pre- , post- and follow - up intervention , the intervention group showed significant improvements post - intervention and at three - month follow - up . however , it would be too simplistic to draw a linear causal relationship between environmental illness and psychological responses . the sheer complexity of the relationship between being human and responding to the physical environment may heighten or accentuate the influence of a variety of stressors . while it is hard to draw a linear relationship , it is possible to delve into some thoughts around the significance of these changes based on the anecdotal reports from our clinical evaluations and the results using scl-90r from this study . post - intervention improvements were observed in the following scl-90r subscales : somatization , depression , phobic anxiety , and paranoid ideation . the stressor of phobic anxiety of the environmental stressors , perceived as threatening can be disproportionate to the offending trigger . this trend showed statistical improvement in the participants of the bmap at post - program to a three - month follow - up . as is the case of a large proportion of the environmentally sensitive patients at the nsehc , they receive a referral to the nsehc following a significant investment of time , energy , and resources in the health care system to find an answer to their problems . often they are met with disdain , disbelief , ridicule , and a referral to psychiatry , which is to say their illness is all in their heads further complicating a poorly understood and difficult - to - treat collection of conditions . disordered thinking traits also changed post - intervention as seen in the positive changes of the somatization subscale of the scl-90r . the characteristics of projective thought of feeling unsafe in ones personal environment , hostility towards health care and insurance providers , suspiciousness , grandiosity , fear of loss of autonomy and delusions were shown to improve . in the bmap , a central component of the training is recognizing thoughts as mental events and not as absolute truths or facts . participants practice becoming aware of the cascade of habits of mind such as catastrophizing , victimizing , and rumination . daily practice encourages a broader perspective of thoughts and images passing through the mind and by turning attention to the breath brings acceptance to the present moment without judgment of pleasant , unpleasant or neutral feelings . in this way resiliency to seeing things as they are , and gaining some distance and perspective on them begin to take hold . this perhaps creates awareness that there may be other ways to think about situations , and thus find freedom from the domination of old thought patterns that pop up automatically . at the three - month follow - up , other changes with the intervention group that showed statistical significance were traits of obsessive compulsiveness , interpersonal sensitivity , anxiety and psychosis . the obsessive nature of thoughts , and safe behaviors ( such as avoidance or isolation ) was greatly improved with the bmap intervention ; feelings of inferiority or inadequacy because of an unexplainable health condition , self - doubt about their health and ability to regain healthy functioning , and self - deprecation are also characteristic features of depressed mood states . the nature of practicing daily homework and attending weekly classes in a group format provided a forum for consistency , discipline , self - responsibility , camaraderie and trust , and an increased sense of self mastery which helped to mitigate the negative side - effects of negative mood states . a number of factors modulate the impact of health and wellness on an individual : personality factors , anxiety , coping skills and the psychological context within which environmental stressors function . the bmap is a means for such individuals to alleviate psychosocial / life stressors that may help change the state of arousal , strengthen inner resiliency to mitigate the negative effects of stressors associated with illness , and develop healthy coping skills for the management of their illness . the study was not conducted in a randomized design which would have enhanced the quality of the research study . however , we considered it unethical for patients to wait to receive treatment following a long wait time to receive the appropriate referral to receive timely care . using wait - list controls with a non - biased selection process which involved using the first 50 women to provide consent was an efficient way to obtain a control group for the study . the wait - list patients typically waited from 46 months to receive treatment at the nsehc , and hence it was convenient to choose this group to measure changes at 0 , 10 weeks , and three - month follow - up time periods . subscales of the scl-90r that have shown trends of change or getting close to statistical significance might have reached statistical significance with a larger sample size . the study was not conducted in a randomized design which would have enhanced the quality of the research study . however , we considered it unethical for patients to wait to receive treatment following a long wait time to receive the appropriate referral to receive timely care . using wait - list controls with a non - biased selection process which involved using the first 50 women to provide consent was an efficient way to obtain a control group for the study . the wait - list patients typically waited from 46 months to receive treatment at the nsehc , and hence it was convenient to choose this group to measure changes at 0 , 10 weeks , and three - month follow - up time periods . subscales of the scl-90r that have shown trends of change or getting close to statistical significance might have reached statistical significance with a larger sample size . the study has shown the importance of a mbsr intervention as a complement to the mainstream management strategies for patients with mcs , cfs , and fm in improving psychological distress and strengthening mental and physical resiliency to disease management . most importantly , with this training format and experiential teaching to embrace the present moment , the locus of control31 shifts back to the patient as they learn to reconstruct their lives independent from external measures such as success , approval and compliancy , and reconnect with the power of choice in their lives and control over their circumstances to live happier and productive lives .

intracameral phenylephrine , an -1 adrenergic receptor agonist , is used in small pupil cataract surgery for mydriasis of the pupil and maintaining dilation throughout the procedure.1 intracameral phenylephrine has also been instrument in preventing complications due to intraoperative floppy iris syndrome ( ifis ) , induced by tamsulosin23 or other -1 adrenergic antagonists.4 although there is evidence for safety of intracameral phenylephrine , some investigators remain concerned and suggest improvements.5 in particular some have reported that complications could theoretically include endothelial cell destruction syndrome , toxic anterior segment syndrome and endophthalmitis.5 recent evidence suggests that intracameral phenylephrine may have an unacceptably high level of free radicals which may cause endothelial cell damage6 and the use of preservatives remains contentious.7 additionally there is no consensus on the correct dosage of intracameral phenylephrine as the dose - response relationship is not linear.8 theoretically , intraoperative intracameral phenylephrine may pose a systemic risk as phenylephrine is a vasopressor . although the effect of intracameral phenylephrine on blood pressure has not been previously studied , topical phenylephrine and intracameral adrenaline / epinephrine have not been noted to have any statistically significant effect.9 due to the increasing indications and use of intracameral phenylephrine for small pupil cataract surgery and the lack of safety data , we elected to perform a comparison of outcomes and complications of patients who did or did not receive intracameral phenylephrine during phacoemulsification . a chart review was performed of 50 patients who had received intracameral phenylephrine , and 50 patients who did not receive intracameral phenylephrine ( control group ) during phacoemulsification . the operating theatre log book was used to select 50 patients who received intracameral phenylephrine during phacoemulsification , in chronological order . january 1 , 2009 was chosen as the starting point for patient selection and the first 50 patients receiving intracameral phenylephrine meant the last patient underwent surgery on may 2010 , a period of 17 months in total . patients who received intracameral phenylephrine were selected purely on the basis of having undergone surgery by one surgeon ( cw ) . in each case the intracameral phenylephrine was composed of 0.3 ml of minims 2.5% phenylephrine hydrochloride ( with sodium metabisulfite 0.075% , sodium edetate 0.0127% , and purified water ) mixed with 0.3 ml of balanced salt solution ( various manufacturers ) . this was mixed by the theatre scrub nurse and the entire 0.6 ml were injected in each case . patients in the control group had also undergone surgery at the same facility by the same surgeon as the study group . the control group received three doses of topical phenylephrine 2.5% and tropicamide 1% ( alcon laboratories , fort worth , tx ) prior to surgery with the phenylephrine group receiving additional intracameral phenylephrine , as detailed above . in order to equally spread the control group over the same 17-month period these 50 patients were each selected as the very next patient in chronological order who did not receive intracameral phenylephrine following the selection of a patient who had . surgical data , data on preoperative ophthalmic and systemic disease , operative details , intraoperative and immediate postoperative complications as well as data from the routine 4-week postoperative visit were collected for both groups . the 4-week postoperative visit included measurement of visual acuity , intraocular pressure as well as documentation of any new complications . any subsequent complications or significant events since as such this study is not registered with the institutional review board ( irb ) and the declaration of helsinki does not apply . the mean age of the two groups were comparable at 74 years ( range , 55 - 87 years ) for those receiving phenylephrine versus 76 years ( range , 59 - 91 years ) for the control group . the reasons for using intracameral phenylephrine were small pupil ( 48 patients ) and tamsulosin therapy ( two patients ) . preoperative ophthalmic and systemic risk factors for complications were also broadly comparable between groups ( p>0.05 all comparisons ; table 1 ) . cardiovascular risk factors include previous myocardial infarction , hypertension , ischemic heart disease and diabetes , while respiratory risk factors included chronic obstructive pulmonary disease and asthma . local and systemic risk factors for complications during cataract surgery for patients who did or did not receive intracameral phenylephrine the operative details were remarkably similar between the groups ( p>0.05 all comparisons ; table 2 ) . intraoperative complications consisted of an anterior capsule rent in one patient in each group and a single posterior capsule rupture in the control group . there were no immediate postoperative complications in both groups in the recovery area and blood pressure readings were stable in all patients . no patients required an interim visit for any concerns between surgery and the 4-week visit . operative details for phacoemulsification power and time for patients who did or did not receive intracameral phenylephrine at 4-weeks postoperatively , intraocular pressure and visual acuity measurements were comparable in the two groups [ table 3 ] . ophthalmic complications noted at the four week visit were a single case of corneal edema in each group , a single case of cystoid macular edema in each group and a case of postoperative uveitis in the group receiving phenylephrine . there were no systemic events noted in the intervening time in either group and no evidence of any attributable systemic event in any patient since the 4-week visit . four - week postoperative data for patients who did or did not receive intracameral phenylephrine during phacoemulsification phenylephrine is a useful adjunct in small pupil cataract surgery as well as in patients susceptible to intraoperative floppy iris syndrome.124 studies have demonstrated increased pupil dilatation following administration of intracameral phenylephrine and thus decreased risks of complications.124 the current study found almost identical intraoperative complications as well as postoperative outcomes suggesting that the addition of phenylephrine has normalized the risk in the small pupil group . currently , there is active discussion of the potential complications of intracameral phenylephrine.569 however , to our knowledge no study has directly compared outcomes between eyes receiving and eyes not receiving intracameral phenylephrine to ascertain any potential adverse events . a prospective , randomized some studies have indicated that sodium metabisulfite , a chemical used to stabilize the phenylephrine molecule prior to use , may be potentially damaging to the cornea.10 in the current study we selected groups with very similar preoperative risk factors and operative details ( apart from pupil size ) indicating that any differences in outcomes would likely be due to the administration of phenylephrine intracamerally . we found no differences intraoperatively or postoperatively ( p>0.05 all comparisons ; table 3 ) . while our outcomes suggest that administration of intracameral phenylephrine was not problematic and normalizes the risks of surgery in small pupil cases , one aspect remains unaddressed . principally , if intracameral phenylephrine had been administered would the complication rate for this group have been higher ? although we can not directly address this question from our outcomes , most ophthalmic surgeons would admit that a small pupil size is associated with higher incidences of operative complications8 in part due to the obstructed view and smaller rhexis . hence a study that directly compares complication rates in eyes with large and small pupils when standardized methods of achieving mydriasis already exist ( including intracameral phenylephrine ) is of dubious ethical validity . hence , the similar rates of complications between groups in the current study must be due to a reduction in the very real risk posed by small pupil sizes , because of the intervention . one drawback of this study is the lack of data on pupil diameters pre- and post- dilation , to determine the significant difference in pupil size before the administration of intracameral phenylephrine . we do not routinely measure corneal pachymetry or endothelial cell counts in postoperative cataract cases but this information would also have been useful . however , the retrospective nature mitigated the likelihood of bias , as the surgeon was not aware at the time of the surgery that the complication rates would be examined in this manner . the operative notes are assumed to be correct as an exhaustive proforma is used by the surgeon that requires yes or no answers to a range of common complications as well as a free text box for the addition of further details . perhaps transcription errors may have arisen in the completion of the operative notes but one would assume that this chance , however small , would be equal for both groups and thus not bias the results . it is the policy of our department to review patients routinely at 4 weeks postoperatively unless an unexpected intraoperative event takes place , although patients are encouraged to call in the intervening time should they notice any signs or symptoms . the fact that none of our patients , from either group , were seen before 4 weeks is indeed encouraging . however , the possibility exists of corneal edema or some other complication developing that cleared by the time of the follow - up visit . we are assuming that the lack of patient contact before 4 weeks indicates no complication took place but concede that there is a source of potential error . a large sample size would have allowed stronger conclusions , but in view of the lack of peer review studies directly comparing outcomes of patients receiving phenylephrine with those not receiving phenylephrine , this paper provides important clinical insights . small pupil diameters can cause problems during phacoemulsification and can be addressed by a variety of techniques , including administration of intracameral phenylephrine . although this intervention is in widespread ophthalmic use , no study has yet been examined the safety of this medication intracamerally , despite the theoretical potential of several problems associated with its use . this retrospective study suggests that intracameral phenylephrine normalizes the intraoperative risk of small pupil cataract surgery and is not associated with any increased risk of systemic or postoperative ophthalmic complications . the volume of published work in this area is small and due to the ubiquitous use of intracameral phenylephrine in cataract surgery , work studies are warranted to properly ascertain the safety of this medication to make cataract surgery safer .

the thalassemias are a group of hemoglobinopathies characterized by a deficient synthesis of protein chains ( either or the ) of globin in the hemoglobin molecule . affected individuals are either heterozygotes , homozygotes , or compound heterozygotes for the - or -chain genes . the heterozygous individual are known as having the - or -thalassemia trait which is the milder form ; the homozygous state is known as a - thalassemia or -thalassemia major ( cooley 's anemia ) which presents with severe manifestations of the disease . the condition was first described by thomas cooley and pearl lee detroit in 1925 . in b thalassemia major , the production of beta globin chains is severely impaired as mutation occurs in the sequence of beta globin gene which results in a severe or total suppression of beta chain synthesis . the patient has anemia and the red blood cells ( rbcs ) show microcytic and hypochromic appearance with an aberrant morphology . this hypertransfusion treatment results in iron overload which is life - limiting complication commonly found in thalassemia patients . it is mainly because of ineffective erythropoiesis and increased absorption of iron in gastrointestinal tract , lack of physiologic mechanism for excreting excess iron , and above all multiple blood transfusions . a unit of packed rbcs contains 250300 mg iron ( 1 mg / ml ) , so that a single transfusion of two units of packed rbcs is about equal to a 12 year intake of iron . the signs of a clinical toxicity become apparent , when the body iron concentration reaches 4001000 mg / kg of the body weight . iron overload occurring due to repeated blood transfusion leads to excessive parenchymal accumulation of this element , causing multiorgan failure and death subsequently . body iron status can be evaluated by assessing serum ferritin level for the diagnosis of iron overload and monitoring the response to treatment . however , serum iron concentration is increased in some diseases even when the body iron stores are within normal limits , as in acute and chronic liver damage , malignancies , infections , and megaloblastic anemia . a liver and bone marrow biopsy is , therefore , required to accurately identify iron overload in parenchymal cells , but these procedures are invasive and , therefore , are not advisable in each case . the present study was designed to assess the utility of perl 's prussian blue in the exfoliated buccal epithelial cells assess the iron overload and its correlation with serum ferritin levels in beta - thalassemia patients undergoing repeated blood transfusions . the present study comprised 35 -thalassemia major patients in the age group of 528 years undergoing repeated blood transfusions and 10 systemically healthy patients in the control group . patients with iron deficiency anemia , megaloblastic anemia , hepatitis , malignancy , and chronic liver damage were excluded from the study . the study subjects were regularly taking oral iron - chelation therapy for more than 1 year . the buccal mucosa of the patients were scraped with a wet wooden spatula after rinsing their mouth with distilled water and smeared onto a glass slide . the smear was fixed immediately in 70% ethanol for 1 h and then stained with perl 's prussian blue . the stained smears were examined under the research microscope ( olympus b 41 ) to study the presence or absence of blue colored intracytoplasmic granules in the oral epithelial cells . the iron overload was assessed using serum ferritin levels of the patients at the time of taking the oral cytosmears . the study group included 21 males ( 60% ) and 14 females ( 40% ) and the mean age was 16.07 6.57 years . the positive cytological smears showed blue colored granules in the cytoplasm of exfoliated oral epithelial cells stained with perl 's prussian blue stain [ figures 1 and 2 ] . twenty - nine ( 82.9% ) cases showed positive reaction for perl 's prussian blue reaction while 6 ( 17.1% ) cases did not show the presence of blue colored granules in the oral cytosmears . the presence of iron by perl 's prussian blue staining reaction was correlated with serum ferritin level and a statistically significant difference ( t - test , p < 0.05 ) was observed for the presence of high iron overload and its detection through perl 's prussian blue in exfoliative cytology [ table 1 ] . however , the results did not correlate statistically with gender of the patients thus indicating that the findings were not related to gender in the study group [ table 2 ] . the number of cases with exfoliative cytosmears showing reaction with perl 's prussian blue correlated with the increasing levels of serum ferritin levels in these patients [ figure 3 ] . photomicrograph showing blue colored intracytoplasmic granules in the exfoliated oral epithelial cells ( perl 's prussian blue , 40 ) photomicrograph showing positive staining for iron in the cytosmears ( perl 's prussian blue , 40 ) correlation of the presence of iron in exfoliated oral epithelial cells with serum ferritin levels in patients with beta - thalassemia correlation of prussian blue reaction with gender in the study group correlation of serum ferritin levels with positivity of oral cytosmears with perl 's prussian blue reaction beta - thalassemia is characterized by increased serum iron levels due to combined effects of increased ferritin synthesis and the release of intracellular ferritin from damaged cells . iron stores in the body show variation depending mainly on the intensity of transfusion and the efficacy of chelation . iron overload can also be seen in various conditions such as hereditary hemochromatosis , thalassemia major , aplastic anemia , and myelodysplasia due to the lack of specific excretory pathways . chemical quantification of iron in liver biopsy tissue has been considered the gold - standard for assessing body iron stores . highlighted the limitations of a single biopsy in predicting long - term complications of transfusional iron overload . oral exfoliative cytology is a simple and noninvasive procedure which can be used to study the cytological and nuclear features in exfoliated oral epithelial cells . the cytosmears were used to assess the iron overload by using perl 's prussian blue staining reaction by histochemical demonstration of iron in the cytoplasm of the oral epithelial cells . perl 's prussian blue reaction is based on the principle that acidified potassium ferrocyanide solution binds to iron in tissue , forming a relatively insoluble blue - purple precipitate . in the present study , 82.9% cases showed positivity for perl 's prussian blue staining reaction in the cytological smears of exfoliated buccal cells . our findings were similar to those observed by nandaprasad et al . who observed 65% , bhat et al . who found ( 71.7% ) , gupta et al . who found 61.6% positivity , and gururaj and sivapathasundaram ( 2003 ) who observed 100% positivity . the presence or absence of these granules is not age or gender specific as previously shown by nandaprasad et al . based on the results of our study , it can be stated that perl 's prussian blue reaction can be utilized as an objective indicator of iron overload in beta - thalassemic patients with high levels of iron overload .

since 1938 endolymphatic hydrops is generally accepted as the histopathological substrate for menire s disease , based on autopsy studies of the ear of menire patients [ 1 , 2 ] . however , it is still unclear if endolymphatic hydrops is the cause of the symptoms , or just a histological marker of the disease . the effect on cochlear functioning of an endolymphatic hydrops has been extensively studied in animals during the last half century [ 4 , 5 ] . although unpredictable attacks of vertigo are the most severe complaint of menire patients , much less information is available on the influence of hydrops on vestibular functioning , most probably because this is more difficult to investigate . reduced or altered vestibular function has been reported after creation of an endolymphatic hydrops in the guinea pig and in the rabbit . the most widely used method for the creation of an endolymphatic hydrops is surgical obliteration or dissection of the endolymphatic sac . with this technique an obvious disadvantage of the model is the destruction of an organ that is essential for inner ear homeostasis . an acute endolymphatic hydrops can be created by directly injecting artificial endolymph into scala media [ 10 , 11 ] . we developed this technique to study inner ear fluid pressure [ 12 , 13 ] and cochlear functioning during an acute hydrops [ 14 , 15 ] in the guinea pig . . showed in the chinchilla that evoked potentials can also be measured in the vestibular system , if the head of the animal is subject to linear acceleration pulses . plotnik confirmed this finding and showed that these short latency vestibular evoked potentials ( vseps ) mainly originate from the otolith organs . in an earlier experiment we combined measurement of vseps with injection of artificial endolymph , to study the effect of an acute endolymphatic hydrops in the guinea pig on the functioning of ( part of ) the vestibular system . apart from a short lasting hydromechanical effect of fluid injection through the basilar membrane , no significant difference in vsep deterioration was found between animals in which an artificial hydrops was created and ( control ) animals in which the injection pipette perforated the basilar membrane and was withdrawn without injecting fluid . this suggested that it was not the ( artificial ) hydrops that caused the slow decrease of vsep amplitude during a time course of hours , but possibly a leakage of potassium ions into the perilymphatic space . intoxication by potassium ions leaking through a ruptured [ 20 , 21 ] or micro - lesioned reissner s membrane is one possible explanation for acute menire attacks . for the present paper the influence of perilymphatic potassium concentration on the vsep was investigated in the guinea pig in two ways : firstly , artificial endolymph was injected slowly through the basilar membrane until reissner s membrane ruptured , causing a massive leakage of high potassium endolymph into the perilymphatic space . secondly , to control the effect of potassium concentration on the vestibular sensory and neural structures in the perilymphatic space , different concentrations of kcl were injected directly into the vestibule . in this study 20 albino guinea pigs , weighing between 350 and 600 g , with normal preyer s reflex were used . the care and use of the animals was in accordance with the principles of the declaration of helsinki and approved by the groningen animal experiment committee . general anaesthesia was induced by intramuscular injection of ketamine / xylazine ( 60/3.6 mg / kg ) and maintained by administering additional anaesthetics every hour ( 40/2.4 mg / kg ) . a tracheostoma was created for artificial ventilation ( columbus instruments , model 7950 ) and intramuscular suxamethoniumchloride ( 2.5 mg / kg ) was given every hour for muscle relaxation . skin electrodes placed on both sides of the thorax monitored the heart rate . to stimulate the vestibular system by acceleration pulses and fixate the head during operation and microinjection , a steel bolt was cemented upside down on the skull of the guinea pig with dental cement . after opening the bulla to expose the round window , a platinum electrode was implanted in both ears in the facial nerve canal , up to the first curvature . acceleration pulses were generated with a bruel and kjaer vibration exciter ( type 4809 ) and monitored with an accelerometer , connected to a bruel and kjaer amplifier ( type 2651 ) . linear acceleration pulses were applied in the direction of the earth vertical axis perpendicular to the top of the skull . the shaker was driven by computer - generated stimuli that consisted of gaussian - shaped pulses , amplified by a power amplifier ( bruel and kjaer type 2712 ) and computer controlled with a programmable attenuator . the vestibular stimulus consisted of 500 alternating pulses with peak amplitude of 40 m / s at 0.5 ms after onset , at a rate of 51/s . the signals of the active electrodes were amplified ( disa , type 15 c01 ) and band - pass filtered ( 100 hz5 khz ) . the first 10 ms of the electrode and accelerometer signals were recorded , averaged and processed , using a tucker - davis biosig - stimulate / record system , version 2.0 . n signal complex in the electrode signal , appearing first between 1 and 2 ms after stimulus onset , is of vestibular origin [ 18 , 23 , 24 ] , and is therefore called vestibular evoked potential ( vsep ; see fig . 1).fig . the first part of it , appearing between 1 and 2 ms after stimulus onset , is of peripheral vestibular origin gross electrode potential evoked by acceleration pulses . the first part of it , appearing between 1 and 2 ms after stimulus onset , is of peripheral vestibular origin in ten guinea pigs artificial endolymph was injected into scala media until one or more membranes surrounding the endolymphatic compartments ruptured . from earlier experiments we knew that this always occurred after injection of more than 3 l of fluid [ 13 , 14 ] . to inject artificial endolymph , the tip of a double barrel micropipette was inserted through the round window and the basilar membrane . the other ear served as control . in a separate set of experiments we evaluated the effect of injecting 4 l of a kcl solution directly into the perilymphatic space , through a small hole that was made in the bony wall of the vestibule next to the oval window . the injected concentrations of k were 0.250 , 0.375 , 0.500 and 3.00 m , in respectively 3 , 3 , 2 and 2 guinea pigs . again the other ear served as control . because the perilymphatic space behind the round window is directly connected to the cerebrospinal fluid space through the cochlear aqueduct we did not inject through the round window membrane . double barrel micropipettes were drawn from borosilicate glass and the tips were bevelled ( narishige eg-40 ) . tip diameters were around 60 m per barrel , which is a compromise between a low enough flow resistance for fluid injection and a small tip size . one of the barrels was used to measure the dc potential ( to monitor tip position ) and inner ear pressure ( wpi 900a micropressure system ) . the other barrel was used to inject artificial endolymph ( 140 mm kcl + 25 mm khco3 ) , by applying pneumatic pressure to the fluid interface ( wpi pv 830 pico pump ) . since the inner diameter of a barrel is precisely known ( 0.84 mm ) the injected volume could be measured by displacement of the fluid interface in the barrel . after opening the bulla and positioning the electrodes in the facial canal the experiment started with measuring vseps in both ears . 1 ) was used as a measure of the functionality of the linear acceleration detecting part of the vestibular organ . this initial value was used as a reference for the vestibular system under investigation , because of interindividual differences of vsep amplitude . directly after rupturing reissner s membrane in the first series of experiments and after injection of kcl in the second series , the vsep amplitude was measured again and then after 1 , 2 , 3 , 4 and 5 h. between the measurements the animal recovered from heavy shaking . it was detached from the shaker during these periods . after the last measurement the animals were terminated by means of intracardial administration of an overdose of pentobarbital . the temporal bones were removed and fixated by immersion in a solution of 2.5% glutaraldehyde in 0.1 m na - cacodylate buffer and 2 mm calcium chloride ( ph 7.4 ; 320 mosm ) for later study . after fixation three specimens of the first series of ( rupture ) experiments were decalcified in 10% edta ( ph 7.4 ) for 5 days , carefully rinsed in distilled water , dehydrated in a graded ethanol series and embedded in plastic . from the bullae sections of the cochlea and the vestibule were cut ( midmodiolar plane ) and stained with toluidine blue for light microscopic examination to check for damage caused by injection of ( too much ) artificial endolymph . time - dependent changes in vsep between injected ears and control ears were compared using ancova ( spss 15.0 ) for repeated measurements . injection of more than 3 l of artificial endolymph in scala media did not only cause a fissure in reissner s membrane in the cochlea , but also in the membrane in the saccule ( fig . 2 ) in all three investigated ears , which makes it very likely that not only the hearing system will be directly affected but also the balance system . each experiment was successful in reaching scala media : dc potential abruptly increased 7080 mv after the basilar membrane was perforated . pressure in scala media increased after starting injection of artificial endolymph and decreased to almost initial pressure after stopping injection ( fig . the endocochlear potential ( ep ) decreased by up to 40% during injection , but recovered almost completely within a few minutes after injection.fig . 2light microscopic picture of a rupture in the membrane of the saccule , created by microinjection of 4 l artificial endolymph into scala media . ( from experience we know that the rupture was not caused by preparing it for light microscopic examination)fig . 3fluid pressure ( cm water ) measured at the tip of the micropipette , before , during and after injection of more than 3 l of artificial endolymph in scala media . this amount of injected fluid ruptures the membrane(s ) surrounding the endolymphatic compartment(s ) , resulting in a sudden small drop of inner ear pressure light microscopic picture of a rupture in the membrane of the saccule , created by microinjection of 4 l artificial endolymph into scala media . ( from experience we know that the rupture was not caused by preparing it for light microscopic examination ) fluid pressure ( cm water ) measured at the tip of the micropipette , before , during and after injection of more than 3 l of artificial endolymph in scala media . this amount of injected fluid ruptures the membrane(s ) surrounding the endolymphatic compartment(s ) , resulting in a sudden small drop of inner ear pressure all rmr ears showed a complete disappearance of vsep after injection with partial recovery subsequently ( fig . differences between vsep amplitudes over time were statistically significant ( p < 0.00).fig . 4average vsep ( 1 se ) for the ruptured and control ears before and at different times after rupture of reissner s membrane by microinjection of artificial endolymph average vsep ( 1 se ) for the ruptured and control ears before and at different times after rupture of reissner s membrane by microinjection of artificial endolymph all ears showed a vsep before intervention . kcl injection resulted in a dose - dependent decrease and subsequent recovery of the vsep . 5average vsep ( 1 se ) for the different concentrations of injected kcl , measured before and at different times after injection . injection resulted in a dose - dependent decrease and subsequent partial recovery of the vsep average vsep ( 1 se ) for the different concentrations of injected kcl , measured before and at different times after injection . injection of more than 3 l of artificial endolymph in scala media did not only cause a fissure in reissner s membrane in the cochlea , but also in the membrane in the saccule ( fig . 2 ) in all three investigated ears , which makes it very likely that not only the hearing system will be directly affected but also the balance system . each experiment was successful in reaching scala media : dc potential abruptly increased 7080 mv after the basilar membrane was perforated . pressure in scala media increased after starting injection of artificial endolymph and decreased to almost initial pressure after stopping injection ( fig . the endocochlear potential ( ep ) decreased by up to 40% during injection , but recovered almost completely within a few minutes after injection.fig . 2light microscopic picture of a rupture in the membrane of the saccule , created by microinjection of 4 l artificial endolymph into scala media . ( from experience we know that the rupture was not caused by preparing it for light microscopic examination)fig . 3fluid pressure ( cm water ) measured at the tip of the micropipette , before , during and after injection of more than 3 l of artificial endolymph in scala media . this amount of injected fluid ruptures the membrane(s ) surrounding the endolymphatic compartment(s ) , resulting in a sudden small drop of inner ear pressure light microscopic picture of a rupture in the membrane of the saccule , created by microinjection of 4 l artificial endolymph into scala media . ( from experience we know that the rupture was not caused by preparing it for light microscopic examination ) fluid pressure ( cm water ) measured at the tip of the micropipette , before , during and after injection of more than 3 l of artificial endolymph in scala media . this amount of injected fluid ruptures the membrane(s ) surrounding the endolymphatic compartment(s ) , resulting in a sudden small drop of inner ear pressure all rmr ears showed a complete disappearance of vsep after injection with partial recovery subsequently ( fig . differences between vsep amplitudes over time were statistically significant ( p < 0.00).fig . 4average vsep ( 1 se ) for the ruptured and control ears before and at different times after rupture of reissner s membrane by microinjection of artificial endolymph average vsep ( 1 se ) for the ruptured and control ears before and at different times after rupture of reissner s membrane by microinjection of artificial endolymph kcl injection resulted in a dose - dependent decrease and subsequent recovery of the vsep . 5average vsep ( 1 se ) for the different concentrations of injected kcl , measured before and at different times after injection . injection resulted in a dose - dependent decrease and subsequent partial recovery of the vsep average vsep ( 1 se ) for the different concentrations of injected kcl , measured before and at different times after injection . multiple studies have demonstrated that potassium has toxic effects on the cochlear function [ 14 , 2528 ] . . a few investigators showed , about 40 years ago , that perfusion of artificial endolymph in the perilymphatic space caused a nystagmus [ 20 , 29 ] . more recently , bhmer et al . demonstrated the toxic effect of potassium on the vseps by introducing potassium chloride crystals in the perilymphatic space of the inner ear of the chinchilla . one case report describes a patient who directly applied potassium iodide solution through a tympanostomy tube into his middle ear , which caused a typical episode of meniere s disease ; vertigo , nausea , vomiting , hearing loss and tinnitus . this shows that potassium intoxication of the inner ear can cause the same clinical symptoms as during an acute meniere s attack . rupture of one or more membranes enclosing the endolymphatic spaces causes an immediate and complete suppression of the vsep , as is shown in fig . the vsep recovers to about 90% of the value in the control ear , most probably due to dilution of the injected artificial endolymph . a similar slow recovery ( from vertigo ) is present during an acute episode of menire s disease . in temporal bone preparations of menire patients hydrops of scala media was sometimes accompanied by a rupture of reissner s membrane , which can be an explanation for the acute character of vestibular attacks in menire s disease . in the first series of experiments ( rmr ) part of the injected fluid will enter the vestibular part of the inner ear through the ductus reuniens and create a vestibular hydrops also , followed by a distention and rupture of one or more membranes ( fig . 2 ) . in the second series of experiments ( kcl injection ) suppression and recovery of the vsep are dose dependent , as is shown in fig . 5 . after injection of the lowest concentration of kcl ( 0.25 m ) the vsep recovers almost completely within 1 h. no recovery is observed after injection of the highest concentration of 3 m , in contrast to the result after membrane rupture ( fig . 4 ) , which best resembles the 0.375 m curve in fig . 5 . immediately after the injection of fluid into the inner ear fluid pressure inside the inner ear compartment increases . but within a few seconds pressure returns to normal , indicating that excess fluid escapes : perilymph through the cochlear aqueduct and endolymph through the ductus endolymphaticus [ 12 , 13 , 30 ] . so , when a kcl solution is directly injected into the perilymphatic space through a small hole in the bony wall of the vestibule , this solution will mix with perilymph and the total fluid volume in the inner ear will not increase . injection of 4 l of 0.25 m kcl solution , assuming a homogeneous distribution of k in 16 l shortly after injection , leads to a k concentration of 63 mm . this is 7 times the normal concentration of k in perilymph in scala vestibuli of the guinea pig , which is 9.0 mm . not much is known about the mechanism of k intoxication of the vestibular system . for the cochlea zenner et al . found that addition of artificial endolymph to the basolateral surface of outer hair cells resulted in inhibition of the physiological repolarizing k efflux from these cells . they also found pathological shortening of outer hair cells ; while long - lasting k intoxication resulted in chronic and complete loss of outer hair cell motility , and finally in cell death , and suggested this to be a pathophysiological basis in some menire patients for chronic hearing loss . very recently nakano et al . mice of this line show a widespread loss of sensory hair cells in the hearing organ , most possibly due to a mutation of the claudin-9 gene that encodes a tight junction protein . claudin - defective tight junctions fail to shield the basolateral side of hair cells from the k -rich endolymph . as a neurotoxic effect of an increased concentration of k in the perilymphatic space during a menire s attack meissner suggested a depolarization of the synapse between the vestibular hair cell and the afferent nerve fiber . this suggestion is supported by the conclusion of kiernan et al . that the toxic effect of k on axons , in patients with hyperkalaemia caused by chronic renal failure , is due to membrane depolarization . both bhmer et al . and plotnik et al . state that the vsep is most probably a compound action potential of the vestibular nerve and pathways . a toxic effect of k on vestibular hair cells will affect the generator potential , leading to less single nerve fiber action potentials , or none at all . a toxic effect on the nerve will affect action potential generation directly . the ( normal ) osmolarity of perilymph in the guinea pig is 0.29 and of endolymph it is 0.30 osmol / l . the number of ions in 4 l of injected 0.250 m kcl is 2 10n ( n is avogadro s number ) . the number of ions in the remaining 12 l of perilymph is 3.5 10n . so , after injection 5.5 10n ions are present in the perilymphatic space , giving an osmolarity of 0.34 osmol / l . ( this is no surprise because the injected fluid is hypertonic with respect to perilymph . ) injection of 0.375 , 0.500 and especially 3.00 m kcl will create a still larger difference in osmolarity between perilymph and endolymph , leading to an osmotic pressure difference between the endolymphatic and the perilymphatic compartments . besides k intoxication this could give an additional explanation for the measured deterioration of the vsep , through hydromechanical effects . however , no hydromechanical effect on the vsep was observed in an earlier experiment in which artificial endolymph was injected in scala media of the guinea pig to create an acute endolymphatic hydrops . in the first series of experiments ( rmr ) k - rich endolymph mixes with perilymph . because the injected artificial endolymph , natural endolymph and perilymph have almost the same osmolarity this will not cause an osmotic pressure difference between the endolymphatic and the perilymphatic compartments . furthermore , if such a pressure difference would occur , pressures would equalize very fast because fluid will flow through the ruptured membrane(s ) . so k intoxication remains as the only explanation for the vsep deterioration shown in fig . , our study shows that an increase of the k concentration in the perilymphatic space of the vestibular system causes a deterioration of the functioning of ( part of ) the vestibular system , followed by a dose - dependent slow recovery . k intoxication is the most likely explanation for this process , which could be an explanation for acute disturbance of vestibular function in menire patients .

gambling disorder ( gd ) is associated with high rates of depression , substance - use disorders , and suicidality ; as well as occupational and legal problems ( bland , newman , orn , & stebelsky , 1993 ; cunningham - williams , cottler , compton , & spitznagel , 1998 ; muelleman , denotter , wadman , tran , & anderson , 2002 ; petry & kiluk , 2002 ; petry , stinson , & grant , 2005 ; toce - gerstein , gerstein , & volberg , 2003 ) . the annual cost of gd is approximately 5 billion us dollars within the united states alone ( national gambling impact study commission & james , 1999 ) . subsyndromal gambling is also a notable concern , and is defined as a significant gambling activity that does not meet full dsm-5 criteria for gd ( grant , derbyshire , leppink , & chamberlain , 2014 ) . subsyndromal gamblers also show high rates of depression , anxiety disorders , substance - use disorders , financial , legal , family , and professional problems ( cunningham - williams et al . , 1998 ; desai , 2004 ; gerstein et al . , 1999 ; grall - bronnec et al . , 2012 ; potenza , maciejewski , & mazure , 2006 ; shaffer & korn , 2002 ; welte , barnes , wieczorek , tidwell , & parker , 2001 ) . therefore , it is likely that gambling symptoms present on a continuum , and that symptoms should be assessed across the spectrum of gambling behavior ( cunningham - williams et al . , 1998 ; eisen et al . , 2001 ; gerstein et al . , 1999 ; grant et al . , 2014 ; shaffer , hall , & vander bilt , 1999 ; shaffer & korn , 2002 ; slutske et al . , 2000 ; toce - gerstein et al . , 2003 ; welte et al . , the previous analyses have highlighted significant associations between gd / subsyndromal gd and increased rates of anxiety symptoms and anxiety disorders relative to the general population ( black & moyer , 1998 ; bland et al . , 1993 ; cunningham - williams et al . , 1998 ; desai & potenza , 2008 ; petry et al . , one large epidemiological study found a lifetime prevalence of anxiety disorders of 41.3% among disordered gamblers ( petry et al . , 2005 ) . a systematic review of epidemiological studies corroborated these findings , with 37% of gamblers reporting a history of a comorbid anxiety disorder ( lorains , cowlishaw , & thomas , 2011 ) . in another analysis , desai and potenza ( 2008 ) also found a high prevalence of panic disorder , generalized anxiety , and social phobia in subsyndromal gd . although anxiety symptoms are common in gamblers , less is known about how anxiety symptoms influence the clinical presentation of gambling problems . various lines of research suggest that anxiety could be a powerful contributor to gambling behavior . first , the research in other addictions ( alcohol - use disorder and substance - use disorder ) suggests that the behavior may be a way to cope with anxiety ( sher , trull , bartholow , & vieth , 1999 ) . the relief of the anxiety , however , tends to be short - lived , and the symptoms may often reappear more intensely . as a result , a harmful cycle is created , and the anxious individual may therefore present with a more frequent and severe form of the addictive behavior . second , attentional bias toward threatening elements is a well - characterized phenomenon in anxious individuals and , as a result of this , other non - threatening elements tend to be less perceived ( bar - haim , lamy , pergamin , bakermans - kranenburg , & van ijzendoorn , 2007 ; bishop , duncan , brett , & lawrence , 2004 ; koster , crombez , van damme , verschuere , & de houwer , 2004 ; koster , crombez , verschuere , van damme , & wiersema , 2006 ; macleod , mathews , & tata , 1986 ; mogg & bradley , 1998 ; schofield , johnson , inhoff , & coles , 2012 ) . therefore , an anxious individual who gambles may have a reduced focus on the gambling behavior ( i.e. , how to bet and how much to bet ) . finally , anxiety symptoms decrease some dimensions of quality of life ( spitzer et al . , 1995 ) . in this context , the increased emotional suffering and the lower level of life satisfaction could also increase the risk of suicide . in light of this background , the objective of this study was to evaluate the association between anxiety symptoms , gambling activity , and neurocognition across the spectrum of gambling behavior ( recreational gamblers , subsyndromal gd , and gd ) in a non - treatment - seeking sample . we assessed the current severity of anxiety symptoms and associations between anxiety symptom severity , gambling symptoms , impulsiveness , and neurocognition . we predicted that anxiety symptoms would be associated with : ( a ) worse overall severity of gd , measured by increased gambling activity and more losses due to gambling and ( b ) higher impulsivity measured with clinical and neurocognitive tasks . in addition , we expected that anxiety would be correlated with : ( c ) worse clinical and neurocognitive attentional performance ; ( d ) lower quality of life ; and ( e ) higher levels of suicidality . if our hypotheses are correct , anxiety symptoms would be associated with variables correlated with poor prognosis and poor overall functioning . therefore , proper management of anxiety should receive greater attention in clinical practice since it could improve the clinical presentation of gamblers in multiple domains . if our hypotheses are not supported , constant assessment and treatment of anxiety would be a secondary approach in the management of gd . the sample consisted of 143 non - treatment - seeking young adults ( aged 1829 years ) [ n = 75 ( 52.1% ) male ; mean age 24.8 ( 2.9 ) years ] with varying levels of gambling severity . also , 63 individuals ( 44.1% ) were classified as recreational gamblers ( see later for definitions ) , 47 ( 32.9% ) as having subsyndromal gd , and 33 ( 23.1% ) met criteria for gd . all assessments were completed as a part of an ongoing study of gambling behavior in young adults . exclusion criteria included the inability to complete the study procedures , inability / unwillingness to provide voluntary written informed consent , and gambling frequency of less than five times in the past year . age , gender , marital status , educational status , professional status , ethnicity , and sexual orientation were recorded . the hamilton anxiety scale ( ham - a ) is a valid and reliable instrument that assesses cross - sectional severity of anxiety symptoms ( hamilton , 1969 ; maier , buller , philipp , & heuser , 1988 ; snaith , baugh , clayden , husain , & sipple , 1982 ) . the ham - a consists of 14 items ( score range 056 ) that evaluate a variety of anxiety symptoms ( beck & steer , 1991 ) . ham - a total score presents an intra - class coefficient of 0.74 and a concurrent validity between 0.63 and 0.75 ( maier et al . , 1988 ) . the ham - a has shown good internal consistency ( cronbach s alpha = .893 ) ( kummer , cardoso , & teixeira , 2010 ) . gambling behavior : we evaluated the age at the start of regular gambling , the amount of money lost with gambling in the last year , and the average gambling frequency ( times per week ) . overall severity of gd : we assessed the total number of dsm-5 gd criteria using the structured clinical interview for gambling disorder ( sci - pg ) ( grant , steinberg , kim , rounsaville , & potenza , 2004 ) . sci - pg was first validated using the criteria of the fourth edition of the diagnostic and statistical manual of mental disorders ( dsm-4 ) . retest reliability on the number of gd criteria endorsed showed r = .97 ( p = .006 ) ( grant et al . , 2004 ) . this procedure was performed deleting the criterion committed illegal acts such as forgery , fraud , theft , or embezzlement to finance gambling regarding illegal acts , which was present in the previous manual , dsm-4 . moreover , we lowered the diagnostic threshold from five to four , consistent with dsm-5 . severity was divided in three categories : recreational gambling ( meets 0 dsm-5 criteria ) , subsyndromal gd ( meets 13 dsm-5 criteria ) and gd ( meets 4 or more dsm-5 criteria ) . in addition , we investigated the overall gambling severity with the pathological gambling yale - brown obsessive - compulsive scale ( pg - ybocs ) . it is a 10-item scale that showed high validity ( r = .895 ) and reliability ( cronbach s = .970 ) ( pallanti , decaria , grant , urpe , & hollander , 2005 ) . this scale provides a total score ( overall severity ) as well as scores in two subscales ( urges and behavior subscales ) . impulsiveness : evaluated by the barratt impulsiveness scale , version 11 ( bis-11 ) ( patton & stanford , 1995 ) , a scale that has been largely used to investigate impulsiveness ( steinberg , sharp , stanford , & tharp , 2013 ) . this scale has shown good internal consistency ( cronbach s between .79 and .83 ) ( patton & stanford , 1995 ) . bis-11 provides scores in three different dimensions , based on previous factor analyses : attentional impulsiveness , motor impulsiveness , and non - planning impulsiveness ( patton & stanford , 1995 ) . prevalence of illegal acts : the commitment of illegal acts to finance gambling activity has been associated with higher severity of gd ( granero et al . , 2015 ; strong & kahler , 2007 ; toce - gerstein et al . , 2003 it was evaluated using an open question using previous dsm-4 criteria : have you committed illegal acts such as forgery , fraud , theft , or embezzlement to finance gambling ? suicidality and psychiatric comorbidity : evaluated by the mini - international neuropsychiatric interview ( mini ) ( sheehan et al . , 1998 ) . quality of life ( quality of life inventory ) : a 17-item scale that evaluates the subject s overall quality of life ( frisch , 1994 ) . alpha coefficients and test retest correlations for this questionnaire have ranged , respectively , from 0.77 to 0.89 and from 0.80 to 0.91 ( mendlowicz & stein , 2014 ) . individuals with gd tend to present with several neurocognitive deficits such as poorer response inhibition , low cognitive flexibility , worse decision making , and problems with sustained attention and executive functioning ( clark , 2010 ; van holst , van den brink , veltman , & goudriaan , 2010 ) . this study evaluated whether anxiety symptoms affected any of these neuropsychological variables . in this context , participants undertook selected tests from the computerized cambridge neuropsychological test automated battery ( cantabeclipse , version 3 , cambridge cognition ltd . , cambridge , uk ) ( cambridge cognition , 2015 ) . task order was fixed and the total duration of cognitive testing was approximately 50 min . response inhibition : assessed by the stop - signal task , which assesses the subject s ability to inhibit / suppress motor responses . individuals react to an arrow stimulus , by touching either a left or right key depending on the direction in which the arrow points . when an audio tone occurs , the participant attempts to suppress their motor response for the particular trial ( morein - zamir & sahakian , 2010 ) . the outcome measure of interest is the stop - signal reaction time , an index of the time taken for the person s brain to stop a response that would normally be made . cognitive flexibility : investigated with the intra - dimensional / extra - dimensional set shifting test , which evaluates rule learning , reversal , and shifting of attentional focus across stimulus dimensions . the test uses visual stimuli ( colorful shapes and white lines ) and gives feedback to the individual so that they are able to learn an underlying rule about which stimulus is correct , based on trial and error . the underlining rule that determines what is correct and incorrect changes several times and assesses the individual s ability to respond with flexibility ( cambrigde cognition , 2015 ) . decision making : assessed using the cambridge gamble task , a test that assesses decision making and risk taking ( cambridge cognition , 2015 ; deakin , aitken , robbins , & sahakian , 2004 ; lawrence , luty , bogdan , sahakian , & clark , 2009 ) . the task simulates gambling activity but uses points for bets , rather than real rewards . the main outcome measures in this test are : quality of decision making , proportion of points gambled , and risk adjustment . this task investigates the ability to detect unpredictable target sequences over prolonged period of time ( sarter , givens , & bruno , 2001 ) . the task consists of a white box in the center of the computer screen , inside which numbers , from 2 to 9 , show up in a pseudo - random manner , at the frequency of 100 digits / min . this test , a variant of a variation of the tower of london ( owen et al . , 1995 ) , investigates goal - directed planning ( cambridge cognition , 2015 ) . the task presents visual problems to the subject and evaluates the individual s ability to plan a solution and answer these problems . we analyzed the association between the severity of anxiety symptoms and demographic , clinical , and neurocognitive variables of the participants using spearman s coefficients for continuous elements and mann whitney tests for categorical variables . to control for multiple comparisons , we divided the usual level of significance ( p = .05 ) by the number of variables evaluated in each group of assessments ( i.e. , clinical variables and neurocognitive testing ) . consequently , significance was defined as p .004 ( .05/13 = .004 ) for clinical variables and p .007 ( .05/7 = .007 ) for neurocognitive variables . to reduce the likelihood of confounding variables contributing to the above analyses , we controlled for current major - depressive disorder , alcohol - use disorder , substance - use disorder , and nicotine use . these controls were included , as all four variables have demonstrated significant overlap with anxiety symptoms and several clinical / neurocognitive variables investigated in this study , in prior work ( clark , 2010 ; maier et al . , this research was approved by the institutional review boards of the university of chicago and the university of minnesota . the study procedures were explained to the participants prior to providing consent , and all participants were given time to ask questions . the sample consisted of 143 non - treatment - seeking young adults ( aged 1829 years ) [ n = 75 ( 52.1% ) male ; mean age 24.8 ( 2.9 ) years ] with varying levels of gambling severity . also , 63 individuals ( 44.1% ) were classified as recreational gamblers ( see later for definitions ) , 47 ( 32.9% ) as having subsyndromal gd , and 33 ( 23.1% ) met criteria for gd . all assessments were completed as a part of an ongoing study of gambling behavior in young adults . exclusion criteria included the inability to complete the study procedures , inability / unwillingness to provide voluntary written informed consent , and gambling frequency of less than five times in the past year . age , gender , marital status , educational status , professional status , ethnicity , and sexual orientation were recorded . the hamilton anxiety scale ( ham - a ) is a valid and reliable instrument that assesses cross - sectional severity of anxiety symptoms ( hamilton , 1969 ; maier , buller , philipp , & heuser , 1988 ; snaith , baugh , clayden , husain , & sipple , 1982 ) . the ham - a consists of 14 items ( score range 056 ) that evaluate a variety of anxiety symptoms ( beck & steer , 1991 ) . ham - a total score presents an intra - class coefficient of 0.74 and a concurrent validity between 0.63 and 0.75 ( maier et al . , 1988 ) . the ham - a has shown good internal consistency ( cronbach s alpha = .893 ) ( kummer , cardoso , & teixeira , 2010 ) . it is probably the most used and accepted scale to evaluate anxiety symptoms . gambling behavior : we evaluated the age at the start of regular gambling , the amount of money lost with gambling in the last year , and the average gambling frequency ( times per week ) . overall severity of gd : we assessed the total number of dsm-5 gd criteria using the structured clinical interview for gambling disorder ( sci - pg ) ( grant , steinberg , kim , rounsaville , & potenza , 2004 ) . sci - pg was first validated using the criteria of the fourth edition of the diagnostic and statistical manual of mental disorders ( dsm-4 ) . retest reliability on the number of gd criteria endorsed showed r = .97 ( p = .006 ) ( grant et al . , 2004 ) . this procedure was performed deleting the criterion committed illegal acts such as forgery , fraud , theft , or embezzlement to finance gambling regarding illegal acts , which was present in the previous manual , dsm-4 . moreover , we lowered the diagnostic threshold from five to four , consistent with dsm-5 . severity was divided in three categories : recreational gambling ( meets 0 dsm-5 criteria ) , subsyndromal gd ( meets 13 dsm-5 criteria ) and gd ( meets 4 or more dsm-5 criteria ) . in addition , we investigated the overall gambling severity with the pathological gambling yale - brown obsessive - compulsive scale ( pg - ybocs ) . it is a 10-item scale that showed high validity ( r = .895 ) and reliability ( cronbach s = .970 ) ( pallanti , decaria , grant , urpe , & hollander , 2005 ) . this scale provides a total score ( overall severity ) as well as scores in two subscales ( urges and behavior subscales ) . impulsiveness : evaluated by the barratt impulsiveness scale , version 11 ( bis-11 ) ( patton & stanford , 1995 ) , a scale that has been largely used to investigate impulsiveness ( steinberg , sharp , stanford , & tharp , 2013 ) . this scale has shown good internal consistency ( cronbach s between .79 and .83 ) ( patton & stanford , 1995 ) . bis-11 provides scores in three different dimensions , based on previous factor analyses : attentional impulsiveness , motor impulsiveness , and non - planning impulsiveness ( patton & stanford , 1995 ) . prevalence of illegal acts : the commitment of illegal acts to finance gambling activity has been associated with higher severity of gd ( granero et al . , 2015 ; strong & kahler , 2007 ; toce - gerstein et al . , 2003 it was evaluated using an open question using previous dsm-4 criteria : have you committed illegal acts such as forgery , fraud , theft , or embezzlement to finance gambling ? suicidality and psychiatric comorbidity : evaluated by the mini - international neuropsychiatric interview ( mini ) ( sheehan et al . quality of life ( quality of life inventory ) : a 17-item scale that evaluates the subject s overall quality of life ( frisch , 1994 ) . alpha coefficients and test retest correlations for this questionnaire have ranged , respectively , from 0.77 to 0.89 and from 0.80 to 0.91 ( mendlowicz & stein , 2014 ) . individuals with gd tend to present with several neurocognitive deficits such as poorer response inhibition , low cognitive flexibility , worse decision making , and problems with sustained attention and executive functioning ( clark , 2010 ; van holst , van den brink , veltman , & goudriaan , 2010 ) . this study evaluated whether anxiety symptoms affected any of these neuropsychological variables . in this context , participants undertook selected tests from the computerized cambridge neuropsychological test automated battery ( cantabeclipse , version 3 , cambridge cognition ltd . , cambridge , uk ) ( cambridge cognition , 2015 ) . task order was fixed and the total duration of cognitive testing was approximately 50 min . response inhibition : assessed by the stop - signal task , which assesses the subject s ability to inhibit / suppress motor responses . individuals react to an arrow stimulus , by touching either a left or right key depending on the direction in which the arrow points . when an audio tone occurs , the participant attempts to suppress their motor response for the particular trial ( morein - zamir & sahakian , 2010 ) . the outcome measure of interest is the stop - signal reaction time , an index of the time taken for the person s brain to stop a response that would normally be made . cognitive flexibility : investigated with the intra - dimensional / extra - dimensional set shifting test , which evaluates rule learning , reversal , and shifting of attentional focus across stimulus dimensions . the test uses visual stimuli ( colorful shapes and white lines ) and gives feedback to the individual so that they are able to learn an underlying rule about which stimulus is correct , based on trial and error . the underlining rule that determines what is correct and incorrect changes several times and assesses the individual s ability to respond with flexibility ( cambrigde cognition , 2015 ) . decision making : assessed using the cambridge gamble task , a test that assesses decision making and risk taking ( cambridge cognition , 2015 ; deakin , aitken , robbins , & sahakian , 2004 ; lawrence , luty , bogdan , sahakian , & clark , 2009 ) . the task simulates gambling activity but uses points for bets , rather than real rewards . the main outcome measures in this test are : quality of decision making , proportion of points gambled , and risk adjustment . this task investigates the ability to detect unpredictable target sequences over prolonged period of time ( sarter , givens , & bruno , 2001 ) . the task consists of a white box in the center of the computer screen , inside which numbers , from 2 to 9 , show up in a pseudo - random manner , at the frequency of 100 digits / min . this test , a variant of a variation of the tower of london ( owen et al . , 1995 ) , investigates goal - directed planning ( cambridge cognition , 2015 ) . the task presents visual problems to the subject and evaluates the individual s ability to plan a solution and answer these problems . age , gender , marital status , educational status , professional status , ethnicity , and sexual orientation were recorded . the hamilton anxiety scale ( ham - a ) is a valid and reliable instrument that assesses cross - sectional severity of anxiety symptoms ( hamilton , 1969 ; maier , buller , philipp , & heuser , 1988 ; snaith , baugh , clayden , husain , & sipple , 1982 ) . the ham - a consists of 14 items ( score range 056 ) that evaluate a variety of anxiety symptoms ( beck & steer , 1991 ) . ham - a total score presents an intra - class coefficient of 0.74 and a concurrent validity between 0.63 and 0.75 ( maier et al . , 1988 ) . the ham - a has shown good internal consistency ( cronbach s alpha = .893 ) ( kummer , cardoso , & teixeira , 2010 ) . gambling behavior : we evaluated the age at the start of regular gambling , the amount of money lost with gambling in the last year , and the average gambling frequency ( times per week ) . overall severity of gd : we assessed the total number of dsm-5 gd criteria using the structured clinical interview for gambling disorder ( sci - pg ) ( grant , steinberg , kim , rounsaville , & potenza , 2004 ) . sci - pg was first validated using the criteria of the fourth edition of the diagnostic and statistical manual of mental disorders ( dsm-4 ) . retest reliability on the number of gd criteria endorsed showed r = .97 ( p = .006 ) ( grant et al . , this procedure was performed deleting the criterion committed illegal acts such as forgery , fraud , theft , or embezzlement to finance gambling regarding illegal acts , which was present in the previous manual , dsm-4 . moreover , we lowered the diagnostic threshold from five to four , consistent with dsm-5 . severity was divided in three categories : recreational gambling ( meets 0 dsm-5 criteria ) , subsyndromal gd ( meets 13 dsm-5 criteria ) and gd ( meets 4 or more dsm-5 criteria ) . in addition , we investigated the overall gambling severity with the pathological gambling yale - brown obsessive - compulsive scale ( pg - ybocs ) . it is a 10-item scale that showed high validity ( r = .895 ) and reliability ( cronbach s = .970 ) ( pallanti , decaria , grant , urpe , & hollander , 2005 ) . this scale provides a total score ( overall severity ) as well as scores in two subscales ( urges and behavior subscales ) . impulsiveness : evaluated by the barratt impulsiveness scale , version 11 ( bis-11 ) ( patton & stanford , 1995 ) , a scale that has been largely used to investigate impulsiveness ( steinberg , sharp , stanford , & tharp , 2013 ) . this scale has shown good internal consistency ( cronbach s between .79 and .83 ) ( patton & stanford , 1995 ) . bis-11 provides scores in three different dimensions , based on previous factor analyses : attentional impulsiveness , motor impulsiveness , and non - planning impulsiveness ( patton & stanford , 1995 ) . prevalence of illegal acts : the commitment of illegal acts to finance gambling activity has been associated with higher severity of gd ( granero et al . , 2015 ; strong & kahler , 2007 ; toce - gerstein et al . , 2003 ) . it was evaluated using an open question using previous dsm-4 criteria : have you committed illegal acts such as forgery , fraud , theft , or embezzlement to finance gambling ? suicidality and psychiatric comorbidity : evaluated by the mini - international neuropsychiatric interview ( mini ) ( sheehan et al . , 1998 ) . quality of life ( quality of life inventory ) : a 17-item scale that evaluates the subject s overall quality of life ( frisch , 1994 ) . alpha coefficients and test retest correlations for this questionnaire have ranged , respectively , from 0.77 to 0.89 and from 0.80 to 0.91 ( mendlowicz & stein , 2014 ) . individuals with gd tend to present with several neurocognitive deficits such as poorer response inhibition , low cognitive flexibility , worse decision making , and problems with sustained attention and executive functioning ( clark , 2010 ; van holst , van den brink , veltman , & goudriaan , 2010 ) . this study evaluated whether anxiety symptoms affected any of these neuropsychological variables . in this context , participants undertook selected tests from the computerized cambridge neuropsychological test automated battery ( cantabeclipse , version 3 , cambridge cognition ltd . , cambridge , uk ) ( cambridge cognition , 2015 ) . task order was fixed and the total duration of cognitive testing was approximately 50 min . response inhibition : assessed by the stop - signal task , which assesses the subject s ability to inhibit / suppress motor responses . individuals react to an arrow stimulus , by touching either a left or right key depending on the direction in which the arrow points . when an audio tone occurs , the participant attempts to suppress their motor response for the particular trial ( morein - zamir & sahakian , 2010 ) . the outcome measure of interest is the stop - signal reaction time , an index of the time taken for the person s brain to stop a response that would normally be made . cognitive flexibility : investigated with the intra - dimensional / extra - dimensional set shifting test , which evaluates rule learning , reversal , and shifting of attentional focus across stimulus dimensions . the test uses visual stimuli ( colorful shapes and white lines ) and gives feedback to the individual so that they are able to learn an underlying rule about which stimulus is correct , based on trial and error . the underlining rule that determines what is correct and incorrect changes several times and assesses the individual s ability to respond with flexibility ( cambrigde cognition , 2015 ) . decision making : assessed using the cambridge gamble task , a test that assesses decision making and risk taking ( cambridge cognition , 2015 ; deakin , aitken , robbins , & sahakian , 2004 ; lawrence , luty , bogdan , sahakian , & clark , 2009 ) . the main outcome measures in this test are : quality of decision making , proportion of points gambled , and risk adjustment . this task investigates the ability to detect unpredictable target sequences over prolonged period of time ( sarter , givens , & bruno , 2001 ) . the task consists of a white box in the center of the computer screen , inside which numbers , from 2 to 9 , show up in a pseudo - random manner , at the frequency of 100 digits / min . this test , a variant of a variation of the tower of london ( owen et al . , 1995 ) , investigates goal - directed planning ( cambridge cognition , 2015 ) . the task presents visual problems to the subject and evaluates the individual s ability to plan a solution and answer these problems . we analyzed the association between the severity of anxiety symptoms and demographic , clinical , and neurocognitive variables of the participants using spearman s coefficients for continuous elements and mann whitney tests for categorical variables . to control for multiple comparisons , we divided the usual level of significance ( p = .05 ) by the number of variables evaluated in each group of assessments ( i.e. , clinical variables and neurocognitive testing ) . consequently , significance was defined as p .004 ( .05/13 = .004 ) for clinical variables and p .007 ( .05/7 = .007 ) for neurocognitive variables . to reduce the likelihood of confounding variables contributing to the above analyses , we controlled for current major - depressive disorder , alcohol - use disorder , substance - use disorder , and nicotine use . these controls were included , as all four variables have demonstrated significant overlap with anxiety symptoms and several clinical / neurocognitive variables investigated in this study , in prior work ( clark , 2010 ; maier et al . , 1988 ) . this research was approved by the institutional review boards of the university of chicago and the university of minnesota . the study procedures were explained to the participants prior to providing consent , and all participants were given time to ask questions . description of demographics and main clinical variables of non - treatment - seeking gamblers ( n = 143 ) note . sd , standard deviation ; dsm-5 , diagnostic and statistical manual of mental disorders fifth edition ; pg - ybocs , gambling adaptation of the yale - brown obsessive - compulsive scale . there was a significant positive correlation between the severity of anxiety symptoms and the number of current dsm-5 gd criteria . in addition , the quality of life was negatively correlated with the severity of anxiety symptoms . the presence of suicidality in gamblers was also associated with significantly higher scores on ham - a , even after controlling for major depression , alcohol - use disorders , substance - use disorder , and use of nicotine ( see also the findings using anova , figure 1 ) . the analysis also showed a direct correlation between anxiety symptoms in gamblers and higher scores on the attentional impulsivity dimension of bis ( i.e. , higher attentional impulsiveness ) ( see table 2 ) . association between the severity of anxiety symptoms and the level of suicidality in non - treatment - seeking gamblers ( n = 138 ) . p value controlled for major depressive disorder , alcohol - use disorder , substance - use disorder , and smoking . the level of suicidality was defined by the mini - score that accesses the risk of suicide in the past month . suicidality is considered low ( scores between 1 and 8 points ) , moderate ( scores between 9 and 16 points ) , and high ( scores 17 ) association between clinical variables and anxiety levels in non - treatment - seeking gamblers ( n = 143 ) note . ham - a , hamilton anxiety scale ; sd , standard deviation ; dsm-5 , diagnostic and statistical manual of mental disorders fifth edition ; pg - ybocs , gambling adaptation of the yale - brown obsessive - compulsive scale . significance was defined as p .004 ( .05/13 = .004 ) for clinical variables . adjusted for current major - depressive , alcohol - use disorder , substance - use disorder , and nicotine use . we did not find significant correlations between neurocognitive variables and anxiety symptoms ( see table 3 ) . association between neurocognitive variables and anxiety level in non - treatment - seeking gamblers ( n = 143 ) significance was defined as p .007 ( .05/7 = .007 ) for neurocognitive variables . adjusted for current major - depressive , alcohol - use disorder , substance - use disorder , and nicotine use . the following measures were used : [ delay at stop - signal test ] = stop - signal reaction time ( ms ) ; [ intra extra dimensional set shifting test ] = total errors ( adjusted ) ; [ rapid visual information processing ] = proportion of targets detected ; [ one touch stockings of cambridge ] = problems solved in minimum possible number of moves . this study analyzed the association of anxiety symptoms , gambling clinical variables , and neurocognitive variables in non - treatment - seeking gamblers . we evaluated subjects with a wide range of gambling severity ( individuals who meet 09 dsm-5 gd criteria ) and with different levels of anxiety ( ham - a scores from 0 to 28 ) . the use of a non - treatment - seeking sample was important to achieve this spread of disease severities , to maximize the ability to detect the relationships between variables . treatment - seeking gamblers tend to be more severe than gamblers in the general population ( petry et al . , 2005 ) and therefore , the range of different levels of gambling activity is narrower in treatment - seeking samples . therefore , the use of a non - treatment - seeking sample enabled us to assess the association between anxiety , gambling , and neurocognition throughout a broad spectrum of gambling and anxiety levels . this study found that the severity of anxiety symptoms was associated with several important clinical variables , even when controlling for major depression , alcohol - use disorder , substance - use disorder , and use of nicotine . there was a positive correlation between anxiety and gambling severities measured by the number of dsm-5 gd criteria met . finally , the severity of anxiety symptoms was negatively correlated with the quality of life . there was no significant correlation , however , between gambling severity , as measured by pg - ybocs , and anxiety . the dsm-5 criteria assess the symptoms over the past year , whereas the pg - ybocs was designed to evaluate severity during the past 7 days . given that the anxiety is often a chronic condition ; the dsm-5 criteria may more accurately capture the impact of these symptoms on gambling behavior . a large epidemiological study suggested that anxiety disorders usually precede gd and appear to trigger gambling problems ( kessler et al . , 2008 ) . the longitudinal relationship between anxiety symptoms and gambling activity , after gambling has started , remains unclear . in this context , important questions remain : do gamblers have a more harmful gambling activity due to higher anxiety levels ? or , does having severe gambling problems and , therefore more negative consequences , make gamblers feel more anxious ? the finding that anxiety levels were positively correlated with gambling severity is clinically important , and proper management of anxiety may improve treatment outcomes in gambling problems . psychological therapies should address anxiety while identifying the reasons for gambling or for worsening of bets . relaxation techniques and alternative ways to deal with anxiety ( other than gambling ) might be useful . pharmacological approaches , such as the use of selective serotonin re - uptake inhibitors , may also enhance the control of anxiety . grant and potenza ( 2006 ) found that the use of escitalopram in a sample of disordered gamblers with co - occurring anxiety significantly improved gambling and anxiety symptoms . this study observed a positive correlation between attentional impulsiveness as indexed by the barratt questionnaire , and severity of anxiety symptoms ; and between worse sustained attention on a computerized task , and severity of anxiety symptoms . attentional impulsiveness has been described as an inability to keep attention / concentration ( stanford et al . , 2009 ) . when the bis was first developed , attentional impulsiveness was believed to be an isolated dimension together with motor impulsiveness and lack of planning ( barratt , 1959 ) . later research suggested that it is an underlying construct that interacts with motor impulsiveness and lack of planning ( patton & stanford , 1995 ) . therefore , attentional impulsiveness may be considered as a dimension that is correlated with impulsiveness in several levels ( patton & stanford , 1995 ) . attentional bias is a well - characterized phenomenon in anxious individuals , who preferably allocate attention / focus on threatening stimuli rather than on neutral stimuli ( bar - haim et al . , 2007 ; this attentional bias to threat also leads to slower disengagement from threatening elements and , as a result of this , other non - threatening elements tend to be less perceived ( bishop et al . , 2004 ; koster et al . , 2004 , 2006 ; schofield et al . , 2012 ) . therefore , an anxious individual who gambles may have a reduced focus on the gambling behavior ( i.e. , how to bet and how much to bet ) . future studies might further investigate how the attentional bias interferes in gambling activity and could , particularly , look at possible threatening stimuli that are over - perceived during gambling behavior . this study also observed that the severity of anxiety symptoms was positively associated with the presence of suicidality ( figure 1 ) . for example , in 2010 , the financial losses associated with suicide were estimated at 44 billion us dollars / year ( american foundation for suicide prevention , 2010 ) . second , suicide is a top 2 leading cause of death in the united states for subjects aged 1534 years ( centers for disease control and prevention , 2011 ) , the population evaluated by this study . third , gd and subsyndromal gd have been associated with higher rates of suicide ideation / attempts ( bland et al . , 1993 ; hodgins , mansley , & thygesen , 2006 therefore , young gamblers appear to have a substantially elevated risk for suicide ( age + gambling activity ) . unlike this research , the majority of previous studies that assessed suicide / risk of suicide in gamblers did not report a significant correlation between anxiety and suicidality . two facts may explain this as follows : ( a ) some studies failed to assess the relationship between anxiety symptoms / anxiety disorders and suicide ( see hodgins et al . , 2006 ) and ( b ) this study evaluated only young adults ( aged 1829 years ) , a group with higher anxiety symptoms , and higher suicidality ( centers for disease control and prevention , 2011 ; martin , 2003 ) . therefore , anxiety symptoms may present a higher impact on suicidality in young gamblers and we evaluated a broad spectrum of gambling activity . ( 2014 ) used a subsyndromal gd sample and found an association between suicidality and anxiety disorders . however , the assessment of suicidality did not control for other mental disorders as this study did . as the correlation between gd and suicide ideation / attempts has been strongly attributed to co - occurring major depressive disorder and alcohol-/substance - use disorders ( crockford & el - guebaly , 1998 ; hodgins et al . , 2006 ) , our study strengthens the possible separate association of anxiety symptoms and suicidality in gamblers . a meta - analysis of 42 studies conducted by kanwar et al . ( 2013 ) suggested that anxiety disorders are associated with higher suicidality ( kanwar et al . , 2013 ) . better assessment of anxiety symptoms , particularly in gamblers with suicidality seems to be important in clinical practice . as anxiety symptoms / disorders are considered relatively treatable ( hofmann & smits , 2008 ) , a more focused management in anxious gamblers may possibly reduce suicide risk . our study found that severity of anxiety was negatively correlated with the quality of life . this finding is consistent with previous epidemiological and clinical studies that have shown that anxiety disorders and subthreshold forms of anxiety disorders are associated with significantly reduced quality of life ( kessler et al . , 1994 ; markowitz , weissman , ouellette , lish , & klerman , 1989 ; mendlowicz & stein , 2014 ; spitzer et al . , 1995 ; weissman , 1991 ; wittchen , 2002 ; wittchen , carter , pfister , montgomery , & kessler , 2000 ; zatzick et al . , 2014 ) . on the other hand , subsyndromal gd and gd have also been associated with poorer life satisfaction when compared with the general population ( black , moyer , & schlosser , 2003 ; grant & kim , 2005 ) . therefore , this study reinforces the negative correlation between anxiety and decreased quality of life in a non - treatment - seeking sample of impulsive patients . quality of life is a core dimension in psychiatry treatments and , as a result of this , this variable has been increasingly recognized as a main outcome measure in clinical trials ( group for the advancement of psychiatry committee on psychopathology , 1994 ; johnson & temple , 1985 ; pietersma , de vries , & van den akker - van , 2014 ; spitzer et al . , 1995 ; wilson & cleary , 1995 ) . cognitive - behavioral therapy focusing on training of social skills may benefit anxious gambling individuals . first , this research used a cross - sectional analysis and , therefore , causal relationships can not be confirmed . second , some of the data collected ( age at the start of recreational gambling and money lost with gambling in the last year ) may be subject to potential recall bias , as it was collected in hindsight . third , our sample consisted of a convenience sample of young adults ( individuals aged 1829 years ) . therefore , caution is needed when generalizing our findings to broader segments of the population . finally , our study used a low significance level [ i.e. , .004 for ( clinical variables ) and .007 for ( neurocognitive variables ) ] . therefore , we increased the likelihood of committing type ii error . on the other hand this study suggests that anxiety may be associated with relevant clinical variables in the broad spectrum of gambling activity . therefore , proper management of anxiety symptoms might improve the clinical presentation of gamblers in different areas : ( a ) reducing the overall gambling severity ; ( b ) improving the attentional deficits and attempting to reduce attentional impulsiveness ; ( c ) reducing the risk / severity of suicidality ; and ( d ) improving the quality of life . the corresponding author affirms that he had access to all data from the study , both what is reported and what is unreported , and also that he had complete freedom to direct its analysis and its reporting , without influence from the sponsors . the corresponding author also affirms that there was no editorial direction or censorship from the sponsors . gcm conducted the literature searches , the statistical analysis , and wrote the first draft of the manuscript . ewl and src wrote the protocol and made edits and amendments to the first draft of the manuscript . jeg designed the study , wrote the protocol , supervised the literature searches and statistical analysis , and reviewed the final version of this paper . his involvement in this research was supported by a grant from the academy of medical sciences ( ams , uk ) . jeg has received research grants from national center for responsible gaming , american foundation for suicide prevention , brainsway , and forest , takeda , and psyadon pharmaceuticals . he receives yearly compensation from springer publishing for acting as editor - in - chief of the journal of gambling studies and has received royalties from oxford university press , american psychiatric publishing , inc .

infantile perianal pyramidal protrusions ( ippp ) as the name suggests either pyramidal , leaf like , or cigar - shaped protrusions which are rare congenital anomalies and most often present at birth or soon after.because of their rarity and inadequate reporting in our literature they are often mistaken for sentinel hemorrhoid , skin tag , or even sexual abuse leading to condyloma . infantile perianal pyramidal protrusions ( ippp ) as the name suggests either pyramidal , leaf like , or cigar - shaped protrusions which are rare congenital anomalies and most often present at birth or soon after . because of their rarity and inadequate reporting in our literature they are often mistaken for sentinel hemorrhoid , skin tag , or even sexual abuse leading to condyloma . one of us ( sv ) has documented two infants with a congenital protrusion located perianally . the first case also had a large segmental facial hemangioma reminiscent of sturge - weber syndrome as well as multiple small hemangiomas on the extremities . the second one was seen by both of us in my clinic in india ( sv and uw ) whereupon both cases were discussed thoroughly and a diagnosis of two different - shaped infantile perianal pyramidal protrusion ( ippp ) has been made . we suggest that the association of hemangiomas in the same location are not coincidental but may be a part of one of the syndromes described in the table below . a 3-month - old female child was seen by one of us ( sv ) in a multi - specialty hospital upon the request of the pediatrician . she was born after a normal full - term vaginal delivery and had no history of consanguinity in her parents . the child was thought to have hemorrhoids with a large hemangioma in the perineal area present since birth . upon examination the child had a skin - colored perianal protrusion that was clearly located posterior to the anus [ figure 1 ] . there was in addition a large midline hemangioma that started from the gluteal cleft all the way to the medial root of the thigh skirting around the vulva [ figure 1 ] . the presence of inflammation around the pyramidal protrusion could not be assessed because of the presence of the large hemangioma . red patch in the area of the hemangioma which evolved over the past 3 months into a hemangiomatous red plaque . the child also had a large hemangioma on the contralateral side of the face with involvement of the lower lip [ figure 2 ] . there were also small hemangiomas found on the ventral aspect of the left forearm and dorsum of the left foot . the hemangiomas were explained in detail to the parents and they decided not to give any oral corticosteroid for the perineal hemangiomas and rather wait for a year . a head and neck as well as spine x - ray was performed that did not show any abnormality . a referral was made to the pediatric surgeon for biopsy of the pyramidal lesion which they refused . the large facial segmental hemangioma was thought to be sturge - weber syndrome with no clinical evidence of internal involvement . the patient was prescribed topical clobetasol cream under monthly supervision after which the lesion had flattened a little and showed a whitish area with pronounced flattening . skin - colored pyramidal perianal protrusion with a large hemangioma facial hemangioma in the same patient a 2-month - old female child was brought to the same clinic where she was examined by both authors ( i.e. , sv and uw ) . the child had a flattened erythematous leaf - like midline protrusion located anterior to the anus which had grown gradually [ figure 3 ] . she too was born after a normal full - term vaginal delivery and had no history of consanguinity in her parents . the parents had noticed the lesion only after 2 weeks of her birth . a hemangiomatous plaque to the left of the anus had been noticed by the parents as a pink - colored macule which had gradually deepened in color but had not grown in size . they called after 2 months saying that the hemangioma was flattening rapidly but the leaf - like protrusion remained unchanged . a diagnosis of ippp was made for both the cases with the diagnosis of the first case done retrospectively . this opens up the strong possibility of the ippp being a part of pelvis syndrome in which a a 3-month - old female child was seen by one of us ( sv ) in a multi - specialty hospital upon the request of the pediatrician . she was born after a normal full - term vaginal delivery and had no history of consanguinity in her parents . the child was thought to have hemorrhoids with a large hemangioma in the perineal area present since birth . upon examination the child had a skin - colored perianal protrusion that was clearly located posterior to the anus [ figure 1 ] . there was in addition a large midline hemangioma that started from the gluteal cleft all the way to the medial root of the thigh skirting around the vulva [ figure 1 ] . the presence of inflammation around the pyramidal protrusion could not be assessed because of the presence of the large hemangioma . red patch in the area of the hemangioma which evolved over the past 3 months into a hemangiomatous red plaque . the child also had a large hemangioma on the contralateral side of the face with involvement of the lower lip [ figure 2 ] . there were also small hemangiomas found on the ventral aspect of the left forearm and dorsum of the left foot . the hemangiomas were explained in detail to the parents and they decided not to give any oral corticosteroid for the perineal hemangiomas and rather wait for a year . a head and neck as well as spine x - ray was performed that did not show any abnormality . a referral was made to the pediatric surgeon for biopsy of the pyramidal lesion which they refused . the large facial segmental hemangioma was thought to be sturge - weber syndrome with no clinical evidence of internal involvement . the patient was prescribed topical clobetasol cream under monthly supervision after which the lesion had flattened a little and showed a whitish area with pronounced flattening . skin - colored pyramidal perianal protrusion with a large hemangioma facial hemangioma in the same patient a 2-month - old female child was brought to the same clinic where she was examined by both authors ( i.e. , sv and uw ) . the child had a flattened erythematous leaf - like midline protrusion located anterior to the anus which had grown gradually [ figure 3 ] . she too was born after a normal full - term vaginal delivery and had no history of consanguinity in her parents . a hemangiomatous plaque to the left of the anus had been noticed by the parents as a pink - colored macule which had gradually deepened in color but had not grown in size . they called after 2 months saying that the hemangioma was flattening rapidly but the leaf - like protrusion remained unchanged . a diagnosis of ippp was made for both the cases with the diagnosis of the first case done retrospectively . this opens up the strong possibility of the ippp being a part of pelvis syndrome in which a the term infantile perianal pyramidal protrusion ( ippp ) was first coined by kayashima in 1996 . , before this description called them skin tags / folds. knowledge of this entity is important as it can be easily mistaken for a sentinel hemorrhoid , anogenital wart or may give rise to an uncalled for alarm regarding sexual abuse . according to one of the largest studies done of this entity comprising 15 patients , as many as 93% of these patients presented with this protrusion at birth . ippp is skin colored or red , located perianally along the medial raphe predominantly in girls but is not necessarily always pyramidal in shape . . it may also rarely present posteriorly and has also been described on both anterior as well as posterior location . because they are not always pyramidal in shape some workers have suggested the term infantile perineal protrusions. however , the term perineal is also not accurate when the lesion is located posterior to the anus . embryologically the lengthening of the urogenital septum during the growth of the fetus forms the perineum . ippp is considered by some to be a residual part of the tip of the urogenital septum . structurally ippp is said to occur due to inherent laxity of the female perineum and that explains the female predilection . there are three different types of ippps described in literature namely , constitutional or congenital , acquired or functional , and the last being associated with lichen sclerosus et atrophicus ( lsa ) . the commonest is the constitutional type of ippp and is described to be occurring often as a leaf like flat protrusion . it is present congenitally and some of the patients have family members who have a similar affliction including a report of its occurrence in two sisters supporting the theory that ippp may be due to a constitutional predisposition . the acquired functional type is thought by some to be associated with constipation and straining during defecation . in some of these patients treatment of constipation resulted in disappearance of the ippp as early as 3 weeks . wiping and cleaning after passing stools , diarrhea , anal fissures , and inflammation in the diaper are also described as etiological factors . however , 93% of patients showing the lesion at birth in one study go against the theory of constipation being a primary causative factor . the classic pyramidal shape is described most often in this particular variant of the ippp . the third type of ippp associated with lsa is described as pyramidal or cigar shaped with porcelain white atrophic appearance . this variant can present with recurrent anogenital erythema or have distinct clinical appearance of lsa . it is proposed that in some patients ippp may be an early clinical feature of lsa . treatment with strong topical corticosteroids helps resolve the recurrent anogenital erythema but not the ippp . the relatively few biopsies performed have shown extension of the surrounding normal skin with some acanthosis , elongation of rete ridges and proliferation of capillaries and lymphatics in the dermis . in only one case in this study upper dermal edema , telangiectasias and mild infiltration of neutrophils and eosinnophils have been described in some . one such patient had significant increase in size of the lesion upon passing hard stools with difficulty and therefore the inflammation documented in some of the patients could be a secondary phenomenon . the ippps in both our patients were in line with all that has been mentioned in literature . one is pyramid shaped and is posterior to the anus which is a rare presentation of ippp ; the other is shaped like a hen 's crest or a both of them have not been studied histologically because of the parents reluctance but they conform to the description of this entity in literature . we would like to present our views , though retrospectively , regarding the presence of accompanying perianal hemangiomas in both our cases . in case 1 girard et al . , have described pelvis syndrome , an acronym for perineal hemangioma , external genitalia malformations , lipomyelomeningocoele , vesicorenal abnormalities , imperforate anus , and skin tag . skin tag. they have also mentioned urorectal septum malformation sequence which could be partial or complete and is very similar to the constellation of signs described by them . they explain that associated hemangiomas may not have been described in previous studies due to a patient recruitment bias in the dermatology and plastic surgery departments . they propose that the association of hemangiomas could be a variant of the urorectal malformation sequence . we propose that both our patients have an incomplete pelvis syndrome with perineal hemangiomas and pelviperineal imaging studies were not done due to financial constraints which is very often a limitation in indian patients . two other syndromes also need to be mentioned with pelvis since they too present with lower body / lumbosacral hemangiomas and anogenital anomalies in addition to other features mentioned in the table [ table 1 ] . it is also felt by some that the two separately described lumbar and pelvis syndrome may be the same due to the major overlap that is seen in the constellation of signs present . syndromes with anorectal malformations and coexisting hemangiomas we believe we are describing ippp for the first time from india . we suggest that ippp with perineal and perianal hemangiomas is part of incomplete pelvis syndrome with ippp being the we further suggest that all cases of ippp should be examined thoroughly and urogenital , renal , and spinal studies should be carried out to rule out the associated abnormalities described in table 1 . it is unfortunate that the vast majority of indian population do not have medical insurance and therefore expensive imaging studies are not routinely done which accounts for probably many missed features associated with such congenital abnormalities . there are certain syndromes reported in literature , particularly pelvis syndrome , comprising multiple congenital visceral and structural anomalies with associated perianal hemangiomas and lesions morphologically identical to ippp.we suggest that ippp with congenital perianal and perineal hemangiomas could be part of incomplete pelvis syndrome in which the skin tag described in the syndrome along with perineal hemangioma , external genitalia malformations , lipomyelomeningocele , vesicorenal abnormalities , imperforate anus is actually ippp . it is noteworthy that ippp was initially described in literature as skin tag by mckann et al.patients coming with such presentations should be carefully screened for other congenital anomalies which may be asymptomatic at the time of presentation by appropriate imaging techniques . there are certain syndromes reported in literature , particularly pelvis syndrome , comprising multiple congenital visceral and structural anomalies with associated perianal hemangiomas and lesions morphologically identical to ippp . we suggest that ippp with congenital perianal and perineal hemangiomas could be part of incomplete pelvis syndrome in which the skin tag described in the syndrome along with perineal hemangioma , external genitalia malformations , lipomyelomeningocele , vesicorenal abnormalities , imperforate anus is actually ippp . it is noteworthy that ippp was initially described in literature as skin tag by mckann et al . patients coming with such presentations should be carefully screened for other congenital anomalies which may be asymptomatic at the time of presentation by appropriate imaging techniques .

glioblastoma multiforme ( gbm ) is the most common form of all primary adult brain tumors . although significant technical advances in surgical and radiation treatments for brain tumors have emerged , their impact on clinical outcome for patients has been modest [ 1 , 2 ] . of the features that characterize gbm , arguably none is more clinically significant than the propensity of glioma cells to aggressively invade the surrounding normal brain tissue . these invasive cells render complete resection impossible , confer strong resistance to chemo- and radiation therapy , and virtually assure the rise of secondary tumors that develop at the resection margins that drive further invasion . meaningful advances in clinical outcomes will require identification and targeting of key signaling effectors mediating glioma invasion . the nonreceptor tyrosine kinase proline - rich tyrosine kinase 2 ( pyk2 ) serves as a point of integration for signaling from cell surface receptors including integrin adhesion receptors , g - protein coupled receptors , and receptor tyrosine kinases [ 57 ] . as such , signaling from pyk2 has been implicated in a variety of cellular processes including migration , cell survival , and proliferation . we have demonstrated in glioblastoma tumor samples that pyk2 expression is upregulated in invasive glioma cells relative to cells in their cognate tumor cores and that increased pyk2 activity positively correlated with increased migration of glioma cells in vitro . furthermore , we established that increased expression of pyk2 simulated glioma cell migration in vitro while specific knockdown of pyk2 expression inhibited glioma cell migration in vitro , impaired invasion in organotypic brain slices , and increased survival and reduced invasion and distant tumor foci in an intracranial xenograft model [ 9 , 10 ] . specific inhibition of pyk2 activity inhibited glioma cell migration in vitro and prolonged survival in a xenograft model [ 11 , 12 ] . pyk2 can be activated by integrin ligation [ 13 , 14 ] and is activated in response to cellular stress and in response to a variety of agonists that raise intracellular calcium [ 7 , 15 , 16 ] . how agonist stimulation ultimately leads to pyk2 activation remains unclear as the intrinsic mechanism of activation for this kinase remains to be defined . pyk2 shares a conserved domain structure with the related focal adhesion kinase fak including an n - terminal ferm domain , a central kinase domain , several proline rich domains , and a c - terminal focal adhesion targeting ( fat ) domain . the fat domain is critically involved in the activation of fak by targeting fak to the focal adhesion [ 17 , 18 ] . similarly , expression of frnk , an alternatively spliced variant corresponding to the c - terminal portion of fak , inhibits fak activation by displacing fak from the focal adhesion [ 19 , 20 ] . interestingly , although pyk2 contains a highly conserved fat domain and can be localized to the focal adhesion , it also exhibits a significant cytoplasmic distribution with perinuclear enrichment in a number of cell types suggesting that focal contact localization is not essential for pyk2 activation . indeed , substitutions within the pyk2 fat domain postulated to disrupt the four - helix bundle structure of the pyk2 fat domain do not result in the loss of pyk2 activity . these data suggest that domains within pyk2 other than the fat domain may function to localize pyk2 to specific cellular locations or in the regulation of pyk2 activity . among the candidates for functional regulatory domains in pyk2 ferm domains are compact protein modules comprised of three distinct subdomains found in a number of proteins . in the prototypical ferm domain proteins ezrin , radixin , and moesin , the ferm domains regulate their activity by mediating protein - protein interactions and membrane targeting . previous studies support an important role for the n - terminal pyk2 ferm domain in the regulation of pyk2 function . we have demonstrated that intracellular expression of an autonomous pyk2 ferm domain potently inhibited pyk2 phosphorylation . subsequently , kohno et al . demonstrated that the ferm domain mediated the formation of ca / calmodulin dependent pyk2 homodimers that facilitated transphosphorylation . structural analysis of several ligand - bound ferm domains has substantiated the importance of a surface formed by 5c-1c of the f3 subdomain in ligand binding [ 2430 ] . select substitutions within this surface of the ferm domain of pyk2 inhibit pyk2 phosphorylation [ 11 , 12 ] . moreover , a monoclonal antibody targeting an epitope localized to the 5c-1c surface of the f3 module of the pyk2 ferm domain effectively inhibits pyk2 phosphorylation when expressed as an intracellular scfv . together , these data support an important role for the ferm domain in the regulation of pyk2 activity perhaps by mediating protein - protein interactions that are required for pyk2 function . similar studies into the regulation of activity of the closely related focal adhesion kinase fak have provided compelling evidence for a functional role of the n - terminal ferm domain . structural studies have demonstrated that the n - terminal ferm domain of fak binds directly to the kinase domain thereby blocking access to the catalytic cleft [ 31 , 32 ] . proposed that fak activation results from displacement of the ferm domain , perhaps mediated by a protein - protein interaction with an activating protein . while the identity of the proposed activating protein remains to be determined , an important role for a conserved cluster of basic amino acids in the f2 subdomain has been described . importantly , we and others have been unable to demonstrate a similar interaction between the pyk2 ferm domain and the pyk2 kinase domain suggesting that the mechanism of intrinsic activation is different for pyk2 than for fak . in the present study , we utilized yeast two - hybrid genetic selection to screen for novel protein - protein interactions mediated by the pyk2 ferm domain . we identified the ste20 homolog map4k4 as a pyk2 binding partner and describe a role for integration of map4k4 with pyk2 function in glioma cell migration . the anti - flag m2 monoclonal antibody was from sigma ( st . louis , mo ) . the rabbit anti - ha monoclonal antibody and the polyclonal anti - pyk2 antibody were from upstate biotechnology ( lake placid , ny ) . the anti - pyk2 monoclonal antibody ot126 was from united states biologicals ( swampscott , ma ) . the anti - phosphotyrosine ( py ) antibody py20 was obtained from bd transduction laboratories ( san jose , ca ) . the irdye conjugated secondary antibodies were from li - cor biosciences ( lincoln , ne ) . the strains , plasmids , and library used in the yeast two - hybrid ( y2h ) interaction screen were obtained from clontech ( mountain view , ca ) and used as recommended by the manufacturer . the y2h pyk2 ferm bait plasmid was constructed by placing the pyk2 ferm dna ( encoding pyk2 e36 through a366 ) between the ncoi and bamhi sites of the bait vector pas2 - 1 . to select for pyk2 ferm prey proteins the saccharomyces cerevisiae strain , ah109 ( mata , trp1 - 901 , leu2 - 3 , 112 , ura3 - 52 , his3 - 200 , gal4d , gal80d , lys : : gal1uas- gal1tatahis3 , gal2uas- gal2tata - ade2 , ura : : mel1uas- mel1tata - lacz ) containing the pyk2 ferm bait plasmid was transformed with a human fetal brain cdna library ( 3.5 10 independent clones ) in the vector pact2 . the transformation reaction was plated onto sc plates minus his , leu , and trp ( sc - his - leu - trp ) . after 46 days at room temperature , the colonies were replica plated onto the more stringent sc - ade - his - leu - trp medium . those colonies appearing on the stringent selection plate were purified and further analyzed . to confirm bait - prey interaction , lacz reporter expression was assayed using the chemiluminescent -galactosidase assay reagent gal - screen ( tropix , bedford , ma ) in 96-well microtiter plates following the manufacturer 's instructions . the interaction of the clone map4k4(143 ) with various proteins was quantified by two - hybrid analysis in diploid strains ( figure 1 ) as follows . strain y189 ( mat , ura3 - 52 , his3 - 200 , ade2 - 101 , trp1 - 901 , leu2 - 3 , 112 , gal4 , met , gal80 , ura3::gal1uas - gal1tata - lacz ) expressing clone map4k4(143 ) as prey was mated with strain ah109 harboring various bait chimeras by mixing the haploid strains on sc plates lacking leucine and tryptophan ( sc - lw ) and incubating the plates at 30c for two days . the resulting diploid strains were cultured overnight in sc - lw broth , and then 100 l was assayed for -galactosidase activity with gal - screen reagent . the relative light unit ( rlu ) signal produced by -galactosidase was normalized for culture density ( optical density , od ) . the human glioblastoma cell line sf767 and the 293 t packaging cells were routinely passaged in dmem ( biowhittaker , walkersville , md ) containing 10% fetal bovine serum , 1% nonessential amino acids , 2 mm glutamine , 100 units / ml penicillin , and 10 g / ml streptomycin . transfections of subconfluent cultures were performed with the effectene reagent ( qiagen , chatsworth , ca ) as previously described . the construction of the following expression plasmids has been previously described : the flag - epitope - tagged pyk2 , the flag - epitope - tagged kinase - deficient pyk2 ( k457a ) , and the ha - epitope - tagged pyk2 ferm ; the ha - epitope - tagged fak ; the ha - epitope - tagged pyk2 ferm , and the ha - epitope - tagged fak ferm domain encoding fak residues r35-p362 [ 11 , 12 ] . the map4k4 sequence contained in clone 143 isolated from the yeast two - hybrid screen ( corresponding to map4k4 residues 9291273 based on the numbering of isoform 2 , the longest isoform ; accession nm_145686 ) was amplified by pcr and ligated in frame downstream of a 3x ha epitope in pcdna or downstream of a 3x flag epitope in p3xflag - cmv ( sigma , st . the coding sequence for full length map4k4 was isolated by rt - pcr of total rna isolated from the sf767 glioma cell line using the titan rt - pcr kit ( roche applied science , indianapolis , in ) according to the manufacturer 's instructions . the final clone contains alternatively spliced modules m1 , m2 , m3 , and m8 and was ligated in frame downstream of a 3x ha epitope in pcdna . two shrnas directed against map4k4 were generated from sirna sequences previously reported to knockdown map4k4 expression . the first shrna , designated 1m4k4i , was based on the rnai sequence described by mack et al . and had the following sequence : 5-gatccgtggttggaaatggcacctttcaagagaaggtgccatttccaaccactttttggaaaa-3. the second shrna , designated 2m4k4i , was based on the rnai sequence described by collins et al . and had the following sequence : 5-gatccgggaaggtctatcctcttattcaagagataagaggatagaccttccctttttggaaaa-3. annealed shrna oligonucleotide duplexes were ligated to bamhi / hindiii digested psilencer 3.0-h1 ( ambion , austin , tx ) and the sequence verified by direct dna sequencing . the h1 promoter and shrna were excised from psilencer by ecori / mlui digestion and ligated to the similarly digested lentiviral transfer vector plvthm ( addgene , cambridge , ma ) . plvthm contains a separate transcriptional cassette in which the ef1- promoter drives gfp expression . vsv - g pseudotyped recombinant lentiviruses were produced by transient transfection of 293 packaging cells . subconfluent cultures of 293 cells were transfected with 20 g of the appropriate lvthm construct , 15 g of pspax2 packaging plasmid , and 5 g of pmd2.g envelope vector by calcium phosphate precipitation . for lentiviral transduction , medium containing recombinant lentiviruses was harvested from the packaging cells after 48 hours , concentrated by peg precipitation and centrifugation , and added to subconfluent cultures of target cells together with 8 g / ml polybrene for 46 hours at 37c . positively transduced cells were enriched by mass sorting the gfp positive cells on a vantage flow cytometer ( bd biosciences , san jose , ca ) . the generation of sf767 glioma cells stably transduced with a shrna targeting pyk2 has been previously described . a monolayer radial migration assay was used as previously described [ 34 , 38 ] . briefly , 2500 control or transduced cells were plated / well of a cell sedimentation manifold ( creative scientific methods , phoenix , az ) on laminin coated slides . cells were incubated for 16 hours , the manifold removed , and a measurement of the diameter of the seeded cells was made with an inverted microscope and image analysis software ( scion image , frederick , md ) . linear migration from the initial seeded area was determined for 10 replicate samples 24 hours after removal of the manifold . transwell assays were performed using a modified boyden chamber ( neuroprobe , cabin john , md ) as previously described . briefly , each well contains an 8 m pore size nucleopore filter coated with 50 g / ml bovine collagen ( purecol , advanced biomatrix , poway , ca ) . control or transduced sf767 glioma cells were seeded at 4.8 10 cells in 100 l of complete media ( dmem containing 10% fetal bovine serum ) to the top well of the chamber , and complete media were added to the lower chamber . after incubation for 48 hours at 37c , nonmigrated cells were scraped off the upper side of the filter , and filters were stained with 4,6-diamidino-2-phenylindole ( dapi ) . nuclei of migrated cells were counted in 5 high - power fields ( hpf ) with a 20x objective . control transduced sf767 cells or sf767 cells stably transduced with a shrna targeting map4k4 ( map4k4i ) were seeded in triplicate in complete media . cell proliferation was measured at 24-hour intervals for 5 days using the mtt assay according to the manufacturer 's instructions ( sigma , st . cells were washed in cold pbs , lysed by addition of 1 ml ipb buffer ( 137 mm nacl , 20 mm tris , ph 7.5 , 1% np-40 , and 10% glycerol containing protease and phosphatase inhibitors ) , and incubated on ice for 30 minutes . detection was performed with hrp - conjugated secondary antibodies and enhanced chemiluminescence ( perkin elmer life sciences , boston , ma ) or by infrared detection using irdye conjugated secondary antibodies with the odyssey infrared imaging system ( li - cor biosciences , lincoln , ne ) . migration data was analyzed using graphpad prism 5.0 ( graphpad software , la jolla , ca ) . independent sample t - tests were used for analysis involving two samples and one - way analysis of variance for tests involving more than two samples . our previous studies supported a role for the pyk2 ferm domain in the regulation of pyk2 activity and function . to identify proteins that interact with the pyk2 ferm domain , we performed a yeast two - hybrid selection from a human fetal brain cdna library using the pyk2 ferm domain ( encoding residues e36-a366 ) as the bait . screening of 1.2 10 transformants resulted in the isolation of clone 143 that interacted with the pyk2 ferm domain . sequence analysis indicated that clone 143 encoded the c - terminal one - third of map kinase kinase kinase kinase 4 ( map4k4 ) . as we were interested in proteins that interacted specifically with the pyk2 ferm domain , we examined the interaction of clone map4k4(143 ) with the closely related fak ferm domain ( fak residues r35-p362 ) by two - hybrid analysis . interaction of map4k4(143 ) with the fak ferm domain was not significantly greater than the interaction of map4k4(143 ) with bait vector alone or with the unrelated bait fkbp51 ( figure 1 ) . the yeast two - hybrid selection also identified two additional independent , overlapping map4k4 clones , designated map4k4(33 ) and map4k4(119 ) . the three overlapping clones , starting at codons h884 , v909 , and a924 , all encompass the c - terminal citron homology domain ( cnh ) of map4k4 and include the alternatively spliced module m8 but lack module m9 ( figure 2 ) . to confirm that map4k4 ( 143 ) interacted with the pyk2 ferm domain in the intracellular environment of cultured cells , cells were cotransfected with flag - tagged pyk2 ferm domain and ha - tagged map4k4 ( 143 ) . immunoblotting of the anti - flag immunoprecipitate demonstrated that the ha - tagged map4k4 ( 143 ) coimmunoprecipitated with the pyk2 ferm domain ( figure 3(a ) ) . to probe the specificity of the interaction , we compared the interaction of map4k4 ( 143 ) with pyk2 and fak . cells were cotransfected with flag - epitope - tagged map4k4 ( 143 ) and either full length ha - tagged pyk2 or full length ha - tagged fak . immunoprecipitation of the cotransfected cell lysates with an anti - flag antibody indicated that pyk2 coimmunoprecipitated with map4k4 ( 143 ) ( figure 3(b ) ) . consistent with the results of the two - hybrid interaction assay , an appreciable amount of fak was not detected in the map4k4 ( 143 ) immunoprecipitate . these results indicated that the c - terminal portion of map4k4 containing the cnh domain , previously reported to function as a protein interaction domain [ 35 , 41 ] , interacted with pyk2 . to examine whether full length pyk2 interacted with full length map4k4 rt - pcr of total rna isolated from sf767 cells resulted in the identification of seven different splice isoforms in sf767 cells consistent with the previous observation that multiple forms of map4k4 can be observed in the same cell . from these seven different isoforms , we assembled a full length map4k4 comparable to the map4k4 isoform previously cloned from a snb19 glioma cell library . the final ha - tagged full length map4k4 clone encoded 1,239 amino acids and contained the alternatively spliced modules m1 , m2 , m3 , and m8 ( figure 2(c ) ) . to examine the interaction of full length map4k4 with pyk2 , cells were cotransfected with flag - tagged pyk2 and either ha - tagged map4k4(143 ) or ha - tagged full length map4k4 . cell lysates were immunoprecipitated with rabbit igg as a control or with an anti - ha antibody ( figure 3(c ) ) . immunoblotting of the immunoprecipitates indicated that pyk2 coimmunoprecipitated with both the c - terminal mapk4(143 ) clone and with full length map4k4 . similarly , endogenous map4k4 coimmunoprecipitated with pyk2 from sf767 glioma cells which express both map4k4 and pyk2 ( figure 3(d ) ) . immunoblotting of sf767 cell lysates indicated the presence of several map4k4 species consistent with our rt - pcr results and the previous observation of multiple isoforms of map4k4 in the same cell . cells were cotransfected with ha - tagged map4k4 and either flag - tagged wild - type pyk2 or a flag - tagged kinase - deficient form of pyk2 ( pyk2kd ) . cells were lysed , immunoprecipitated with an anti - pyk2 antibody or an anti - map4k4 antibody , and the precipitates immunoblotted with the anti - phosphotyrosine antibody py20 ( figure 4(a ) ) . immunoblotting of pyk2 precipitated from cells cotransfected with wild - type pyk2 and map4k4 demonstrated positive staining of pyk2 with py20 . immunoprecipitation of map4k4 from these cotransfected cells demonstrated that map4k4 co - immunoprecipitated with wild - type pyk2 and was phosphorylated on tyrosine as indicated by positive py20 staining . in contrast , immunoprecipitation of pyk2 from cells cotransfected with a kinase - deficient pyk2 and map4k4 demonstrated that only minimal py20 staining was evident on the pyk2kd variant verifying the loss of pyk2 catalytic activity . no py20 staining was observed on map4k4 immunoprecipitated from cells cotransfected with map4k4 and the kinase - deficient pyk2 . notably , the loss of pyk2 catalytic activity significantly reduced the amount of pyk2 that coimmunoprecipitated with map4k4 suggesting that increased pyk2 activity improved the association of map4k4 with pyk2 . next , we examined the effect of map4k4 activity on the interaction with pyk2 ( figure 4(b ) ) . cells cotransfected with wild - type pyk2 and either wild - type map4k4 or a kinase - deficient map4k4 variant ( k54a ) were lysed and immunoprecipitated with an anti - map4k4 antibody . analysis of the map4k4 immunoprecipitates indicated that equivalent amounts of pyk2 co - precipitated with the wild - type and kinase - deficient map4k4 indicating that the catalytic activity of map4k4 did not appear to be required for its interaction with pyk2 . to determine the effect of map4k4 expression on glioma cell migration two different shrnas , 1m4k4i and 2m4k4i , were tested for their capacity to knockdown map4k4 expression ( figure 5(a ) ) . transfection of sf767 cells with 1m4k4i produced a small reduction of map4k4 expression while transfection with 2m4k4i shrna achieved a greater amount of knockdown . the 2m4k4i oligonucleotide duplex was assembled into a lentiviral transfer vector for stable transduction of sf767 glioma cells . transduced sf767 cells were enriched by mass sorting , and immunoblotting of the positively transduced cell population indicated significant reduction of endogenous map4k4 expression ( figure 5(b ) ) . the effect of knockdown of map4k4 on the cell migration was examined by radial migration assay ( figure 5(c ) ) . the observed reduction of migration in the map4k4 knockdown cells was not due to a reduction of proliferation as there was not a significant difference in the proliferation of map4k4 knockdown cells relative to sf767 control cells over the 24-hour time course of the migration assay as determined by mtt assay ( data not shown ) . knockdown of map4k4 expression also significantly inhibited glioma cell invasion in a transwell assay ( figure 5(d ) ) . we have previously shown that increased expression of pyk2 stimulated glioma cell migration [ 9 , 34 ] . to determine the effect of knockdown of map4k4 on pyk2 stimulated glioma cell migration , sf767 cells with stable knockdown of map4k4 were transfected with flag - epitope - tagged pyk2 . immunoblotting of cell lysates indicated that knockdown of map4k4 did not alter the expression of pyk2 . notably , increased expression of pyk2 in the map4k4 knockdown cells knockdown was unable to circumvent the inhibition of migration imposed by map4k4 knockdown . while increased expression of pyk2 stimulated glioma cell migration in a dose - dependent manner , knockdown of pyk2 expression by shrna significantly inhibited glioma cell migration in vitro , invasion ex vivo , and increased survival of intracranial xenografts in vivo [ 9 , 10 ] . to further examine the relationship between map4k4 and pyk2 stimulated glioma cell migration , control sf767 glioma cells or sf767 glioma cells stably transduced with a shrna targeting pyk2 were transfected with map4k4 , and the effect on cell migration was assayed with a radial migration assay ( figure 6 ) . increased expression of map4k4 in sf767 cells stimulated glioma cell migration relative to control sf767 cells . notably , increased expression of map4k4 in the pyk2 knockdown cells did not rescue glioma cell migration . in previous studies , we have described a role for the pyk2 ferm in the regulation of pyk2 function . pyk2 stimulated glioma cell migration in vitro and increased survival in an intracranial xenograft model . substitution of residues that map to a shallow groove on the surface of the f3 subdomain of the pyk2 ferm or targeting this surface with a monoclonal antibody similarly inhibited pyk2 phosphorylation and function . these data suggest that pyk2 ferm domain mediated interactions are important for pyk2 function in glioma cell migration . in the present study , we sought to identify novel protein - protein interactions mediated by the pyk2 ferm domain . the major findings of this report are as follows : ( 1 ) the c - terminal cnh domain of the ste20 homolog map4k4 was identified as a binding partner for the pyk2 ferm domain , ( 2 ) the map4k4 cnh domain and full length map4k4 coimmunoprecipitated with pyk2 from cell lysates , ( 3 ) knockdown of map4k4 expression inhibited glioma cell migration and blocked pyk2 stimulation of migration , ( 4 ) increased expression of map4k4 stimulated glioma cell migration , ( 5 ) map4k4 stimulation of glioma cell migration was blocked by knockdown of pyk2 expression . together these data suggest a role for the integration of map4k4 and pyk2 in glioma cell migration . increased intracellular expression of pyk2 although the exact mechanism for this increased phosphorylation remains to be defined , we have demonstrated that increased expression of pyk2 results in the formation of pyk2 oligomers that facilitate transphosphorylation of pyk2 and the recruitment of src . it has been reported that increased intracellular ca induces the formation of ca / calmodulin dependent pyk2 homodimers resulting in increased pyk2 phosphorylation . mutations within a putative ca / calmodulin binding site in f2 subdomain of the pyk2 ferm significantly reduced the formation of pyk2 homodimers and pyk2 phosphorylation . consistent with our previous results , it was also demonstrated that expression of an autonomous ferm domain inhibited pyk2 phosphorylation through the formation of a heterodimer between the ferm domain and full length pyk2 blocking the formation of pyk2 homodimers . these data demonstrate the mechanistic importance of ca binding to the ferm domain to the activation of pyk2 . we have demonstrated that mutations within the f3 subdomain of the pyk2 ferm domain result in the loss of pyk2 phosphorylation . notably , the effect of these f3 mutations is independent of the capacity of pyk2 to form oligomers suggesting that in addition to its requirement for binding ca / calmodulin , the ferm domain might mediate protein - protein interactions that are required for protein re - arrangement and efficient phosphorylation . the ferm domain is well appreciated as a protein - protein interaction domain , and the pyk2 ferm domain has been reported to interact with several proteins including nir1 , fip200 , and the tumor suppressor p53 [ 4345 ] . the relationship of the interaction of pyk2 with these proteins to the regulation of pyk2 kinase activity or in its larger role as a protein scaffold remains an area of active investigation . that the pyk2 ferm domain is involved in the regulation of pyk2 activity and could act as a scaffold for functionally important interactions led us to look for specific protein interactions mediated by the ferm domain . utilizing a yeast two - hybrid selection assay and the pyk2 ferm domain as the bait , we identified three overlapping clones that corresponded to the c - terminal cnh domain of map4k4 . interestingly , these clones did not interact significantly with the ferm domain from the related fak kinase in the yeast interaction assay nor did they co - immunoprecipitate with fak from cell lysates . in contrast , the cnh domain containing map4k4(143 ) clone coimmunoprecipitated with the pyk2 ferm domain as well as full length pyk2 from cell lysates . the cnh domain was first described in citron rho - interacting kinase where it mediates an interaction with rho gtpases . notably , this domain has been reported to mediate the interaction of murine map4k4 with mekk1 and the cytoplasmic domain of integrin 1 linking map4k4 to cytoskeletal organization and cell migration [ 41 , 47 ] . map4k4 is expressed at low levels in normal tissues but highly expressed in brain [ 41 , 48 ] . map4k4 is overexpressed in many tumor cell lines , has been shown to modulate cellular transformation , invasion , and adhesion , and is highly expressed in gbm tumor samples . map4k4 was identified as a promigratory kinase in a genome wide sirna screen for modulators of tumor cell motility and is part of a five - gene signature that is a positive predictor of metastasis and negative survival in colorectal cancer . knockout studies in drosophila , c. elegans , and mice demonstrated that map4k4 is essential for proper cell migration in embryonic development [ 47 , 50 , 51 ] . interestingly , the phenotype of the map4k4 mouse shares significant similarities to the fibronectin and integrin 5 knockout mice suggesting a potential role for map4k4 in adhesion receptor signaling pathways consistent with its interaction with 1 integrin . notably , map4k4 expression is upregulated by egfrviii expression in glioma cells , and it is known to interact with the ferm domain of classical erm proteins and phosphorylates them to promote f - actin anchoring and stabilization of lamellipodia in response to egf and pdgf . map4k4 also binds to the sh3 domain of the adapter protein nck promoting actin assembly and membrane protrusion [ 54 , 55 ] . we confirmed the interaction of map4k4 with pyk2 by co - immunoprecipitation suggesting that map4k4 may play a role in pyk2 stimulated migration of glioma cells . association of map4k4 with pyk2 altered the phosphorylation of map4k4 as map4k4 that coimmunoprecipitated with wild - type pyk2 was tyrosine phosphorylated . in contrast , phosphorylation of map4k4 was not observed when map4k4 was immunoprecipitated from cells cotransfected with a kinase deficient pyk2 . the functional significance of this phosphorylation is unknown and an area of current investigation . knockdown of map4k4 significantly inhibited glioma cell migration and effectively blocked pyk2 stimulated glioma migration suggesting that map4k4 may integrate with pyk2 signaling . increased expression of map4k4 stimulated glioma cell migration consistent with the previous observation that increased expression of map4k4 in rat intestinal epithelial cells reduced cell spreading and adhesion and increased cell invasion through matrigel . interestingly , increased expression of map4k4 was unable to rescue the inhibition of glioma cell migration following pyk2 knockdown suggesting that an interaction between pyk2 and map4k4 is integrated with the stimulation of glioma cell migration . alternatively , the interaction of pyk2 and map4k4 may take place within the context of a larger protein scaffold of signaling effectors involved in cell migration . in this regard , it has been reported that the small gtp - binding protein rap2 interacts with map4k4 and enhanced map4k4 mediated jnk activation . it has also been reported that rap activation downstream of integrin engagement induced pyk2 phosphorylation . thus , pyk2 could function as a component of a protein scaffold to colocalize rap and map4k4 and promote translation of receptor mediated adhesive interaction into changes in actin polymerization and migration . future studies will seek to identify the signaling pathways associated with this interaction and to substantiate the requirement for this interaction in glioma cell invasion in orthotopic mouse models . the interaction of map4k4 with pyk2 appears to be part of a signaling pathway associated with glioma cell migration . increased expression of map4k4 stimulated glioma cell migration that was blocked by knockdown of pyk2 expression . conversely , knockdown of map4k4 expression significantly inhibited glioma cell migration that could not be rescued by increased pyk2 expression . therefore , the current results suggest that inhibition of the interaction of map4k4 and pyk2 may represent a potential therapeutic strategy to limit glioblastoma tumor dispersion .

rapid rise of noncommunicable diseases has been considered as a major health challenge in the present century , which threatens social and economic development of communities and people health . such diseases impose half of the burden of diseases cost in the world [ 24 ] . following the present trend , it is predicted that patients will be responsible for over 70 percent of diseases by 2020 [ 1 , 4 , 5 ] . in this regard , hypertension has been considered as the first and the most common risk factor to cardiovascular diseases , stroke , and renal diseases ; it has been known as a main modulated disability cause around the world [ 6 , 7 ] so that hypertension caused at least 45 percent of mortality due to cardiovascular problems and 51 percent of stroke mortality rate . among 17 million deaths due to cardiovascular problems in the world , 9.4 million occur as a consequence of complications of hypertension . in this regard , in eastern mediterranean region where iran is located , 26 percent of mortality results from hypertension . on the other hand the number of patients with hypertension has increased from 600 million cases in 1980 to 1 billion in 2008 ; over 40 percent of adults were known to have hypertension . also , it was suggested that , by 2025 , 1.54 billion adults will suffer from hypertension . in this regard , it was reported to be 14 to 34 percent in iran [ 1318 ] and various studies in other countries reported high rates of hypertension from 18 to 72 percent among males and females [ 1927 ] . it is essential to control hypertension to minimize the side effects of hypertension . rates reported for hypertension control were disappointing , which were suggested to be 13 to 56 percent around the world [ 23 , 26 , 29 , 30 ] . an important component to control hypertension is knowledge , which is relative to lower rates of ceasing interventions , following the interventions behavior and better control on disease by patients . as a result , careful evaluation of hypertension has been considered as an inseparable part of general care of the patients . studies suggested low levels of knowledge on hypertension among patients [ 23 , 24 , 32 ] , and lack of correct information and improper understanding of hypertension did not appertain to rural sites ; it has been widely reported in urban environments and industrial countries , too [ 33 , 34 ] . in iran , studies on knowledge , treatment , and control of hypertension suggested low levels of the mentioned factors [ 15 , 16 ] . in isfahan healthy heart program , the effects of comprehensive self - care programs on improving knowledge , attitude , and treatment among patients with hypertension were investigated . the results from the program showed that factors such as knowledge , treatment pursuit , and hypertension control were 49.9 , 43.8 , and 15.8 percent , respectively . considering factors related to knowledge and hypertension management is an essential starting point to preventing high rates of cardiovascular mortality due to hypertension . however , there is little information available on knowledge , treatment , and control of hypertension in southern iran . noticing the importance of the topic , the aim of this study was to determine the factors relevant to hypertension knowledge , treatment , and control in southern iran . this cross - sectional study was conducted on a sample of men and women with hypertension in urban and rural health centres in kohgiluyeh and boyer - ahmad province in southern iran , in 2014 . patients met the inclusion criteria ( six months past the definitive diagnosis disease doctor , a record in health centres , over the age of 30 ) . to participate in the study the patients participants who could not communicate effectively with the study personnel or provide informed consent were excluded . finally , out of the 2,400 patients invited to study , 1836 ( 76.52% ) signed the consent form and voluntarily agreed to participate in the study . questionnaire included three sections that comprised the following questions : sociodemographic characteristics , hypertension - related information , and hypertension knowledge . sociodemographic data included data on age , gender , educational level ( illiterate , elementary school , middle school , high school , diploma , and university ) , monthly family income ( > 5 million rials , 5 million rials to 10 million rials , 10 million rials to 15 million rials , and < 15 million rials ) , marital status ( single , married , divorced , or widowed / widower ) , employment status which was coded in 5 categories ( employed , unemployed , farmer , retired , and housekeeper ) , physical activity , and smoking . height ( height was evaluated with an error close to 0.1 cm , using a seca portable stadiometer with the individual in the standing position ) and weight ( weight measurements were taken with participants wearing light clothing and no shoes ; weight was measured with an error close to 0.5 kg using a seca digital scale ) were recorded by a nurse . body mass index ( bmi ; calculated as weight / height ) was divided into 4 categories : below normal weight ( bmi < 18.5 ) , normal weight ( 18.5 bmi < 25.0 ) , overweight ( 25.0 bmi < 30.0 ) , and obese ( bmi 30.0 ) . hypertension - related questions included duration of hypertension , family history of htn , and treatment of htn which was defined as self - reported current use of antihypertensive medication . hg and dbp < 90 mm hg among hypertensive participants who treated their htn with drugs ; that is , control in treatment was also analyzed . hypertension was defined as systolic blood pressure ( sbp ) c140 mm hg or diastolic blood pressure ( dbp ) c90 mm hg according to the who criteria or history of previously diagnosed disease ; bp was measured in a sitting position after a minimum of 5 minutes of rest by using a standardized digital bp measuring machine ( omron digital ) . hypertension knowledge was ascertained by using hypertension knowledge level scale ( hk - ls ) . this tool was developed from the hypertension knowledge level scale ( hk - ls ) , a 22-item scale prepared by erkoc et al . . hypertension knowledge level scale ( hk - ls ) assessed respondents ' knowledge in defining hypertension , lifestyle , medical treatment , drug compliance , diet , and complication of hypertension . each item was prepared as part of a standard answer ( correct , incorrect , or do not know ) . to assess the content validity of the scale to identify whether items were or were not representative of the knowledge level of hypertension , the opinions of experts were requested via an assessment form . all items were evaluated in terms of clarity and expression by considering the expert opinions , and relevant changes ( definition ( 1 item ) , medical treatment ( 4 items ) , drug compliance ( 4 items ) , lifestyle ( 4 items ) , diet ( 2 items ) , and complications ( 5 items ) ) were made . statistical analyses were performed using spss windows version 21.0 software ( spss inc . , chicago , illinois , usa ) . the mean age of respondents was 66.26 years [ sd : 12.36 ] , ranging from 30 to 93 years . almost , 722 participants were male ( 39.3% ) and 1114 were female ( 60.7% ) . furthermore , 822 participants ( 44.8% ) were living in cities and 1014 ( 55.2% ) were living in villages . regarding education , 1149 ( 66.2% ) were illiterate , 362 ( 19.7% ) were at elementary levels , 114 ( 6.2% ) were at guidance level , 46 ( 2.5% ) were at high school level , 90 ( 4.9% ) finished high school , and 75 ( 4.1% ) had academic education . in addition , 45.5 percent reported family history of hypertension . also , 27.5 percent of the respondents reported regular physical activity ( table 1 ) . as shown in table 1 , 222 male ( 30.7% ) and 350 ( 31.4% ) participants reported self - control on their hypertension . there was no significant difference between male and female participants ( p = 0.796 ) , though it was higher among females . there was a statistically significant correlation between habitat ( p = 0.000 ) , education ( p = 0.000 ) , income ( p = 0.002 ) , family history on hypertension ( p = 0.003 ) , smoking ( p = 0.006 ) , and diagnosis time ( p = 0.045 ) . participants living in cities , with higher education and income levels , showed better control on their hypertension . the treatment rates were 75.5% and 37.7% for females and males , respectively , while there was no statistically significant correlation between male and female participants ( p = 0.383 ) . there was a statistically significant correlation between age ( p = 0.000 ) , habitat ( p = 0.019 ) , education ( p = 0.004 ) , and diagnosis time ( p = 0.045 ) . table 2 represents patient 's knowledge about hypertension . in general , 25.2 percent of participants showed high knowledge on hypertension ( definition of hypertension , treatment pursuit , lifestyle , nutrition , and hypertension side effects ) . this parameter was higher among male in comparison to females , participants with higher income in comparison to those with low incomes , among employees in comparison to other occupations , and among those with regular physical activities which was statistically meaningful . improving knowledge , treatment , and control on hypertension could decrease high rates of mortality by cardiovascular diseases . results from our study showed that over half of the participants in the study were over 60 years old . several studies reported a direct significance in the prevalence of hypertension and age , which corresponded to the results from the present study [ 14 , 3942 ] . therefore , it seems essential to plan preventive interventions to control hypertension among older people . results from the present study reported that hypertension remains mostly undiagnosed during the early years of prevalence so that over 45 percent of patients over 60 were diagnosed for hypertension less than 5 years ago , which suggest the careful consideration of screening and increasing public knowledge on hypertension and its symptoms . our results indicated treatment and control rates on hypertension to be 74.8 and 31.2 percent , which were higher in comparison to the results by esteghamati et al . ( 2007 ) in isfahan where treatment and control on hypertension were 43.8 and 15.8 percent , respectively . this increase could result from implementing family physician program and offering health care services to the patients in iran . ( 2013 ) reported that the rate of treatment and control of hypertension in high- , average- , and poor - income countries is low ; results from the studies in asia suggested similar findings [ 26 , 44 , 45 ] . low rates of control and treatment of hypertension have been considered as a major risk to increase cardiovascular diseases and stroke , which suggested the necessity of efficient implementation of proper training programs and offering essential services to health care services by doctors and health experts , while majority of patients with diagnosed hypertension showed poor control on their disease . this gap has also been reported in several other studies [ 41 , 43 , 47 ] . it seemed that the only way used to control hypertension by patients was taking prescribed medicines ; they avoided self - care behaviors such as regular physical activities , good nutrition , and weight control . results from the present study reported higher treatment and control of hypertension in females in comparison to males , which was in accordance with the results from other studies [ 14 , 43 , 47 , 48 ] . it seems that patients were more sensitive to their disease when they grew older and tried medicines to treat their disease . it corresponded to the results from other studies [ 41 , 43 , 47 ] . another finding from the present study suggested a statistically significant correlation between hypertension control and patients education . the higher their education level , the higher the control rate among patients so that patients having university degrees showed higher control on their hypertension which was in accordance with results from other studies [ 14 , 26 , 47 , 49 ] , though it was different from study by chpsne which reported lower control on hypertension among individuals with lower education . more than half of the participants reported average knowledge on hypertension , treatment pursuit , lifestyle , nutrition , and complication of hypertension . only 25.2 percent of participants showed high knowledge on the disease . in comparison with other studies , another finding from the present study considered statistically significant correlation between patient 's age and knowledge on the disease . it reported that the older the participants the less their knowledge on the issue , which was in accordance with results from other studies [ 34 , 52 ] . it was concluded that age group of 30 to 39 had the higher knowledge on hypertension in comparison to other age groups , which could result from their higher education levels relative to other groups . results from the present study in this regard suggested a meaningful relationship between education and knowledge : as the education among the patients improves , their knowledge on hypertension increases . low knowledge levels on hypertension were in relation to old age , low levels of education , and low income . lack of training materials , poor screening , and poor consistency of health team were reported in these groups . it seems necessary to create proper training materials by health experts to improve patients ' knowledge and , also , increase treatment rates and control on patients . the strength of the present study was investigating a large urban and rural population with high rates of responding . however , as over 80 percent of the participants were over 50 years old and there was not a normal coverage to all possible age groups , it was considered as a limitation to the study . considering the low rate of knowledge and control of patients on hypertension , health care providers should reinforce their activities to help to improve patients ' knowledge level , especially among elders , through focusing on identifying risk factors to hypertension , regular drug intake , good nutrition , physical activity , and changing and informing lifestyles of patients with hypertension .

the microscope is composed of two excitation arms : a video - rate laser - scanning two - photon imaging arm and a point - detection two - photon phosphorescence lifetime sensing arm ( extended data fig . 1 ) . output from a 80 mhz femtosecond laser source ( maitai , newport , mountain view , ca ) tuned to 840 or 913 nm is split into s- and p - polarizations by a polarizing beamsplitter ( pbs1 ) . the s - polarized beam passes through pbs1 and enters the imaging arm whereas the p - polarized beam is deflected 90 into the point detection arm . the power for each arm can be adjusted by rotating a half - wave plate in front of pbs1 . these polarized beams are later recombined by a second polarizing beamsplitter ( pbs2 ) immediately before entering the objective . for the point - detection arm , the beam is swept across a slit aperture ( nt38 - 560 , edmund optics ) using a galvanometric scanner ( 6220h , cambridge technology ) and imaged onto the sample to form a ~3.5 m scan line . first , it generated an excitation gate with adjustable pulse duration ( 1540 sec ) , determined by the scanning speed across the slit aperture . secondly , it served to reduce the triplet state saturation effects that may accompany stationary excitation , thus helping to prevent degradation of the point spread function in the axial dimension . after application of the excitation gate , the arrival time of individual phosphorescence photons was recorded using a custom photon counting circuit . the histogram of the photon arrival times was then analyzed to obtain the triplet state lifetime . we found that the standard deviation of the po2 measurement was typically under 4 mmhg and inversely proportional to the signal - to - noise ratio ( snr , data not shown ) . for the imaging arm , we use a similar scan engine as described previously and combine the beam with the point - detection arm using pbs2 . the beam is then focused by a 60 1.2 na water immersion , infinity corrected , near - infrared coated objective ( uplsapo 60xw , olympus america inc . ) with a working distance of 280 m . the two - photon antenna , coumarin 343 , within ptp - c343 ( extended data fig . 3 ) has a two - photon absorption cross section ( 2 ) of ~28 gm at 840 nm and a fluorescent quantum yield fl ) of ~0.90 . this means that the two - photon action cross - section ( = 2fl ) of ptp - c343 is ~25.2 gm . the phosphorescence lifetime of ptp - c343 in the absence of oxygen ( 0 ) is ~60 s with a dynamic range well suited for the physiological range of po2 . for calibration , we recorded lifetime measurements in a ph 7.4 buffered solution of ptp - c343 at 38 c in vitro over a range of po2 recorded independently by an oxygen electrode ( ox-500 , unisense a / s , extended data fig . our results match a published curve for the same batch of dye ( extended data fig . all procedures were approved by the institutional animal care and use committee ( iacuc ) at massachusetts general hospital . male wild - type c57bl/6j ( jackson laboratory ) and c57bl/6 nes - gfp mice < 6 months of age were used for all in vivo ptp - c343 experiments . in a typical experiment , anesthesia was slowly induced in mice via inhalation of a mixture of 1.352% isoflurane and o2 . to maintain anesthesia the mixture calvarial bone was exposed through a u - incision in the scalp to create a skin flap and 2% methocellulose gel was placed on the scalp for refractive index matching . the mouse then received an intravenous injection of 100180 l of 1.7 mm ptp - c343 suspended in 0.9% phosphate buffered saline ( 1x pbs , invitrogen ) and 4060 l of 10 mg / ml 70 kda rhodamineb - dextran ( life technologies corporation ) . we used relatively high amounts of ptp - c343 in order to accomplish a sufficient concentration outside of the blood vessels for interstitial measurement of po2 . the mouse was placed on a heated stage with the skull positioned under the objective as described previously . an approximately 46 mm area of the calvarium encompassing most of the parasagittal bm cavities within the left and right frontal bones was scanned in each imaging session and appropriate locations were selected for po2 measurements . in agreement with previous in vivo studies , no apparent toxicity or phototoxicity was observed using the ptp - c343 probe . after imaging , the scalp was closed with 6 - 0 sutures and the mouse allowed to wake up . for terminal experiments , the mice were either sacrificed by dislodgement of the spinal cord or by euthanasia with co2 . when analyzing vessel size as a function of distance to the endosteal surface , we noticed that vessels 2040 m and > 40 m away from the bone surface were generally larger than the 020 m zone with average diameters of 27.0 and 26.9 m , respectively ( p < 0.002 and 0.06 , respectively , fig . the region > 40 m away from the bone surface constituted a dense network of sinusoidal vessels , which were more irregular in shape compared to vessels near the bone surface ( fig . mice were conditioned on day 0 with either irradiation ( 4.5 gy or 9.5 gy ) or chemotherapy ( busulfan , 35 mg / kg ) and imaged on day 2 . for hspc transplantation , ~10 facs sorted hspcs ( lin-/c - kit+/sca1 + ) were stained with cell - tracker green ( life technologies corporation ) and injected intravenously on day 1 . for whole bm transplantation , four - month old male c57bl/6j mice ( jackson laboratory , bar harbor , me ) or male actin - dsred knock - in mice ( cg - tg(cag - dsredmstnagy / j , jackson laboratory , bar harbor , me ) were lethally irradiated with a single dose of 9.5 gy gamma irradiation from a cs137 source ( gammacel exactor , nordion , ottawa , ca ) 4 hours before rescue with 2510 whole bone marrow cells from a donor actin - gfp knock - in mouse ( c57bl/6-tg(cag - egfp)131osb / leysopj , jackson laboratory , bar harbor , me ) . on the fifth day following irradiation and transplantation , the actin - dsred recipient mice were imaged and po2 measurements acquired in the bm . c57bl/6j mice were sacrificed , femurs and tibias extracted , and the marrow prepared for analysis of cycling cells . rbcs were lysed on ice using red blood cell lysing buffer ( sigma chemical co. , st . the remaining bone marrow cell fraction was counted , fixed , permeabilized and stained with pe - conjugated ki-67 according to the manufacturer s recommendations ( bd pharmingen / biosciences , san diego , ca ) . analysis of cycling cells was performed on a sorp facsaria ii instrument ( bd biosciences , san diego , ca ) . for vascular mapping , we extracted blood from a donor c57bl/6 mouse , isolated the rbcs , labeled 710 rbcs with cfda - se ( life technologies corporation ) @ 15 m for 12 min @ 37 c ( labeling concentration ~2010 rbc / ml , in pbs without ca + 0.2% bovine serum albumin + 0.2 g / l glucose ) , and washed 2x . we then suspended the rbcs in 200 l of 400 nmqtracker 655 ( life technologies corporation ) vascular label - pbs solution or 200 l of 3 mg / ml 70 kda rhodamineb - dextran vascular label - pbs solution and intravenously injected the solution into the donor nes - gfp mice immediately prior to imaging . for in vivo anti - sca-1 staining , we intravenously injected 20 g of trustainfcx ( biolegend catalog # b164564 ) into a c57bl/6j mouse to block non - specific fc binding . two hours later , we intravenously injected ~15 g of pe - cy7 anti - sca-1 antibody ( bd biosciences catalog # 108114 ) and ~15 g of percp - cy5.5 rat igg2a isotype control antibody ( ebioscience catalog # 45 - 4321 - 80 ) . we then imaged the bm 24 hours later and found that anti - sca-1 antibody had high specificity compared to the isotype control ( data not shown ) . we then repeated the same protocol in a nes - gfp mouse . using the axial - stacks , the distance of each po2 measurement to the nearest bone surface ( endosteum ) briefly , the shortest distance to the endosteum was determined in three dimensions using the pythagorean theorem . this measurement was only performed in the x - y plane and is the diameter of the lumen of the vessel . both the distance and vessel diameter measurements were performed in imagej v. 1.47p . for display purposes , the brightness and contrast of figure 1a b , 1e , 4a b , extended data figures 4 , 6 , and 7 , and extended data video 1 were adjusted . all image analysis was performed on raw images . in figure 1a , the maximum intensity projection image displayed only includes ~23 m of depth in the blue channel . we did not include the full thickness for the blue channel because the bone shg signal covers over the majority of the image at the most shallow depths . for distance measurements to blood vessels , 3d stacks were re - sliced using fiji ( image ja v. 1.45b ) into the vertical planes ( y - z , x - z ) and digitally rotated to remove the tilt of the skull relative to the imaging plane . subsequently , for each r / g / b channel , the grayscale intensity profile along the z direction was measured and fitted into an empirical exponential decay curve , the inverse of which was then multiplied to each stack image to equalize the image intensity as a function of depth . next , the 3d stack was converted into binary images and manually verified for structural accuracy . applying the exact euclidean distance transform ( eedt ) function on the binarized red ( blood vessel ) channel stack returned the 3d distance of each non - vascular pixel to the nearest blood vessel wall as a 32-bit grayscale image stack . pixels outside the bone marrow space were excluded using binarized blue ( bone ) channel stack . a histogram of eedt distances hence described the distribution of distances from each pixel in the intra - marrow , non - vascular space to its nearest blood vessel . the signal - to - noise ratio ( snr ) of the po2 measurements was calculated using the following formula : snr = n/(n+2nbd ) , where n is the number of photon counts and nbd is the background count determined using the background value described above . nbd is the combined signal from dark counts and background from stray light with the majority arising from dark counts . all p - values were determined using a two - tailed , unequal variance student s t test except for fig . 1f and 4c where a two - tailed , paired students s t test and a one - tailed , paired student s t test were used , respectively . for cases where multiple statistical tests were performed on the same data set , the microscope is composed of two excitation arms : a video - rate laser - scanning two - photon imaging arm and a point - detection two - photon phosphorescence lifetime sensing arm ( extended data fig . 1 ) . output from a 80 mhz femtosecond laser source ( maitai , newport , mountain view , ca ) tuned to 840 or 913 nm is split into s- and p - polarizations by a polarizing beamsplitter ( pbs1 ) . the s - polarized beam passes through pbs1 and enters the imaging arm whereas the p - polarized beam is deflected 90 into the point detection arm . the power for each arm can be adjusted by rotating a half - wave plate in front of pbs1 . these polarized beams are later recombined by a second polarizing beamsplitter ( pbs2 ) immediately before entering the objective . for the point - detection arm , the beam is swept across a slit aperture ( nt38 - 560 , edmund optics ) using a galvanometric scanner ( 6220h , cambridge technology ) and imaged onto the sample to form a ~3.5 m scan line . first , it generated an excitation gate with adjustable pulse duration ( 1540 sec ) , determined by the scanning speed across the slit aperture . secondly , it served to reduce the triplet state saturation effects that may accompany stationary excitation , thus helping to prevent degradation of the point spread function in the axial dimension . after application of the excitation gate , the arrival time of individual phosphorescence photons was recorded using a custom photon counting circuit . the histogram of the photon arrival times was then analyzed to obtain the triplet state lifetime . we found that the standard deviation of the po2 measurement was typically under 4 mmhg and inversely proportional to the signal - to - noise ratio ( snr , data not shown ) . for the imaging arm , we use a similar scan engine as described previously and combine the beam with the point - detection arm using pbs2 . the beam is then focused by a 60 1.2 na water immersion , infinity corrected , near - infrared coated objective ( uplsapo 60xw , olympus america inc . ) with a working distance of 280 m . the two - photon antenna , coumarin 343 , within ptp - c343 ( extended data fig . 3 ) has a two - photon absorption cross section ( 2 ) of ~28 gm at 840 nm and a fluorescent quantum yield fl ) of ~0.90 . this means that the two - photon action cross - section ( = 2fl ) of ptp - c343 is ~25.2 gm . the phosphorescence lifetime of ptp - c343 in the absence of oxygen ( 0 ) is ~60 s with a dynamic range well suited for the physiological range of po2 . for calibration , we recorded lifetime measurements in a ph 7.4 buffered solution of ptp - c343 at 38 c in vitro over a range of po2 recorded independently by an oxygen electrode ( ox-500 , unisense a / s , extended data fig . 2 ) . our results match a published curve for the same batch of dye ( extended data fig . all procedures were approved by the institutional animal care and use committee ( iacuc ) at massachusetts general hospital . male wild - type c57bl/6j ( jackson laboratory ) and c57bl/6 nes - gfp mice < 6 months of age were used for all in vivo ptp - c343 experiments . in a typical experiment , anesthesia was slowly induced in mice via inhalation of a mixture of 1.352% isoflurane and o2 . to maintain anesthesia the mixture calvarial bone was exposed through a u - incision in the scalp to create a skin flap and 2% methocellulose gel was placed on the scalp for refractive index matching . the mouse then received an intravenous injection of 100180 l of 1.7 mm ptp - c343 suspended in 0.9% phosphate buffered saline ( 1x pbs , invitrogen ) and 4060 l of 10 mg / ml 70 kda rhodamineb - dextran ( life technologies corporation ) . we used relatively high amounts of ptp - c343 in order to accomplish a sufficient concentration outside of the blood vessels for interstitial measurement of po2 . the mouse was placed on a heated stage with the skull positioned under the objective as described previously . an approximately 46 mm area of the calvarium encompassing most of the parasagittal bm cavities within the left and right frontal bones was scanned in each imaging session and appropriate locations were selected for po2 measurements . in agreement with previous in vivo studies , no apparent toxicity or phototoxicity was observed using the ptp - c343 probe . after imaging , the scalp was closed with 6 - 0 sutures and the mouse allowed to wake up . for terminal experiments , the mice were either sacrificed by dislodgement of the spinal cord or by euthanasia with co2 . when analyzing vessel size as a function of distance to the endosteal surface , we noticed that vessels 2040 m and > 40 m away from the bone surface were generally larger than the 020 m zone with average diameters of 27.0 and 26.9 m , respectively ( p < 0.002 and 0.06 , respectively , fig . the region > 40 m away from the bone surface constituted a dense network of sinusoidal vessels , which were more irregular in shape compared to vessels near the bone surface ( fig . mice were conditioned on day 0 with either irradiation ( 4.5 gy or 9.5 gy ) or chemotherapy ( busulfan , 35 mg / kg ) and imaged on day 2 . for hspc transplantation , ~10 facs sorted hspcs ( lin-/c - kit+/sca1 + ) were stained with cell - tracker green ( life technologies corporation ) and injected intravenously on day 1 . for whole bm transplantation , four - month old male c57bl/6j mice ( jackson laboratory , bar harbor , me ) or male actin - dsred knock - in mice ( cg - tg(cag - dsredmstnagy / j , jackson laboratory , bar harbor , me ) were lethally irradiated with a single dose of 9.5 gy gamma irradiation from a cs137 source ( gammacel exactor , nordion , ottawa , ca ) 4 hours before rescue with 2510 whole bone marrow cells from a donor actin - gfp knock - in mouse ( c57bl/6-tg(cag - egfp)131osb / leysopj , jackson laboratory , bar harbor , me ) . on the fifth day following irradiation and transplantation , the actin - dsred recipient mice were imaged and po2 measurements acquired in the bm . c57bl/6j mice were sacrificed , femurs and tibias extracted , and the marrow prepared for analysis of cycling cells . rbcs were lysed on ice using red blood cell lysing buffer ( sigma chemical co. , st . the remaining bone marrow cell fraction was counted , fixed , permeabilized and stained with pe - conjugated ki-67 according to the manufacturer s recommendations ( bd pharmingen / biosciences , san diego , ca ) . analysis of cycling cells was performed on a sorp facsaria ii instrument ( bd biosciences , san diego , ca ) . for vascular mapping , we extracted blood from a donor c57bl/6 mouse , isolated the rbcs , labeled 710 rbcs with cfda - se ( life technologies corporation ) @ 15 m for 12 min @ 37 c ( labeling concentration ~2010 rbc / ml , in pbs without ca + 0.2% bovine serum albumin we then suspended the rbcs in 200 l of 400 nmqtracker 655 ( life technologies corporation ) vascular label - pbs solution or 200 l of 3 mg / ml 70 kda rhodamineb - dextran vascular label - pbs solution and intravenously injected the solution into the donor nes - gfp mice immediately prior to imaging . for in vivo anti - sca-1 staining , we first demonstrated the in vivo specificity of the anti - sca-1 antibody . we intravenously injected 20 g of trustainfcx ( biolegend catalog # b164564 ) into a c57bl/6j mouse to block non - specific fc binding . two hours later , we intravenously injected ~15 g of pe - cy7 anti - sca-1 antibody ( bd biosciences catalog # 108114 ) and ~15 g of percp - cy5.5 rat igg2a isotype control antibody ( ebioscience catalog # 45 - 4321 - 80 ) . we then imaged the bm 24 hours later and found that anti - sca-1 antibody had high specificity compared to the isotype control ( data not shown ) . using the axial - stacks , the distance of each po2 measurement to the nearest bone surface ( endosteum ) was computed as described previously . briefly , the shortest distance to the endosteum was determined in three dimensions using the pythagorean theorem . this measurement was only performed in the x - y plane and is the diameter of the lumen of the vessel . both the distance and vessel diameter measurements were performed in imagej v. 1.47p . for display purposes , the brightness and contrast of figure 1a b , 1e , 4a b , extended data figures 4 , 6 , and 7 , and extended data video 1 were adjusted . all image analysis was performed on raw images . in figure 1a , the maximum intensity projection image displayed only includes ~23 m of depth in the blue channel . we did not include the full thickness for the blue channel because the bone shg signal covers over the majority of the image at the most shallow depths . for distance measurements to blood vessels , 3d stacks were re - sliced using fiji ( image ja v. 1.45b ) into the vertical planes ( y - z , x - z ) and digitally rotated to remove the tilt of the skull relative to the imaging plane . subsequently , for each r / g / b channel , the grayscale intensity profile along the z direction was measured and fitted into an empirical exponential decay curve , the inverse of which was then multiplied to each stack image to equalize the image intensity as a function of depth . next , the 3d stack was converted into binary images and manually verified for structural accuracy . applying the exact euclidean distance transform ( eedt ) function on the binarized red ( blood vessel ) channel stack returned the 3d distance of each non - vascular pixel to the nearest blood vessel wall as a 32-bit grayscale image stack . pixels outside the bone marrow space were excluded using binarized blue ( bone ) channel stack . a histogram of eedt distances hence described the distribution of distances from each pixel in the intra - marrow , non - vascular space to its nearest blood vessel . the signal - to - noise ratio ( snr ) of the po2 measurements was calculated using the following formula : snr = n/(n+2nbd ) , where n is the number of photon counts and nbd is the background count determined using the background value described above . nbd is the combined signal from dark counts and background from stray light with the majority arising from dark counts . phosphorescent measurements with a snr below 20 were excluded from this study . sample size required for the experiments all p - values were determined using a two - tailed , unequal variance student s t test except for fig . 1f and 4c where a two - tailed , paired students s t test and a one - tailed , paired student s t test were used , respectively . for cases where multiple statistical tests were performed on the same data set ,

medications have been cited as a primary or contributing cause of hyperkalemia in 35% to 75% of hospitalized patients . nonsteroidal anti - inflammatory drugs ( nsaids ) may be associated with more than 10% of these cases . cox-2 inhibitors appear to have similar effects to nsaids via the inhibition of renal prostaglandin synthesis . the susceptibility to drug - induced hyperkalemia is higher in patients with impaired renal function , diabetes mellitus and states of impaired potassium homeostasis such as hypoaldosteronism . a 75-year - old male patient presented to the emergency department in acute respiratory distress . the patient had a history of diabetes , hypertension and hyperthyroidism lasting for several years . his blood pressure , blood sugar ( including hb1ac ) and thyroid status were well controlled with medicines ( table 1 ) . for the last 5 years the light salt diet is primarily composed of potassium chloride salt ( approximate consumption 3.5 g / day ) . he had not been on any herbal medications , potassium supplements , potassium - sparing diuretics or over - the - counter analgesics . he had documented evidence of diabetic neuropathy and microalbuminurea ( normal creatinine clearance by the cockroft - gault formula ) , which had not been deteriorating up to his last routine examination 1 month prior to admission ( table 2 ) . an electrocardiogram done 15 days prior to this emergency admission did not show any changes suggestive of hyperkalemia such as tall t waves , increased pr interval or broad qrs complex . table 1patient medicationsmedicationsdoseroutefrequencynifedipine20 mgorallytwice a daytelmisartan / hydrochlorthiazide40 mgoralonce a daytorsemide10 mgoralonce a dayatorvastatin10 mgoralonce a dayecospirin75 mgoralonce a daylevothyroxine50 goralonce a dayinsulin mixtard ( 30:70)34u/24 usubcutaneoustable 2biochemical parameters-3 m-1 md1d2d3d4d5serum urea ( 1225 mg / dl)38426278635245serum creatinine(0.51.2 mg / dl)1.31.21.82.31.91.51.2serum sodium ( 136145 meq / dl)-132103109117123130serum potassium ( 3.55.3 meq / dl)-3.87.74.73.63.83.5 - 3 m : 3 months prior to admission , -1 m : 1 month prior to admission , d : day biochemical parameters -3 m : 3 months prior to admission , -1 m : 1 month prior to admission , d : day the patient was treated 5 days prior to admission for low backache of musculoskeletal origin by his regular physician who prescribed etoricoxib 90 mg / day for 3 days . he had complaints of nausea , weakness , lassitude , swelling of the legs and dyspnea for the last 72 h with gradually increasing respiratory distress . on presentation our patient was afebrile , with a pulse rate of 60/min , blood pressure 180/90 mmhg , respiratory rate 30 per minute and oxygen saturation of 82% on room air . examination showed a moist tongue , bilateral pitting pedal edema , raised jugular venous pressure and crepitations at the lung bases bilaterally . oxygen inhalation and 40 mg intravenous furosemide were given , and the patient was shifted to the intensive care unit promptly as his restlessness increased . the electrocardiogram done here showed a heart rate of 38/min with a near sine wave pattern that was broad qrs complex , with absent p waves and tall tented t waves in lead ii ( fig . 1 ) . 1the initial 12-lead ecg ( 25 mm / s , 5 mm / mv ) obtained on presentation to the emergency department demonstrates a sinus bradycardia with prolonged atrial conduction ( flattened p waves ) and an intraventricular conduction delay ( qrs , 154 ms ) the initial 12-lead ecg ( 25 mm / s , 5 mm / mv ) obtained on presentation to the emergency department demonstrates a sinus bradycardia with prolonged atrial conduction ( flattened p waves ) and an intraventricular conduction delay ( qrs , 154 ms ) aggressive medical management of hyperkalemia ( revealed in the blood gas electrolyte study ) with 10 ml of 10% calcium gluconate , hourly nebulization with salbutamol and glucose with insulin infusion administered intravenously ( 25% dextrose , 100 ml , with 10 units of regular insulin ) reverted the electrocardiogram changes ( fig . 2 ) and decreased the serum potassium level after 8 h to 6.5 meq / dl . parenteral furesemide ( 40 mg twice a day ) , tramadol ( an opioid analgesic ) for pain relief and the addition of salt to the diet were instituted . respiratory status improved , and pedal edema resolved within 24 h. biochemical parameters ( table 2 ) revealed life - threatening hyperkalemia , deranged renal function and hyponatremia . complete blood count with a differential , serum - free t3 , t4 , tsh and lipid profile was within normal limits . urinalysis on admission showed a ph of 5.0 , specific gravity 1.005 , 2 + protein , 2 + glucose , negative ketones and no microbiological growth on culture . 2lead ii of the ecg done after an hour of initiation of medical management showing improvement of heart rate and reappearance of the p waves lead ii of the ecg done after an hour of initiation of medical management showing improvement of heart rate and reappearance of the p waves echocardiogram showed a left ventricular hypertrophy with a good left ventricular systolic function . there was a return of serum potassium to normal levels within 24 h. serum sodium gradually improved over 6 days to 130 meq / dl . serum urea decreased to pre - admission values by day 5 when he was discharged . the case was discussed with his primary physician , and with the consent of the patient , telmisartan and a low salt supplement were carefully restarted one at a time . on follow - up at 2 months , he had restarted all his previous medications including telmisartan , and his electrolyte and renal parameters were at his usual baseline levels . the nsaid etoricoxib is a selective inhibitor of cyclo - oxygenase 2 ( cox-2 ) approved for treatment of patients with chronic arthropathies and musculoskeletal and dental pain . like parecoxib , celecoxib and other selective cox-2 inhibitors , it has demonstrated a significant reduction in gastrointestinal toxicity , although its renal adverse effects appear to be similar to those of other nsaids . the cardiovascular adverse effects of the selective cox-2 inhibitors like myocardial infarction , hypertension , fluid retention and congestive heart failure , among others , have led to a revoking of merck s vioxx ( rofecoxib ) and the unavailability of arcoxia ( etoricoxib ) in the usa [ 6 , 7 ] . they are preferred over other nsaids mainly due to the favorable gastrointestinal profile and convenience of single - day dosing . specific cox-2 inhibition may induce renal ischemia , electrolyte imbalance and increased blood pressure , ultimately leading to fluid and sodium retention and decreased gfr . in elderly patients on a sodium - depleted diet , a drop in gfr may be significant after a single dose of cox-2 inhibitor . in our case , acute renal insult and inhibition of prostaglandin i2 ( which increases potassium secretion ) as a result of cox-2 inhibition is the proposed mechanism precipitating hyperkalemia in our patient who was already on low salt supplements rich in potassium and telmisartan , which causes hyperkalemia by inducing a state of hypoaldosteronism . the time course of events , absence of other precipitating causes of renal dysfunction or hyperkalemia , rapid improvement on stopping the drug with no recurrence on reintroduction of the other medications and diet make etoricoxib the most probable cause ( naranjo score 7 ) . similar to non - selective nsaids , selective cox-2 inhibition may cause edema , hypertension and acute renal failure in a minority of patients . the number and spectrum of these side effects have been greater with rofecoxib than with the other cox-2 inhibitors , presumably due to the non - class effects of these drugs as their chemical structures are different . in 14 cases of cox-2 inhibitor ( celecoxib and rofecoxib ) associated acute renal failure reported by perazella et al . , all the patients had several risk factors for nsaid - induced nephrotoxicity , including renal compromise and multiple medications . acute renal failure , disturbances in volume status ( heart failure , edema ) , acidosis and hyponatremia were common . twelve patients developed hyperkalemia ( > 5.1 meq / dl ) and four required hemodialysis . similar to our case , treatment before the development of clinically recognized renal failure ranged from 4 days to 3 weeks , and resolution of the renal dysfunction took 2 days to 3 weeks after discontinuation of drugs and supportive therapy . more specifically for etoricoxib , recent data from the large medal ( multinational etoricoxib vs. diclofenac arthritis long term ) program , which enrolled 34,000 arthritis patients over 18 months , show clinically important increases in renal function endpoints . unlike in healthy people , patients with edematous conditions requiring dietary sodium restriction and diuretics , such as those with chronic renal insufficiency , congestive heart failure , chronic liver disease and hypertension , should be treated cautiously with cox-2 inhibitors . electrolytes and renal function should be checked 12 weeks after initiation of cox-2 inhibitors in these patients to screen for hyperkalemia and renal insufficiency [ 14 , 15 ] . hyperkalemia/acute renal failure/cox-2 inhibitors/adverse drug reactions and etoricoxib did not yield any results , making our case possibly the first such in the literature . even a short duration of treatment with the new cox-2 inhibitor etoricoxib may have the potential to precipitate renal failure and life - threatening hyperkalemia when administered to selected patients . the importance of considering a patient s existing diet , medications and systemic status before writing a new prescription that may interfere with the potassium physiology can not be overstressed . written informed consent was obtained from the patient ( or the patient s relatives ) for publication of this case report .

respiratory syncytial virus ( rsv ) is the leading viral pathogen responsible for bronchiolitis and pneumonia in infants during the first year of life in the united states . the risk of serious rsv illness is highest among those with prematurity , bronchopulmonary dysplasia ( bpd ) , chronic lung disease ( cld ) , congenital heart disease ( chd ) , congenital abnormalities of the airway or neuromuscular disease , and certain immunodeficiencies . eighty to ninety percent of infant hospitalizations are rsv - related and the majority of these occur in children younger than 6 months of age . the prevention of this infection is available with intramuscular humanized monoclonal antibody ( palivizumab ) . palivizumab has been found to be safe in doses of 15 mg / kg administered by intramuscular injection every 2830 days during the rsv season . in 1998 , the results of a multicenter , multinational , phase iii trial ( impact - rsv ) to evaluate the safety and effectiveness of monthly administration of palivizumab as prophylaxis for serious rsv illness in high - risk infants was published . palivizumab reduced the incidence of hospitalization due to rsv compared to placebo by 55 % [ 2 , 3 ] . children randomized to palivizumab had fewer days of rsv hospitalization and fewer days of supplemental oxygen therapy . the authors reported that intramuscular injections were well tolerated and there were no toxicities associated . in puerto rico , rsv infections are seen throughout the year with a peak season starting in july and ending in march . in 2009 , the american academy of pediatrics ( aap ) published new guidelines for rsv prophylaxis recommending fewer doses for premature infants born at 3235 weeks of gestation in all geographical areas . recommendations for initiation and termination of prophylaxis were modified to reflect current descriptions from the centers for disease control and prevention ( cdc ) of rsv seasonality in different geographic locations within the united states . regardless of the month in which the first dose is administered , the recommendation for a maximum number of 5 doses for all geographic locations is emphasized for infants with hemodynamically significant chd , cld , or birth before 32 weeks 0 days gestation . a maximum number of three doses were recommended for infants with a gestational age of 32 weeks 0 days to 34 weeks 6 days without hemodynamically significant chd or cld who qualify for prophylaxis . due to the year round prevalence of rsv in puerto rico infants in the island after the new aap statement was released , the puerto rico health department made the resolution to follow their recommendations , and decreased the number of doses given . we studied the data of all the infants who received rsv prophylaxis during the 20092010 rsv season in puerto rico , distributed by one specific specialty pharmacy with the objectives of assessing if infants at risk of rsv infection were receiving palivizumab as recommended by the 2009 aap guidelines and evaluating the reasons for noncompliance . the data was collected from the special care pharmacy and compounding services database for patients who were eligible to receive rsv prophylaxis during the 20092010 rsv season in puerto rico ( from july 2009 to march 2010 ) . special care pharmacy was the main distributor of palivizumab in puerto rico providing services to approximately 90 % of the infants referred for rsv prophylaxis . they collected data prospectively during the season which was made available to us without identifiers . data included demographics , eligibility for palivizumab , medical insurance approval , number of doses received and interval between doses . subjects from all over the island and from every medical insurance , private and government were included . the statistical analysis of collected data was performed by using frequency , means , median , and ranges . the study was approved by the university of puerto rico medical sciences campus institutional review board . the data of 868 infants ( 47 % females , 53 % males ) who qualified to receive rsv prophylaxis during the 20092010 rsv season were evaluated . the mean gestational age of the infants was 33 weeks ( range 2341 ) , and the mean birth weight was 1,767 g ( range 5094,120 ) . figure 1 shows the distribution of subjects by risk group who were eligible to receive palivizumab prophylaxis . most of them ( 55 % ) were premature infants born at 32 1/735 weeks of gestation followed by those born at 2932 weeks of gestation . infants were equally distributed with regard to government ( public ) medical insurance ( 50 % ) and private insurances ( 50 % ) .fig . 1distribution of risk groups eligible to receive palivizumab in the studied population ( n = 868 ) distribution of risk groups eligible to receive palivizumab in the studied population ( n = 868 ) seventy - four percent of the infants ( n = 640 ) referred to the specialty pharmacy received at least one dose of palivizumab prophylaxis in the studied season . the median number of doses administered was 2 ( range 19 ) , and the median interval between the doses was 32 days ( range 23123 ) . the total number of palivizumab doses received by all infants was 1,163 and 69 % of the doses ( n = 806 ) were administered in intervals not > 35 days . six percent of the doses ( n = 68 ) were administered in intervals of < 28 days . of those patients that qualified to receive a maximum of five doses , infants born at < 32 weeks of gestation and those with chd received a median of three doses . infants with cld received a median of 4 doses . of those patients that qualified to receive a maximum of three doses , infants born at 32 1/735 weeks of gestation received a median of two doses . the difference in maximum doses between these groups was significant ( p < 0.01 ) . the maximum number of five doses was received by 27 % of infants born at < 32 weeks of gestation , 31 % of infants with chd , and 47 % of those with cld . the maximum number of three doses was received by 36 % of infants born at 32 1/735 weeks of gestation . only 16 % of the patients received their first dose before discharge from the hospital . the reasons for noncompliance with the recommended regimen of rsv prophylaxis were non - approval by the medical insurance ( 53 % ) followed by unavailability of the parents ( 31 % ) , sick infant ( 11 % ) , not being able to afford co - pay ( 3 % ) , lack of transportation ( 1 % ) , and no interest in the prophylaxis ( 1 % ) . after the implementation of the 2009 aap guidelines , infants born at 32 1/735 weeks of gestation were more likely to be denied prophylaxis ( p < 0.01 ) . rsv prophylaxis was not approved by medical insurance in 9 % of the infants born at < 29 weeks of gestation , 12 % of the infants 2932 weeks of gestation , 42 % of the infants 32 1/735 weeks of gestation , 28 % of the infants with chd , and 10 % of the infants with cld . infants with government medical insurance were more likely to be denied prophylaxis ( 36 % denials vs. 24 % denials , p < 0.01 ) and to receive fewer doses ( two doses vs. three doses , p < 0.01).fig . 2palivizumab coverage denial by eligible risk group and medical insurance palivizumab coverage denial by eligible risk group and medical insurance palivizumab prophylaxis has been shown to reduce the incidence of rsv hospitalization in at - risk infants [ 3 , 5 ] , but effective prophylaxis requires full compliance with the monthly dosing schedule . noncompliance is common and may be the most controllable barrier to pharmacologic prevention of rsv infection and its potentially severe sequelae in high - risk infants . studies analyzing compliance rates of palivizumab , defined in various ways across the studies , have shown rates as low as 25 % to as high as 100 % [ 810 ] . the compliance rates of patients in clinical studies demonstrating reduced hospitalization rates in high risk infants involved in palivizumab licensure were 92 and 93 % [ 3 , 11 ] . in our study 74 % of the infants received at least one dose of palivizumab , and < 50 % received the recommended maximum number of doses , much less than in studies that demonstrated efficacy of palivizumab . berger and colleagues assessed palivizumab use in 10,390 infants based on dispensing records for a pharmacy benefits management company that provided follow - up telephone contact to ensure the prescribed dose was administered . a total of 9,675 ( 93 % ) of 10,390 infants were found to be compliant , defined as having no more than 35 days between shipment of doses . rsv hospitalization rates were 1.4 % in the compliant group versus 3.1 % in the noncompliant group ( or 2.2 , 95 % ci 1.43.5 , p < 0.001 ) . frogel and coworkers also examined compliance and rsv hospitalization rates , suggesting that improved compliance was associated with a reduced risk of rsv hospitalization , consistent with the clinical efficacy of palivizumab . these studies show that despite the progress that has been made in reducing rsv hospitalization rates , infants whose parents are noncompliant with palivizumab continue to have higher hospitalization rates . however , due to the nature of the study design no clinical outcome data of the patients were available and noncompliance can not be correlated with worse outcomes . a systematic review of compliance with palivizumab administration showed that the most common barriers that influence or predict noncompliance with the recommended regimen of rsv prophylaxis were parental smoking , medicaid enrollment , lower parental expectations for the benefits of rsv prophylaxis , lack of transportation , and language difficulties . our study showed similar results with respect to medical insurance where infants with government medical insurance were more likely to be denied prophylaxis and to receive fewer doses . this finding needs attention since palivizumab , when dosed consistent with food and drug administration ( fda ) approved labeling has been shown to be cost - effective among infants enrolled in medicaid . the support services provided by specialty pharmacies are now widely used to streamline the drug distribution , delivery , and management process in ways that engage patients in their care . in a recently published article the patients who were eligible to receive palivizumab prophylaxis had a 18 % higher likelihood of receiving the 2009 aap recommended doses when they were administered through a specialty pharmacy rather than a traditional pharmacy . the analysis found that 83 % of infants who obtained their palivizumab from a specialty pharmacy received the recommended doses , compared to 66 % who received their medication from a non - specialty pharmacy , thus emphasizing how specialty pharmacies can improve patient compliance and treatment course completion . in puerto rico , specialty pharmacies have distributed , managed insurance approvals , and delivered palivizumab since rsv prophylaxis started in 1999 , but compliance remains poor . the reasons for this are unclear and should be investigated . a procedure involving extensive counseling of parents , reminder telephone calls on the day prior to the appointment , calendars with reminder stickers , and tracking charts in the nursing medication rooms was used to improve compliance in a hospital - based clinic . results showed that an increased percentage of infants ( 71 % ) received the appropriate number of injections after implementation of the new interventions compared with 25 % before the new interventions . some authors have reported that a home care strategy for administration of palivizumab was associated with better compliance with therapy by offering consistent delivery of medication and ongoing parent / caregiver education . based on these studies , a home - based program to administer rsv immunoprophylaxis was proposed as a key component in ensuring compliance during the rsv season . most barriers that affect compliance could be overcome , providing greater opportunity to educate parents or caregivers on the risks of severe rsv disease and the benefits of prophylaxis . a home - based delivery system might offer some additional benefit of decreasing exposure of the infant to pathogens , including rsv , in the clinic or office setting . a strategy like this one may help puerto rican infants improve their compliance by eliminating the intermediary between the specialty pharmacy and the patient . at present , the specialty pharmacies deliver prophylaxis to the pediatricians office and they administer it . with this approach doses may be missed or administered at longer intervals as they depend on the parents taking the child to the office for administration . it is critically important that parents of children at high risk for severe rsv disease be empowered with clear information about the seriousness of rsv disease and the benefits of palivizumab prophylaxis to enable them to make informed choices . physicians and other health professionals are primary sources of information for patients or their caregivers , and play a key role in guiding families in their choices about rsv prophylaxis and may play an important role in improving compliance [ 6 , 18 ] . our study emphasizes the need to educate parents on the serious consequences of rsv infection in high - risk patients and the importance of administration of prophylaxis in the recommended interval during the rsv season to prevent rsv hospitalizations and their complications . physicians should be aware that many infants are not being dosed appropriately and that strategies need to be established in order to improve compliance . these strategies should include working with payors to ensure that all infants who qualify for prophylaxis are given approval for its use .

the prevalence of low back pain ( medscimonit-21 - 1934 ) ranges from 6.0% to 15.7% across the world ; it accounts for 49.6% of the global disability burden and causes an enormous economic impact [ 13 ] . it was estimated that 41.8% of lbp can be attributed to intervertebral disk degeneration ( ivdd ) . several radiographic findings , such as internal disk dehydration , end - plate sclerosis , and loss of disk height , have been identified on conventional magnetic resonance imaging ( mri ) in patients with ivdd . described a grading system to assess the morphologic changes of ivdd based on standard t2-weighted mri for research and clinical purposes . although pfirrmann grading system is useful , it is subjective and insufficient to detect early - onset ivdd ; thus , quantification of the degeneration process is needed to promote disc health evaluation . over the past decade , new quantitative mri techniques such as t2- [ 810 ] , t1- , t2 * -relaxation time , diffusion - weighted imaging , spectroscopy , and delayed gadolinium enhanced mri have been explored in hopes of revealing early biochemical changes in ivdd , elucidating the degeneration progress , and possibly helping clinicians initiate timely biologic therapies with injected growth factors , genetic material , and stem cells . in t1 sequence , pulse is applied to the magnetization transversely and t1 relaxation time is considered to be sensitive to protons on macromolecules such as glycosaminoglycan ( gag ) , providing a potential to detect the loss of proteoglycan in ivdd . in vitro studies have reported a correlation between t1 and gag content in the nucleus pulposus ( np ) . several investigators have found that t1 is correlated with pfirrmann degenerative grade and clinical symptoms assessed by health questionnaires . t2 * relaxation times provide information regarding the spatial macromolecule architecture in conjunction with water molecule mobility , which has been reported to be a reliable tool for quantitative assessment of various cartilaginous tissues [ 2426 ] . recent studies have established the relationship between t2 * value and pfirrmann scoring system , as well as macroscopic and histological grading in ivdd , where a decrease in t2 * relaxation time was significantly correlated with a higher degree of disc degeneration . animal and human cadaveric studies showed a good correlation between t2 * relaxation time and gag content throughout the discs , as well as between t2 * relaxation time and mechanical health . however , little is known about the relative performance of t1 and t2 * relaxation times in their assessment of disc degeneration . the purpose of the current in vivo 3.0-t mri study was to assess and compare t1 and t2 * relaxation time measurements in ivdd with reference to the morphological pfirrmann grading systems . this prospective study was approved by our institute s research ethics committee , and written informed consent was obtained from each participant prior to the study . from october 2013 to september 2014 , 50 consecutive subjects with single or recurrent episodes of low back pain presented initially to the outpatient spine clinic and then were referred for a lumbar mri . inclusion criteria were patients with single or recurrent episodes of low back pain , and radiologic screening and conventional mri confirmation of no other spine diseases except disc degeneration . exclusion criteria were a body mass index greater than 25 , spinal malignancy , spine fractures , spinal infection , lumbar scoliosis , sacroiliac arthritis , rheumatoid arthritis , metabolic bone disease , previous spine surgery or interventional treatment , and contraindications for mr imaging . mri was performed in the morning for all subjects to reduce the potential influence of diurnal variation in intervertebral disks . all mri examinations were performed by a 3.0 t scanner ( achieva , philips healthcare , best , the netherlands ) and a dedicated 15-channel sense spine coil . first , conventional sagittal t2-weighted images were acquired by using a turbo spin echo ( tse ) sequence with the following parameters : tr / te=2500 ms/90 ms , flip angle=90 , field of view=220 mm , nsa=2 , and scan time=1 min 40 s. next , a series of sagittal t1 and t2*quantification sequences were performed . the t1-weighted images were obtained with a 3d balanced fast field echo ( b - ffe ) sequence with the following parameters : tr / te=4.8 ms/2.4 ms , flip angle=50 , nsa=1 . five subsequent t1-weighted scans were performed with spin lock durations of 0 , 10 , 20 , 30 , and 40 ms , spin - lock frequency of 500hz , and with scan time of 1 min 9 s for each scan . the t2 * relaxation time measurements were acquired by a fast field echo ( ffe ) sequence with the following parameters : tr / tes = 310ms/5.1 ; 10.0 ; 14.9 ; 19.8 ; 24.7ms , flip angle=25 , nsa=4 , and acquisition time=2min46s . to guarantee the same in - plane and out - of - plane resolution for better comparability between the t1 and t2 * sequences , the following parameters were kept consistent : field of view=220 mm , matrix=432432 , slice thickness=5 mm , slices=9 , voxel size=0.570.575.00 mm . mr images were transferred to a workstation ( extended mr workspace , version 2.6.3.2 , philips healthcare ) . two radiologists ( with 5 and 10 years of experience in musculoskeletal radiology ) independently assessed the five lumbar discs in each subject on the mid - sagittal t2-weighted images according to the pfirrmann grading system : grade i , homogeneous shape , bright hyperintense white signal intensity , normal disc height , and clear distinction of annulus fibrosus ( af ) and np ; grade ii , nonhomogeneous shape with or without horizontal gray bands , hyperintense white signal , normal disc height , clear distinction of af and np ; grade iii , nonhomogeneous shape , intermediate gray signal intensity , normal or slightly decreased height , and unclear distinction of af and np ; grade iv , nonhomogeneous shape , hypointense dark gray signal intensity , normal to moderate decrease in height , and lost distinction of af and np ; and grade v , same as grade iv but collapsed disc space . in cases of discrepancy , a consensus reading was obtained . the t1-weighted images were fitted on a pixel by pixel basis to the exponentially decaying t1 function using idl 6.3 ( itt visual information solutions , boulder , co ) to generate a t1 relaxation map , and the t2 * -weighed image analyses were conducted by using image j software ( national institutes of health , bethesda , md ) . regions of interest ( rois ) were set manually on the t1 and t2 * mappings with reference to the t2 images . the rois were drawn on the np , anterior af ( aaf ) and posterior af ( paf ) . the roi sizes for np were 10~55 mm , while the roi sizes for af ( anterior and posterior ) were 10~45 mm . all the measurements were performed twice by a single observer with an interval of four weeks . the intraclass correlation coefficients ( icc ) with 95% confidence intervals ( 95% cis ) was used to assess the reproducibility in two measurements of t1 and t2 * relaxation times , and icc values that were 0.75 were considered excellent agreement . the differences of t1 , t2 * relaxation times between np and af were tested using the wilcoxon signed rank test , as well as between aaf and paf . kruskal - wallis test and post - hoc tests were applied to compare the difference in t1 and t2 values of various disc degeneration grades of np and af . associations between t1 , t2 * relaxation times and pfirrmann grade were tested using spearman rank correlation . correlations were considered strong for r>0.7 , moderate for 0.5 < r0.7 , and weak for r0.5 . from october 2013 to september 2014 , 50 consecutive subjects with single or recurrent episodes of low back pain presented initially to the outpatient spine clinic and then were referred for a lumbar mri . inclusion criteria were patients with single or recurrent episodes of low back pain , and radiologic screening and conventional mri confirmation of no other spine diseases except disc degeneration . exclusion criteria were a body mass index greater than 25 , spinal malignancy , spine fractures , spinal infection , lumbar scoliosis , sacroiliac arthritis , rheumatoid arthritis , metabolic bone disease , previous spine surgery or interventional treatment , and contraindications for mr imaging . mri was performed in the morning for all subjects to reduce the potential influence of diurnal variation in intervertebral disks . all mri examinations were performed by a 3.0 t scanner ( achieva , philips healthcare , best , the netherlands ) and a dedicated 15-channel sense spine coil . first , conventional sagittal t2-weighted images were acquired by using a turbo spin echo ( tse ) sequence with the following parameters : tr / te=2500 ms/90 ms , flip angle=90 , field of view=220 mm , nsa=2 , and scan time=1 min 40 s. next , a series of sagittal t1 and t2*quantification sequences were performed . the t1-weighted images were obtained with a 3d balanced fast field echo ( b - ffe ) sequence with the following parameters : tr / te=4.8 ms/2.4 ms , flip angle=50 , nsa=1 . five subsequent t1-weighted scans were performed with spin lock durations of 0 , 10 , 20 , 30 , and 40 ms , spin - lock frequency of 500hz , and with scan time of 1 min 9 s for each scan . the t2 * relaxation time measurements were acquired by a fast field echo ( ffe ) sequence with the following parameters : tr / tes = 310ms/5.1 ; 10.0 ; 14.9 ; 19.8 ; 24.7ms , flip angle=25 , nsa=4 , and acquisition time=2min46s . to guarantee the same in - plane and out - of - plane resolution for better comparability between the t1 and t2 * sequences , the following parameters were kept consistent : field of view=220 mm , matrix=432432 , slice thickness=5 mm , slices=9 , voxel size=0.570.575.00 mm . mr images were transferred to a workstation ( extended mr workspace , version 2.6.3.2 , philips healthcare ) . two radiologists ( with 5 and 10 years of experience in musculoskeletal radiology ) independently assessed the five lumbar discs in each subject on the mid - sagittal t2-weighted images according to the pfirrmann grading system : grade i , homogeneous shape , bright hyperintense white signal intensity , normal disc height , and clear distinction of annulus fibrosus ( af ) and np ; grade ii , nonhomogeneous shape with or without horizontal gray bands , hyperintense white signal , normal disc height , clear distinction of af and np ; grade iii , nonhomogeneous shape , intermediate gray signal intensity , normal or slightly decreased height , and unclear distinction of af and np ; grade iv , nonhomogeneous shape , hypointense dark gray signal intensity , normal to moderate decrease in height , and lost distinction of af and np ; and grade v , same as grade iv but collapsed disc space . in cases of discrepancy , a consensus reading was obtained . the t1-weighted images were fitted on a pixel by pixel basis to the exponentially decaying t1 function using idl 6.3 ( itt visual information solutions , boulder , co ) to generate a t1 relaxation map , and the t2 * -weighed image analyses were conducted by using image j software ( national institutes of health , bethesda , md ) . regions of interest ( rois ) were set manually on the t1 and t2 * mappings with reference to the t2 images . the rois were drawn on the np , anterior af ( aaf ) and posterior af ( paf ) . the roi sizes for np were 10~55 mm , while the roi sizes for af ( anterior and posterior ) were 10~45 mm . all the measurements were performed twice by a single observer with an interval of four weeks . the intraclass correlation coefficients ( icc ) with 95% confidence intervals ( 95% cis ) was used to assess the reproducibility in two measurements of t1 and t2 * relaxation times , and icc values that were 0.75 were considered excellent agreement . the differences of t1 , t2 * relaxation times between np and af were tested using the wilcoxon signed rank test , as well as between aaf and paf . kruskal - wallis test and post - hoc tests were applied to compare the difference in t1 and t2 values of various disc degeneration grades of np and af . associations between t1 , t2 * relaxation times and pfirrmann grade were tested using spearman rank correlation . correlations were considered strong for r>0.7 , moderate for 0.5 < r0.7 , and weak for r0.5 . all statistical analysis was performed by pasw statistics software ( v17.0 , spss inc . , of the 50 patients recruited during the study period , 8 patients were excluded for the following reasons : body mass index greater than 25 ( n=3 ) ; sacroiliac arthritis ( n=2 ) , and motion artifacts ( n=3 ) . a total of 42 subjects ( 30 men , 12 women ; rang , 21~80 years ; mean age , 44.2 years ) with 210 intervertebral lumbar disks from l12 to l5s1 were included in this study . the distribution of the t1 and t2 * values of the 210 intervertebral disks with respect to the pfirrmann grading is provided in table 1 and figure 1a , 1b . representative t1 and t2 * maps of subjects with different grades of degeneration are shown in figure 2 . in the reliability of measurements , iccs with 95% cis for each technique at different rois are shown in table 2 ; the reproducibility between the two measurements was excellent ( icc=0.909~0.969 ) . t1 relaxation time ranged from 73.8 to 235.1ms for np , and from 55.7 to 173.8 ms for af , while t2 * relaxation times ranged from 9.6 to 185.8 ms for np , and from 12.0 to 52.7 ms for af . the difference in t1 and t2 * values between np and af were highly significant ( both p<0.001 ) , and the difference in t1 and t2 * values between aaf and paf were also highly significant ( both p<0.01 ) . boxplots of t1 and t2 * values in np and af are shown in figure 3 , respectively , which showed trends of decreasing t1 and t2 * values with increasing pfirrmann grade . the kruskal - wallis test showed a significant difference in t1 and t2 * relaxation times of np and af among the pfirrmann grade groups ( p<0.01 ) . for both t1 ( figure 3a ) and t2 * relaxation times ( figure 3b ) in the nps , a significant difference was found between pfirrmann grade ii and pfirrmann grades greater than ii ( p<0.001 ) , especially between pfirrmann grade ii and iii , and the difference was not statistically significant between pfirrmann grade i and ii ( p>0.05 ) , nor between pfirrmann grade iv and v ( p>0.05 ) . for afs , the t1 ( figure 3c ) values also showed pfirrmann grade ii was significantly different from pfirrmann grades higher than ii ( p<0.001 ) , particularly notable between pfirrmann grades ii and iii ; the t2 * values ( figure 3d ) demonstrated significant difference between pfirrmann grade i and all the other grades ( p<0.05 ) . spearman correlation analysis demonstrated that pfirrmann grades were inversely significantly correlated with both t1 and t2 * values in the np ( r=0.69 , p<0.001 ( figure 4a ) ; r=0.56 , p<0.001 ( figure 4b ) ) and af(r=0.45 , p<0.001 ( figure 4c ) ; r=0.26 , p<0.001 ( figure 4d ) ) . of the 50 patients recruited during the study period , 8 patients were excluded for the following reasons : body mass index greater than 25 ( n=3 ) ; sacroiliac arthritis ( n=2 ) , and motion artifacts ( n=3 ) . a total of 42 subjects ( 30 men , 12 women ; rang , 21~80 years ; mean age , 44.2 years ) with 210 intervertebral lumbar disks from l12 to l5s1 were included in this study . the distribution of the t1 and t2 * values of the 210 intervertebral disks with respect to the pfirrmann grading is provided in table 1 and figure 1a , 1b . representative t1 and t2 * maps of subjects with different grades of degeneration are shown in figure 2 . in the reliability of measurements , iccs with 95% cis for each technique at different rois are shown in table 2 ; the reproducibility between the two measurements was excellent ( icc=0.909~0.969 ) . t1 relaxation time ranged from 73.8 to 235.1ms for np , and from 55.7 to 173.8 ms for af , while t2 * relaxation times ranged from 9.6 to 185.8 ms for np , and from 12.0 to 52.7 ms for af . the difference in t1 and t2 * values between np and af were highly significant ( both p<0.001 ) , and the difference in t1 and t2 * values between aaf and paf were also highly significant ( both p<0.01 ) . boxplots of t1 and t2 * values in np and af are shown in figure 3 , respectively , which showed trends of decreasing t1 and t2 * values with increasing pfirrmann grade . the kruskal - wallis test showed a significant difference in t1 and t2 * relaxation times of np and af among the pfirrmann grade groups ( p<0.01 ) . for both t1 ( figure 3a ) and t2 * relaxation times ( figure 3b ) in the nps , a significant difference was found between pfirrmann grade ii and pfirrmann grades greater than ii ( p<0.001 ) , especially between pfirrmann grade ii and iii , and the difference was not statistically significant between pfirrmann grade i and ii ( p>0.05 ) , nor between pfirrmann grade iv and v ( p>0.05 ) . for afs , the t1 ( figure 3c ) values also showed pfirrmann grade ii was significantly different from pfirrmann grades higher than ii ( p<0.001 ) , particularly notable between pfirrmann grades ii and iii ; the t2 * values ( figure 3d ) demonstrated significant difference between pfirrmann grade i and all the other grades ( p<0.05 ) . spearman correlation analysis demonstrated that pfirrmann grades were inversely significantly correlated with both t1 and t2 * values in the np ( r=0.69 , p<0.001 ( figure 4a ) ; r=0.56 , p<0.001 ( figure 4b ) ) and af(r=0.45 , p<0.001 ( figure 4c ) ; r=0.26 , p<0.001 ( figure 4d ) ) . in this study , significant negative correlations were observed between the t1 , t2 * relaxation times and the pfirrmann grades . moreover , both methodologies displayed roughly comparable performance . to the best of our knowledge , this is the first comparison of t1 and t2 * relaxation times at 3 t mr in patients experiencing low back pain with different grades of disc degeneration . the human intervertebral disc is composed of the central np , the peripheral af , and the endplates connecting to the vertebrae . normally , the np is gelatinous , well - hydrated , and mainly consists of proteoglycans enclosed by the type ii collagen network , which produces a high internal hydrostatic pressure that allows the disk to support compressive load in the spine . the af is characterized by concentric lamellae primarily of bundles of type i and iii collagen , which serves as the limiting capsule of the np . in a healthy young adult , proteoglycan accounts for about 50% of the np and 1020% of the af dry weight , while collagen accounts for approximately 1520% of the np and 6570% of the af dry weight . at the earliest stages of ivdd , the proteoglycan degrades and its content decreases ; thus , it is of great importance to probe this change in subjects with ivdd noninvasively . new mr techniques such as t1 and t2 * mapping provide information of biochemical composition in disc and thus allow detection of early changes in ivdd . t1 relaxation time was obtained by using spin - lock radiofrequency field , which is related to slow motional interactions between macromolecules of extracellular matrix and bulk water . biochemical analysis on cadaveric human discs revealed that the t1 relaxation time was strongly correlated with proteoglycan content and was moderately correlated with water content . our results showed a decreasing trend in t1 values of both np and af with the increase of pfirrmann grade , and the association between t1 values of np and af and pfirrmann grade was significant ( r=0.69 , p<0.001 ; r=0.45 , p<0.001 ) , which is in agreement with previous reports that t1 values of af decline significantly over disc degeneration and that t1 values of np are moderately correlated with pfirrmann grade i t2 relaxation and additional relaxation because of magnetic inhomogeneities , and is sensitive to the spatial macromolecule architecture and its interaction with water molecule mobility . recent studies have demonstrated the low - to - moderate correlation between t2 * value and pfirrmann grade , as well as t2 * value and gag content in ivdd . in the present study , t2 * relaxation times on np were significantly higher than af , and both t2 * values were negatively correlated with pfirrmann grades ( r=0.56 , p<0.001 ; r=0.26 , p<0.001 ) . welsch et al . reported an increase in t2 * values from the aaf to the np and the subsequent decline from the np to the paf , and a decrease in t2 * values on np and paf was significantly correlated with a higher pfirrmann grade ( r=0.220~0.334 , p<0.01 ) in patients experiencing low back pain , which was comparable to our results . with regard to the discernment of the single pfirrmann grade , t1 and t2 * mapping displayed roughly comparable performance . in particular , both t1 and t2 * mapping seemed to be able to differentiate between pfirrmann grade ii and iii , as well as between pfirrmann grade iii and iv . a relatively larger decline in t1 and t2 * values between pfirrmann grades ii and iii was observed , which probably was due to rapid degeneration of lumbar intervertebral discs confirmed in an animal model by zhou et al . . moreover , t2 * values of af demonstrated a significant difference between pfirrmann grade i and ii , indicting t2 * relaxation time might not only be sensitive to gag content , but also to collagen integrity . probing the early changes in the degeneration process of intervertebral discs would be beneficial for timely developing and applying new therapeutics . this deficit may be filled by t1 and t2 * relaxation time measurements . particularly in the longitudinal follow - up of patients with ivdd , t1 and t2 * values might provide a reliable quantification of longitudinal biochemical changes of degeneration . t1 and t2 * mapping in this work showed roughly comparable performance in assessing the biochemical content of the ivdd . however , t1-weighted imaging is not yet easily applicable on standard mri scanners , while t2 * mapping could be routinely available on a clinical mr scanner . additionally , the acquisition time on t2 * mapping was almost half of that on the t1 mapping ( 2 min 46 s vs. 5 min 45 s ) . hence , t2 * mapping is more promising than t1 mapping in diagnosing ivdd in routine clinical practice . on the one hand , the present results were not verified by histological and biochemical findings , partly because of inability in obtaining specimens from human subjects . on the other hand , the relation between the t1 or t2 * mapping and physical symptoms was not investigated , particularly the degree and duration of lbp , as various causes account for discogenic lbp ( such as innervations and tear of the annulus ) and the mechanism by which ivdd leads to lbp remains unclear . besides , comparison of the sex difference in t1 values at different levels showed a significant lower value in the female discs at l3l4 and l4l5 discs of the np , and at l3l4 disc of the af , but the assessments of t1 and t2 * relation times according to the anatomic level of each disc and sex were not performed in this study because the size of women was small and the emphasis of current study was on the relative performance of t1 and t2 * mapping . to draw more detailed and definitive conclusions during the process of disc degeneration , further studies including a large number of patients with multiple follow - up mri and in vitro investigations are needed . in conclusion , our results show that the t1 and t2 * values significantly and negatively correlate with the pfirrmann grades of disc degeneration , and both methodologies displayed roughly comparable performance . t1 and t2 * relaxation time measurements may promote the detection of ivdd at a relatively early stage .

parkinson s disease ( pd ) is characterized by bradykinesia , tremors , rigidity , and postural instability . a meta - analysis of global data demonstrated an increasing prevalence of pd with age,1 and a higher prevalence of pd was observed in the chinese male population.2 in addition to age and sex , other potential risk factors for pd , including a family history of pd,3 depression,4 constipation,5 and sleep disorders requiring zolpidem,6 have been reported in epidemiological studies . the prevalence of sleep disorders in pd is 40%90%.7 sleep disorders negatively affect cognitive functions and quality of life in patients with pd . insomnia , daytime sleepiness with sleep attacks , restless legs syndrome , and rapid eye movement sleep behavior disorder are the most frequent sleep disorders associated with pd.8 furthermore , snoring was observed in up to 70% of patients with pd,9 and 14.5%60% of patients with pd were estimated to have an apnea hypopnea index of > 15.10 therefore , a close association between sleep - disordered breathing and pd can be considered . sleep apnea ( sa ) , one of the most common forms of sleep - disordered breathing , is characterized by repeated periods of hypoxia and reoxygenation during sleep . sa may be mild , moderate , or severe , based on the number of apneas and hypopneas occurring per hour.11 the clinical significance of sa in pd has been discussed but remains controversial.12,13 sa - induced chronic intermittent hypoxia increases oxidative stress and inflammation,14 which are involved in pd pathophysiology.15 experimental data have demonstrated the role of oxidative stress in -synuclein aggregation and dopaminergic cell death.16 therefore , sa - induced oxidative stress and inflammation may increase the risk of pd . the present study investigated the hypothesis that patients with sa are at an increased risk of pd . a nationwide , population - based study was conducted to assess the relationship between sa and subsequent diagnosis of pd within a 3-year follow - up period . in addition , we attempted to determine whether sa is an age- and sex - dependent risk factor for pd . this retrospective cohort study was conducted using claims data from the national health insurance research database ( nhird ) , taiwan . the national health insurance ( nhi ) program , which was implemented on march 1 , 1995 , has reimbursed the health care costs of 99.9% of taiwan s population as of 2014 . the nhi program provides comprehensive health care information , including demographic data of the insured , clinical visit dates , diagnostic codes , and prescription details . under the nhi program , the national health research institutes ( nhri ) several studies using nhird data have been published in peer - reviewed journals . in the present study , data were obtained from the longitudinal health insurance database ( lhid ) 2005 , a subset of the nhird . the lhid 2005 dataset contains historical ambulatory data and inpatient care data for 1 million randomly sampled beneficiaries who enrolled in the nhi program in 2005 . currently , the lhid 2005 allows researchers to explore the medical service utilization data of these 1 million patients during 19962012 . the nhri claims that there are no statistically significant differences in the data on age , sex , geographic region , and health care costs from the lhid 2005 and all claims data . this study was approved by the institutional review board of kaohsiung medical university hospital , taiwan ( kmuhirb - exempt[ii]-20160001 ) . therefore , the review board waived the requirement of obtaining written informed consent from the study patients . we retrospectively examined the sa and matched non - sa cohorts to investigate the relationship between sa and the risk of pd . the sa cohort included patients aged 40 years and diagnosed as having sa ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] codes 780.51 , 780.53 , 780.57 , and 327.23 ) between january 1 , 1997 , and december 31 , 2005 , during inpatient or outpatient care . patients with a history of sa before january 1 , 1997 , were excluded from the present study . sa diagnosis was defined according to validated definitions from previous studies.17,18 the date of initial sa diagnosis for each study patient was considered the index date . the matched non - sa cohort comprised five non - sa patients who were matched to each patient with sa based on age , sex , and index year , and randomly selected from the remaining population in the same database . patients who were diagnosed as having sa during follow - up were excluded from the present study . furthermore , patients with a history of pd ( icd-9-cm 332 ) before enrollment were excluded from the sa and non - sa cohorts . the study patients were individually followed for 3 years from their index date to evaluate pd development . we identified several comorbidities before the index date as potential confounding factors for pd , including hypertension ( icd-9-cm 401405 ) , diabetes mellitus ( icd-9-cm 250 ) , hyperlipidemia ( icd-9-cm 272 ) , concussion and head trauma ( icd-9-cm 310.2 , 852 , and 853 ) , copd ( icd-9-cm 496 ) , and asthma ( icd-9-cm 493 ) . the outcome of interest was defined as receiving a diagnosis of pd after the index date . pd diagnosis was defined as receiving any outpatient or inpatient care for pd ( icd-9-cm 332 ) . a validation study reported that this definition had favorable diagnostic accuracy.19 to avoid the misdiagnosis of secondary parkinsonism as idiopathic pd , patients diagnosed as having carbon monoxide intoxication ( icd-9-cm 986 ) , major psychiatric diseases ( icd-9-cm 291 , 292 , 293.0 , 293.1 , 293.81 , 293.82 , and 295299 ) , and dementia ( icd-9-cm 290.11 , 290.12 , 290.20 , 290.3 , 290.41 , 290.42 , 290.8 , 290.9 , 294.11 , and 294.21 ) before the index date and during follow - up were excluded from the present study . a pearson chi - squared test was performed to evaluate the differences in the categorical variables , including urbanization level , monthly income , geographic region , and comorbidities , of the sa and non - sa cohorts . a kaplan meier survival test was performed to estimate pd - free survival rates in the sa and non - sa cohorts , and a log - rank test was used to analyze the differences between the survival curves . cox proportional hazards regression analysis was conducted to examine the crude and adjusted hazard ratios ( hrs ) for pd in the sa cohort , compared to the non - sa cohort during the 3-year follow - up period , after adjustments for sex , age , urbanization level , geographic region , monthly income , hypertension , hyperlipidemia , diabetes mellitus , asthma , copd , and head injury . furthermore , an age- and sex - stratified analysis using the cox proportional hazards regression model was conducted to evaluate the risk of pd in the sa cohort across different age groups and sex . this retrospective cohort study was conducted using claims data from the national health insurance research database ( nhird ) , taiwan . the national health insurance ( nhi ) program , which was implemented on march 1 , 1995 , has reimbursed the health care costs of 99.9% of taiwan s population as of 2014 . the nhi program provides comprehensive health care information , including demographic data of the insured , clinical visit dates , diagnostic codes , and prescription details . under the nhi program , the national health research institutes ( nhri ) several studies using nhird data have been published in peer - reviewed journals . in the present study , data were obtained from the longitudinal health insurance database ( lhid ) 2005 , a subset of the nhird . the lhid 2005 dataset contains historical ambulatory data and inpatient care data for 1 million randomly sampled beneficiaries who enrolled in the nhi program in 2005 . currently , the lhid 2005 allows researchers to explore the medical service utilization data of these 1 million patients during 19962012 . the nhri claims that there are no statistically significant differences in the data on age , sex , geographic region , and health care costs from the lhid 2005 and all claims data . this study was approved by the institutional review board of kaohsiung medical university hospital , taiwan ( kmuhirb - exempt[ii]-20160001 ) . therefore , the review board waived the requirement of obtaining written informed consent from the study patients . we retrospectively examined the sa and matched non - sa cohorts to investigate the relationship between sa and the risk of pd . the sa cohort included patients aged 40 years and diagnosed as having sa ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] codes 780.51 , 780.53 , 780.57 , and 327.23 ) between january 1 , 1997 , and december 31 , 2005 , during inpatient or outpatient care . patients with a history of sa before january 1 , 1997 , were excluded from the present study . sa diagnosis was defined according to validated definitions from previous studies.17,18 the date of initial sa diagnosis for each study patient was considered the index date . the matched non - sa cohort comprised five non - sa patients who were matched to each patient with sa based on age , sex , and index year , and randomly selected from the remaining population in the same database . patients who were diagnosed as having sa during follow - up were excluded from the present study . furthermore , patients with a history of pd ( icd-9-cm 332 ) before enrollment were excluded from the sa and non - sa cohorts . the study patients were individually followed for 3 years from their index date to evaluate pd development . we identified several comorbidities before the index date as potential confounding factors for pd , including hypertension ( icd-9-cm 401405 ) , diabetes mellitus ( icd-9-cm 250 ) , hyperlipidemia ( icd-9-cm 272 ) , concussion and head trauma ( icd-9-cm 310.2 , 852 , and 853 ) , copd ( icd-9-cm 496 ) , and asthma ( icd-9-cm 493 ) . the outcome of interest was defined as receiving a diagnosis of pd after the index date . pd diagnosis was defined as receiving any outpatient or inpatient care for pd ( icd-9-cm 332 ) . a validation study reported that this definition had favorable diagnostic accuracy.19 to avoid the misdiagnosis of secondary parkinsonism as idiopathic pd , patients diagnosed as having carbon monoxide intoxication ( icd-9-cm 986 ) , major psychiatric diseases ( icd-9-cm 291 , 292 , 293.0 , 293.1 , 293.81 , 293.82 , and 295299 ) , and dementia ( icd-9-cm 290.11 , 290.12 , 290.20 , 290.3 , 290.41 , 290.42 , 290.8 , 290.9 , 294.11 , and 294.21 ) before the index date and during follow - up were excluded from the present study . a pearson chi - squared test was performed to evaluate the differences in the categorical variables , including urbanization level , monthly income , geographic region , and comorbidities , of the sa and non - sa cohorts . a kaplan meier survival test was performed to estimate pd - free survival rates in the sa and non - sa cohorts , and a log - rank test was used to analyze the differences between the survival curves . cox proportional hazards regression analysis was conducted to examine the crude and adjusted hazard ratios ( hrs ) for pd in the sa cohort , compared to the non - sa cohort during the 3-year follow - up period , after adjustments for sex , age , urbanization level , geographic region , monthly income , hypertension , hyperlipidemia , diabetes mellitus , asthma , copd , and head injury . furthermore , an age- and sex - stratified analysis using the cox proportional hazards regression model was conducted to evaluate the risk of pd in the sa cohort across different age groups and sex . a total of 1,944 and 9,720 patients were included in the sa and non - sa cohorts , respectively . of the 11,664 patients , 42.6% , 30.2% , and 27.1% belonged to the 4049 , 5059 , and 60 years of age groups , respectively ; 63.1% of all patients were men . after adjustments for sex , age , and index year , the sa cohort was more likely to reside in urbanized communities and had a higher monthly income than the non - sa cohort . in addition , the sa cohort tended to have more comorbidities , such as hypertension , hyperlipidemia , diabetes mellitus , asthma , copd , and head injury , than the non - sa cohort . table 1 presents the demographic data and comorbidities of the sa and non - sa cohorts . of the 11,664 patients , 55 ( 0.5% ) patients received a diagnosis of pd during the 3-year follow - up period ; 17 ( 0.9% ) and 38 ( 0.4% ) patients in the sa and non - sa cohorts , respectively , developed pd ( table 2 ) . cox proportional hazards regression analysis demonstrated that the crude hr for pd was 2.25 times higher ( 95% confidence interval [ ci ] = 1.273.98 ; p<0.01 ) in the sa cohort than in the non - sa cohort . the risk of pd remained significant after adjustments for potential confounders , including sex , age , urbanization level , geographic region , monthly income , hypertension , hyperlipidemia , diabetes mellitus , asthma , copd , and head injury ( adjusted hr , 1.85 ; 95% ci = 1.023.35 ; p=0.042 ) . kaplan meier analysis demonstrated that the sa cohort had a significantly lower 3-year pd - free survival rate than the non - sa cohort ( log - rank p<0.01 ; figure 1 ) . table 3 presents the risk for pd between sa and non - sa cohorts after sex stratification . men with sa had a significantly higher risk of pd than those without sa before ( hr , 2.97 ; 95% ci = 1.505.90 ; p<0.01 ) and after ( adjusted hr , 2.26 ; 95% ci = 1.114.63 ; p<0.05 ) adjustments for potential confounders . in contrast , women did not exhibit a significantly increased risk of pd in the sa cohort compared to those in the non - sa cohort during the 3-year follow - up period ( adjusted hr , 1.10 , 95% ci = 0.363.40 ) . in addition , age stratification demonstrated that only elderly patients ( age 60 years ) in the sa cohort had a significantly higher risk of pd than those in the non - sa cohort before ( hr , 2.29 ; 95% ci = 1.224.30 ; p<0.05 ) and after ( adjusted hr , 1.93 ; 95% ci = 1.013.71 ; p<0.05 ) adjustments for potential confounders ( table 4 ) . our study demonstrated an association between sa and the risk of pd using nhird data during a 3-year follow - up period . the main study finding was that after adjustments for possible confounders , the sa cohort had a 1.85-fold higher risk of pd than the non - sa cohort . separate analyses revealed that sa is an age- and sex - dependent risk factor for pd . men and elderly patients ( age 60 years ) were at a particularly high risk of pd . this finding is in accordance with our hypothesis that patients with sa are at an increased longitudinal risk of pd . whether sa is an independent risk factor for pd or merely an early symptom during the prodromal phase of pd remains unclear . the cohort design of this study enabled the identification of a temporal association between sa and pd . to clearly demonstrate the temporal association between sa and pd , patients with a history of pd before the index date we observed that the sa cohort had an increased risk of pd , indicating that sa may be an independent risk factor for pd . the pathophysiological mechanisms underlying the association between sa and the risk of pd remain unclear ; however , several theories can be discussed . first , sa - induced chronic intermittent hypoxia can activate inflammatory pathways , leading to increased levels of systemic inflammatory markers.2022 the brains of patients with pd exhibited signs of neuroinflammatory reactions , contributing to a cascade of events and subsequent neuron death.23 furthermore , increased oxidative stress was previously reported in patients with sa.24 a postmortem study indicated that oxidative stress plays an important role in the pathological processes underlying pd development.25 therefore , increased oxidative stress and inflammation due to sa may directly contribute to an increased risk of pd . in this study , men and elderly patients ( age 60 years ) with sa were at a significantly increased risk of pd . the prevalence of severe sa is higher in adult men than in adult women.26 in another study , male sex and age were also independent risk factors for severe sa in the elderly group.27 moreover , a strong correlation has been reported between sa severity and biomarkers of systemic inflammation and oxidative stress.28,29 therefore , it is reasonable to conclude that men and elderly patients have an increased risk of pd , assuming inflammation and oxidative stress play key roles in pd pathophysiology . furthermore , findings from brain imaging studies in patients with sa have demonstrated that sa is associated with the risk of pd . diffusion tensor imaging studies in patients with sa revealed decreased mean diffusivity in the basal ganglia.30 magnetic resonance spectroscopy demonstrated that hypoxia - related metabolites and microstructural changes in the putamen are more pronounced with increasing sa severity.31 furthermore , decreased cerebral blood flow was reported in the right red nucleus and right midbrain of patients with sa.32 these abnormal findings in the basal ganglia and midbrain of patients with sa may indicate an association between sa and the risk of pd . several studies have reported that sa is associated with subsequent pd.3335 however , the present study findings were different and had stronger implications than those of previous studies . second , we excluded patients with several conditions that may contribute to secondary parkinsonism , such as carbon monoxide intoxication and neuropsychiatric diseases with psychotic features , to appropriately identify patients with idiopathic pd . third , we identified and adjusted for common confounding comorbidities , including hypertension , hyperlipidemia , diabetes mellitus , asthma , copd , and head injury , which may affect the subsequent risk of pd , to minimize their influence on pd development . nevertheless , the present study had some limitations . first , the diagnoses of sa and pd were completely based on the diagnostic codes recorded by physicians in the database , but information regarding these specialists was unavailable . in addition , we were unable to distinguish between primary and secondary parkinsonism . to maximize case ascertainment , we adopted validated definitions of sa and pd from previous studies.1719 furthermore , we excluded patients who were at a possible risk of secondary parkinsonism , including those who had been diagnosed with carbon monoxide intoxication , major psychiatric diseases , and dementia , before the index date and during follow - up to improve the diagnostic accuracy . therefore , the duration , severity , and treatment status of sa could not be determined . third , individual patient information , including family history , environmental factors , toxin exposure , and lifestyle factors , were not available from the nhird . fourth , because of the relatively short follow - up period of 3 years , it was difficult to determine whether sa occurred during the prodromal phase of pd before the onset of motor symptoms . previous studies with longer follow - up periods have supported our findings , despite using different inclusion and exclusion criteria.3335 fifth , the actual number of patients with sa who developed pd was small , particularly in women and young patients , thus limiting the statistical power in subgroup analysis based on age and sex . we assumed that most patients had obstructive sa based on the literature about patients with polysomnography - confirmed sa in taiwan , which states that only 0.8% of patients have pure central sa.18,36 further investigation is required to elucidate whether obstructive and central sa differ in the risk of pd . finally , despite the temporal relationship between sa and pd , the underlying causal mechanism was not definitively confirmed in this retrospective study . pd may remain asymptomatic for many years , and sa may be regarded as an initial premotor symptom of pd . therefore , based on our study results , we can not obviate the possibility that sa may be an early symptom of pd . moreover , future studies considering these indices must evaluate the association between sa and the risk of pd and investigate the longitudinal effect of treatment on continuous positive airway pressure . the sa cohort had a higher longitudinal risk of pd than the non - sa cohort , particularly elderly people and men . further prospective studies are warranted to evaluate the causality and underlying mechanisms of the association between sa and the risk of pd .

eight mixed - breed adult dogs , aged between 1 and 6 years old and weighing between 11 and 18 kg , were studied . the dogs were healthy based on physical examination and exhibited no clinical signs or biochemical laboratory abnormalities ( amylase , lipase and c - reactive protein ) suggesting pancreatic disease . prior to ceus studies , fundamental b - mode us was performed on the pancreas and duodenum of each dog , and no evidence of focal or diffused abnormalities was observed . all procedures were approved by the hokkaido university animal care and use committee . an us scanner ( aplio xg , toshiba medical systems , tochigi , japan ) with a 511 mhz broadband linear probe ( plt-704 at , toshiba medical systems ) suitable for pulse subtracting imaging was used . the b - mode and contrast imaging gain were set at 100 and 75 db , respectively . us imaging was set at 3031 frames / sec , and the images were recorded in 40-sec cine - loops to a hard disk for off - line analysis . perfusion of the pancreatic parenchyma and duodenal mucosa was evaluated after intravenous bolus injection and continuous infusion of microbubble contrast agent ( sonazoid , daiichi - sankyo , tokyo , japan ) . for bolus injection , we estimated that a microbubble contrast agent dose of 0.01 ml / kg would be suitable based on manufacturer s recommendation ( 0.015 ml / kg ) and clinical experiences with canine liver and spleen imaging in our facility . for continuous infusion of microbubble contrast agent , based on preliminary studies ( unpublished data ) , a dose 5 times of the bolus injection dose that was reported to be safe was used . the experiments using bolus injection and continuous infusion were carried out on separate days for each dog in order to eliminate residual effects of microbubble contrast agent in the organs of interest or blood circulation . for bolus injection , a single bolus of contrast agent was administered by hand through a 21 g butterfly catheter attached to a 22 g intravenous catheter placed in the cephalic vein , flushed by 3 ml of heparinized saline . for continuous infusion model , a single infusion of contrast agent diluted to 5 ml of saline was administered using a syringe pump ( top-5300 , top , tokyo , japan ) at a rate of 5 ml / min . the injections were given in a standardized manner by the same person throughout the study . the timer on the us machine was manually started when the milky - white contrast agent entered the intravenous catheter . the animal was positioned on left lateral recumbency , and the us probe was placed longitudinally between 2 ribs to image the transverse view of the right pancreatic limb and proximal portion of the descending duodenum . scanning was done continuously for 5 min after bolus injection and at the start of continuous infusion of microbubble contrast agent . for quantitative analysis , us images were analyzed using an off - line image analysis ( imagej , us national institutes of health , bethesda , md , u.s.a . ) . in this system , the gray - scale level ranged from 0 to 255 mean pixel value ( mpv ) . for bolus injection , one image per sec for the first 60 sec was analyzed . for continuous infusion , one image per sec for the first 120 sec followed by one image every 10 sec interval until 300 sec from start of infusion was analyzed . tissue intensity was observed and evaluated for each of the region of interest ( roi ) typically containing 300600 pixels placed in the pancreatic parenchyma and duodenal mucosa for bolus injection and continuous infusion ( fig . right intercostal approach imaging the transverse view of the normal right pancreatic limb ( circle , outlined by a dashed line ) and proximal descending duodenal mucosa ( square ) in a representative adult dog ( dorsal is to the left , ventral is to the right and medial is to the bottom ) . baseline tissue echo components of the pancreatic parenchyma and duodenal mucosa were minimal in the ceus mode . ( b ) corresponding twin view b - mode image of ( a ) . ( c ) crpda ( white arrow ) was more enhanced compared to the pancreatic parenchyma at 8 sec after bolus injection of contrast agent . ( d ) both the pancreatic parenchyma and duodenum reached its pi ( shown here 12 sec after bolus injection of contrast agent ) . region of interests ( rois ) are manually placed in the pancreatic parenchyma ( solid box ) and duodenal mucosa ( dashed box ) to measure the tissue intensity . ( e ) contrast washing out of the pancreatic parenchyma and duodenal mucosa at 20 sec . ) . a time intensity curve ( tic ) the wash - in as reflected by time to initial upslope ( ttu ) and peak time ( tp ) ; peak intensity ( pi ) and the wash - out as reflected by time to wash - out ( ttw ) were measured in all rois ( fig . baseline intensity remained unchanged until time to initial upslope ( ttu ) when intensity first reached 30% of peak intensity ( pi ) . time to wash - out ( ttw ) was measured from contrast injection to when intensity dropped to 30% of pi . ) . ttu and ttw were defined as the time when the gray - scale level increased to and decreased to 30% of pi . right intercostal approach imaging the transverse view of the normal right pancreatic limb ( circle , outlined by a dashed line ) and proximal descending duodenal mucosa ( square ) in a representative adult dog ( dorsal is to the left , ventral is to the right and medial is to the bottom ) . baseline tissue echo components of the pancreatic parenchyma and duodenal mucosa were minimal in the ceus mode . ( b ) corresponding twin view b - mode image of ( a ) . ( c ) crpda ( white arrow ) was more enhanced compared to the pancreatic parenchyma at 8 sec after bolus injection of contrast agent . ( d ) both the pancreatic parenchyma and duodenum reached its pi ( shown here 12 sec after bolus injection of contrast agent ) . region of interests ( rois ) are manually placed in the pancreatic parenchyma ( solid box ) and duodenal mucosa ( dashed box ) to measure the tissue intensity . ( e ) contrast washing out of the pancreatic parenchyma and duodenal mucosa at 20 sec . baseline intensity remained unchanged until time to initial upslope ( ttu ) when intensity first reached 30% of peak intensity ( pi ) . time to wash - out ( ttw ) was measured from contrast injection to when intensity dropped to 30% of pi . because the data were not normally distributed , the wilcoxon signed rank test was used to test for significant differences between pancreas and duodenum during bolus injection and continuous infusion , between pancreas during bolus injection and continuous infusion and between duodenum during bolus injection and continuous infusion for the four measured parameters . statistical analyses were performed with a statistical analysis program ( jmp 8 , sas institute inc . , cary , nc , u.s.a . ) . pancreatic images from all eight dogs were included for analysis after bolus injection and continuous infusion . images of the duodenum from one dog were excluded from the continuous infusion group due to inability to image both organs simultaneously in one field throughout the imaging period of 5 min . no adverse effects , such as dyspnea or anaphylaxis , were noticed in any dogs during or after bolus injection or continuous infusion of microbubble contrast agent . before contrast agent administration , the baseline tissue echo components of the pancreatic parenchyma and duodenal mucosa were minimal in the pulse subtraction ceus mode . after bolus injection , the cranial pancreaticoduodenal artery ( crpda ) was enhanced earliest ( fig . soon after , enhancement of the pancreatic parenchyma was seen followed almost simultaneously by the duodenal mucosa ( fig . 3fig . 3.tic showing the mean pixel intensity in the pancreatic parenchyma ( solid line , , n=8 ) and duodenal mucosa ( dashed line , , n=8 ) 60 sec after bolus injection of contrast agent . notice the fast wash - in to sharp peak , followed by fast and then gradual decline in tissue intensity . the pancreatic parenchyma reached an intense peak several seconds later , slightly before or at the same time as the duodenal mucosa . the pancreas was more enhanced than the duodenum and was clearly delineated in the ceus mode ( fig . contrast effect of the pancreas and duodenum decreased sharply followed by a gradual loss of enhancement ( fig . subjectively , the parenchyma of the pancreas and duodenum can not be seen clearly when the intensity dropped to around 30 mpv . tic showing the mean pixel intensity in the pancreatic parenchyma ( solid line , , n=8 ) and duodenal mucosa ( dashed line , , n=8 ) 60 sec after bolus injection of contrast agent . notice the fast wash - in to sharp peak , followed by fast and then gradual decline in tissue intensity . 4.tic showing the mean pixel intensity in the pancreatic parenchyma ( solid line , , n=8 ) and duodenal mucosa ( dashed line , , n=7 ) 300 sec after continuous infusion of contrast agent . notice a more gradual wash - in to peak followed by a long plateau and slow wash - out of tissue intensity . initial enhancement of the crpda and pancreatic parenchyma was delayed compared to bolus injection . initial enhancement of the duodenal mucosa was slightly slower compared to the pancreatic parenchyma . image enhancement of the pancreatic parenchyma and duodenal mucosa was more gradual until its peak enhancement ( fig . 5afig . right pancreatic limb ( circle , outlined by a dashed line ) and duodenal mucosa ( square ) in the same representative adult dog as fig . 1 ( dorsal is to the left , ventral is to the right and medial is to the bottom ) . ( b ) contrast enhancement of the pancreatic parenchyma persisted at 108 sec with ( c ) gradual wash - out as seen here at 172 sec . ) ; thereafter , the pancreas was continuously enhanced ( fig . this period of enhancement lasted longer than the period of contrast agent infusion ( 1 min ) . subjectively , the enhancement of the pancreas and duodenum was similar at pi , but was less homogenously enhanced when compared to bolus injection . tic showing the mean pixel intensity in the pancreatic parenchyma ( solid line , , n=8 ) and duodenal mucosa ( dashed line , , n=7 ) 300 sec after continuous infusion of contrast agent . notice a more gradual wash - in to peak followed by a long plateau and slow wash - out of tissue intensity . right pancreatic limb ( circle , outlined by a dashed line ) and duodenal mucosa ( square ) in the same representative adult dog as fig . 1 ( dorsal is to the left , ventral is to the right and medial is to the bottom ) . ( b ) contrast enhancement of the pancreatic parenchyma persisted at 108 sec with ( c ) gradual wash - out as seen here at 172 sec . the measured parameters for bolus injection and continuous infusion are summarized ( table 1table 1.results ( median , range ) of the characteristic parameters of the time intensity curve measured after bolus injection and continuous infusion in the normal pancreas and duodenum of healthy dogsbolus injectioncontinuous infusionpancreasduodenumpancreasduodenumtime to initial upslope ( sec)6.0 ( 4.08.0)7.5 ( 4.09.0)20.0 ( 16.025.0)22.0 ( 18.026.0)peak time ( sec)8.5 ( 8.010.0)10.5 ( 6.012.0)33.0 ( 19.052.0)52.0 ( 25.066.0)peak intensity ( mpv ) 100.9 ( 80.2124.3)86.5 ( 36.6120.2)77.6 ( 58.299.5)70.2 ( 60.990.3)time to wash - out ( sec)17.0 ( 15.030.0)23.5 ( 10.049.0)225.0 ( 190.0280.0)215.0 ( 169.0216.0)a ) significant ( p<0.01 ) difference versus duodenum during bolus injection . d ) significant ( p<0.05 ) difference versus bolus injection for corresponding organ . median contrast enhancement durations ( ttw ttu ) of the pancreas and duodenum were prolonged by continuous infusion from 11 sec ( range , 10 to 23 sec ) and 16 sec ( range , 3 to 43 sec ) at bolus injection to 205 sec ( range , 170 to 264 sec , p<0.01 ) and 193 sec ( range , 169 to 216 sec , p<0.05 ) , respectively . median pi of the pancreas at bolus injection ( 100.9 mpv ; range , 80.2 to 124.3 mpv ) was significantly ( p<0.01 ) greater than median pi of the duodenum at bolus injection ( 86.5 mpv ; range , 36.6 to 120.2 mpv ) and median pi of the pancreas at continuous infusion ( 77.6 mpv ; range , 58.2 to 99.5 mpv , p<0.05 ) . contrast enhancement of the pancreatic parenchyma was subjectively adequate with continuous infusion ( fig . d ) significant ( p<0.05 ) difference versus bolus injection for corresponding organ . in our study , after microbubble contrast agent administration as bolus or continuous infusion , the enhanced pancreas was clearly delineated from surrounding organs enabling good visualization . for bolus injection , the pancreas and duodenum had a similar early , intense and uniform enhancement , as could be expected in organs receiving all its blood and therefore microbubbles directly from the arterial supply . the enhancement was followed by a sharp peak and then a fast initial decline ; followed by a gradual washout . the patterns of enhancement of these organs were similar to another study on the canine pancreas and duodenum . for continuous infusion , the enhancement of the pancreas and duodenum was initially delayed and was more gradual until it reached its peak and then plateaued . the order at which pancreas and duodenum were enhanced did not change with bolus injection or continuous infusion . perfusion parameters ttu , tp and ttw were significantly prolonged in the pancreas and duodenum using continuous infusion method when compared to bolus injection . this result can be expected , because in continuous infusion , microbubbles were infused steadily and continuously over a minute in comparison to the quick bolus injection . reasonably , it took a longer time for the enhancement of the pancreas and duodenum to be appreciable subjectively , thus a longer ttu and tp . the contrast enhancement was prolonged , because of this continuous inflow of microbubbles into the roi leading to slow wash - out as shown by prolonged ttw . this effect was beneficial , because the duration of diagnostically useful enhancement for the pancreas and duodenum was prolonged to 18 and 12 times , respectively when compared to bolus injection , even though the continuous infusion dose used was only 5 times the bolus injection dose . preliminary studies performed with lower dose ( 3 times of bolus dose ) showed poor enhancement subjectively and were inadequate for objective analysis . the pancreatic parenchyma was less homogenously enhanced during continuous infusion when compared to bolus injection . this could be explained by the lesser amount of microbubbles at pi due to the dilution of microbubbles during continuous infusion in contrast to bolus injection where all microbubbles injected intravenously perfuse the roi almost simultaneously in high concentration . lower pi was significantly evident in the pancreas ; however , imaging of the pancreas was subjectively adequate . using continuous infusion , a reduction of 23% in the pi of the pancreatic parenchyma was comparable to 16% reduction in a previous continuous infusion ceus study of the liver in which enhancement was deemed adequate despite that reduction . in human medicine , ceus of the pancreas is applied for identification of pancreatic tumors , such as pancreatic ductal adenocarcinoma and neuroendocrine tumor , based on its vascularization . in severe acute pancreatitis , areas of necrosis are delineated as hypoechoic areas due to lack of vascularization . however , with bolus injection of microbubble contrast agent , enhancement of the pancreas is brief due to the absence of venous blood supply unlike the portal blood supply for the liver . the same limitation was seen in our study with the bolus injection method as the pancreas and duodenum were enhanced for only a brief period . a statistical difference was noted for ttu of the pancreas and duodenum for continuous infusion , although this time difference was small . more clinically observable was the prolonged tp of the duodenum when compared to the pancreas in continuous infusion , but not in bolus injection . the pancreatic blood supply originates from the celiac artery through the splenic and hepatic arteries . the splenic artery is the primary blood supply to the left limb of the pancreas . the hepatic artery terminates as the crpda , which enters the body of the pancreas and courses through the proximal portion of the right limb of the pancreas . branches from the crpda exit the pancreatic tissue and supply the closely associated duodenum . in bolus injection , because of the fast speed of microbubbles washing in , this order of vascular supply was not appreciable . however , because the buildup of microbubbles to pi was slower in continuous infusion , the order of this vascular supply became appreciable , although not statistically significant . the crpda courses through and supplies the distal portion of the pancreatic right limb . the cranial and caudal pancreaticoduodenal arteries anastomose within the right limb of the pancreas . the right pancreatic limb was analyzed in this study along with the neighboring proximal descending duodenum . further studies will need to be conducted on the left limb and body of the pancreas due to the tripartite blood supply of the pancreas and the distal portion of the descending duodenum . the obvious advantage of bolus injection is that it is easy to administer , less cumbersome , more repeatable , gives good tissue to tumor contrast and is the conventional method employed in characterizing nodular lesions [ 9 , 12 , 16 , 20 , 21 , 27 , 29 ] . however , contrast enhancement is too rapid and brief in the pancreas . continuous infusion method provides a gradual increase in tissue intensity and longer period of tissue enhancement , however , necessitating a higher dose in our study to achieve adequate imaging . the continuous infusion method may therefore be potentially useful in detecting differences in pancreatic perfusion in diffuse pancreatic disease that may otherwise be too subtle for detection using bolus injection method . the prolonged enhancement of the pancreas may also allow screening of the pancreas for hypovascular areas , such as necrosis . for other applications , such as characterization of nodular lesions , bolus injection should be applied so that the lesion can be studied dynamically in comparison with surrounding normal parenchyma . however , because no lesions were included into this study , further research is needed to determine when the bolus or continuous infusion method is preferable . wash - in time can be taken as the time from contrast agent injection when a certain percentage of increase in baseline tissue intensity is observed . however , as our baseline tissue intensity was set to a minimum ( nearing zero ) , this method was not feasible . another method of determining wash - in time is when tissue intensity reaches a certain percentage of pi . in our study , ttu and ttw were defined as time when the gray - scale level increased and decreased to 30% of pi . this percentage resulted in tissue intensity values of above 20 mpv for both bolus and continuous infusion methods . in the gray - scale , previous ceus studies performed in veterinary medicine employed the usage of sedatives or general anesthesia [ 10 , 11 , 26 ] . in our study , no sedatives were used to eliminate its confounding effects on patterns of contrast enhancement . however , without sedatives , imaging was affected by greater motion and respiratory artifact . these movements can affect the tic , because of difficulty in maintaining a similar roi throughout the entire imaging period . other than that , because of the placement of us beam parallel to the ribs , excessive movements might cause acoustic shadowing effects from the ribs resulting in compromised images with lowered tissue intensity or that can not be analyzed . therefore , great care was taken to place rois manually at similar locations whenever possible and large blood vessels were avoided . applying these precautions resulted in a more homogenous tic ; however , large variation was seen in pi both in bolus injection and continuous infusion . looking at individual data , all dogs had higher pi in pancreas compared to duodenum , except for one dog receiving continuous infusion . the different pi could be attributed to individual differences and perfusion status of an individual animal at any one time . therefore , the duodenum could be used as an internal control when investigating ceus pattern of the pancreas . however , it is important to remember that diseases of the pancreas , such as pancreatitis , may affect the surrounding organs including the duodenum . the present results indicate that ceus using bolus injection and continuous infusion can be used in dogs to image the pancreas and duodenum . the obtained baseline data using bolus injection and continuous infusion may serve as a reference in future assessment of canine pancreatic diseases .

schizophrenia is a complex and clinically heterogeneous psychiatric disorder with unknown etiology , age at onset in late adolescence / early adulthood , and a lifetime prevalence of approximately 1% [ 1 , 2 ] . one of the hallmark characteristics of this devastating disorder is a disturbance in emotion processing , which has been demonstrated in numerous behavioral , physiological , and functional neuroimaging investigations that employed tasks ranging from passive viewing of emotional material , through to facial emotion identification and emotional memory [ 38 ] . although widely investigated , the neural correlates of atypical emotion processing in schizophrenia patients are still not well understood . for instance , while the majority of studies report diminished activations in patients relative to healthy subjects in several regions implicated in affect ( e.g. , hippocampus , amygdala , medial prefrontal , orbitofrontal cortex , and cingulate cortices ) ( e.g. , [ 912 ] ) , others have found no effect or increased neural reactivity to emotionally charged material ( e.g. , [ 1315 ] ) . in our recent study we have observed diminished activations during retrieval of negatively valenced emotional material but enhanced activations during positively valenced condition in clinically stable schizophrenia patients relative to controls . another important variable is gender of tested individuals , as numerous studies in the general population have demonstrated significant differences between men and women in brain activations during performance of emotional tasks ( e.g. , [ 1620 ] ) . despite the large number of functional neuroimaging investigations of emotion processing in schizophrenia , very few have included women and , even when they did , the sample sizes were typically too small to allow for examination of brain activations specifically in women . this is not surprising given that the prevalence and incidence of schizophrenia is greater in men than in women during the first half of life ( below 4045 years of age ) , but this should not prevent us from studying female patients . in one of our previous studies of sex differences in emotion processing in schizophrenia , we observed a different pattern of cerebral activity between male and female patients , and we have subsequently found that the symptom profiles correlated differently with brain activations . these results point to the importance of investigating two sexes separately while evaluating emotion processing in psychoses . the purpose of the present study was to examine brain activations associated with emotion processing in female patients relative to a female comparison group . given the implication of sex steroid hormones in emotion processing ( e.g. , [ 2426 ] ) and in schizophrenia [ 2729 ] , we compared patterns of brain activations during two different stages of the menstrual cycle : follicular ( associated with high estradiol and low progesterone levels ) and luteal ( characterized by a lower estradiol to progesterone ratio ) . based on majority of studies in males with schizophrenia ( e.g. , [ 912 ] ) , we hypothesized that , relative to healthy women , female patients will demonstrate decreased brain activations during emotion processing regardless of the menstrual cycle phase or valence of presented stimuli . several recent reports in healthy premenopausal women have suggested that elevated levels of estradiol diminish , while progesterone enhances neural responsiveness to emotional situations ( particularly highly arousing negative stimulation ) [ 24 , 3032 ] . thus , in the present study , we have performed correlational analyses between brain activations and levels of estradiol and progesterone . consistently with the literature in healthy women , we predicted to find negative correlations between cerebral activations and estradiol , and positive correlations with progesterone ( especially during processing of aversive images ) . in comparison , because there have been some reports of the overall diminished levels of ovarian hormones in women with schizophrenia ( e.g. , [ 3335 ] ) , we expected to replicate this finding and to see attenuated relationship between hormones and brain activations in patients relative to healthy women . twenty - one women were meeting dsm - iv criteria for schizophrenia , in a stable phase of their illness ( defined as no relapse within the last two months and no change in their antipsychotic treatment within the last month ) , and 23 healthy premenopausal women participated in the study . the groups were matched for age , handedness ( edinburgh inventory ) , and parental socioeconomic status ( national occupational classification ; noc ) ( table 1 ) . all participants reported having regular menses ( cycles ranging from 25 to 33 days ) . all patients were reevaluated by experienced psychiatrists before being assigned to the research group ( dsm - iv , criteria a - e ) ; affective , schizoaffective , and schizophreniform psychoses were excluded . symptom severity was rated according to the positive and negative syndrome scale ( panss ) . all the patients received at least one atypical antipsychotic ( 15 patients received one and 6 received two ) : clozapine : n = 8 ; mean dosage = 350.00 124.64 mgs ; olanzapine : n = 6 , mean dosage = 15 5.48 mgs ; risperidone : n = 8 , mean dosage = 2.63 1.41 mgs ; quetiapine : n = 3 , mean dosage = 587.50 271.95 mgs ; ziprasidone : n = 1 , dosage = 200 mgs . control participants were screened with the nonpatients edition of the clinical interview for dsm - iv ( scid ) to exclude presence of any axis - i disorders . general exclusion criteria included age below 18 or above 45 years , lack of menstrual cycle , any past or present neurological disorder , alcoholism or drug abuse , abnormal uncorrected vision , or any contraindication for mri , such as a cardiac pacemaker , an aneurysm clip , a metal prostheses or cardiac valve replacement , the presence of metal in an eye or any part of the body , certain dental work , or claustrophobia . in agreement with the declaration of helsinki , the ability of schizophrenia patients to give informed consent was established using the guidelines of the canadian psychiatric association . the ethics committees of the fernand - seguin research center of the louis - h lafontaine hospital and the regroupement neuroimagerie qubec approved the study . each woman was scanned twice approximately two weeks apart ( 2 days ) to examine the cerebral activations associated with the processing of emotional material at two different phases of the menstrual cycle . prior to each scan , participants were asked about the history of their menstrual cycle . however , due to the high rate of unreliability in the reports , especially in patients who had difficulty keeping track of the first day of their last cycle , we calculated the estradiol to progesterone ratio ( e : p ratio ) to better differentiate between the follicular and luteal phases of the cycle . progesterone is significantly higher during the luteal phase of the cycle , while estradiol is the main hormone controlling the follicular phase . thus , out of the two blood samples taken in the span of two weeks , the one with the significantly higher e : p ratio was indicative of the follicular phase while the blood sample with the lower e : p ratio was indicative of the luteal phase . women , whose e : p ratio was not significantly different between the two fmri sessions , were excluded from the final analyses . moreover , the hormone levels of a few women during the second scan could not be ascertained due to laboratory errors ; as a result these women were also excluded from the final analysis . in the end , a total of 15 healthy women and 17 schizophrenia women were included in the analysis . a blood sample of 10 ml was taken approximately 30 minutes prior to each scanning session to evaluate the levels of estradiol and progesterone in all participants . the samples were stored ( 40c ) and later transported and analyzed at the laboratory of maisonneuve - rosemont hospital . serum levels of estradiol and progesterone were determined using the automated chemiluminescence assay system ( synchron lx i 725 , beckman coulter , usa ) . for estradiol the analytical sensitivity was 20 pg / ml ( 73 pmol / l ) and dynamic range 204800 pg / ml ( 7317621 pmol / l ) . for progesterone the analytical sensitivity was 0.08 ng / ml and dynamic range : 0.0840.0 ng / ml . while in the fmri scanner , participants passively viewed 48.5-second blocks of emotionally positive , negative , and neutral pictures . the stimuli were selected from the international affective picture system ( iaps ) based on normative valence and arousal ratings and were matched for content ( e.g. , people , animals , and landscapes ) . each image category was presented in separate blocks , and there were 16-second periods of rest separating the blocks from one another . each picture appeared for 3000 ms followed by a blank screen with a fixation point for an average of 1.75 s ( ranging from 1 to 2.5 s and giving an average interstimulus interval ( isi ) of 4.75 s ) . as a means to ensure that participants were attentive to the presented images during this emotion task , they were asked to indicate with the press of a button whenever they saw a person or part of a person in the picture . to evaluate the participants subjective emotional responses to the presented images , immediately at the end of the first fmri session , participants were represented with the images of each block and were asked to rate the block of images as whole on a scale ranging from 0 ( absence of any emotional reaction ) to 8 ( strongest emotion ever felt in one 's lifetime ) the intensity of experienced emotion for each block of stimuli . blood oxygenated dependent level ( bold ) signals were recorded using a single - shot , gradient - recalled echo - planar imaging sequence ( repetition time ( tr ) = 3000 ms , echo time ( te ) = 30 ms , flip angle = 90 degrees , matrix 64 64 voxels ) on a mri siemens trio system at 3.0 tesla , which is operational at the functional neuroimaging unit at the university of montreal geriatric institute . the functional volumes were then registered to individual high - resolution coplanar anatomical images taken during the same scanning session ( three - dimensional , spoiled gradient echo sequence ; 28 slices , slice thickness = 5 mm , tr = 22 ms , te = 4 ms , flip angle = 30 ; matrix 256 256 voxels ) to better identify activated structures . the demographic data ( for the entire sample ; table 1 ) , hormonal levels ( for the entire sample and for the subgroup of women tested during two phases of their menstrual cycle ; table 2 ) , as well as subjective ratings of presented stimuli ( obtained during the first scan only ; table 3 ) were analyzed with the statistical package for the social sciences ( spss ) , version 15.0 . the fmri data were analyzed using statistical parametric mapping software ( spm5 ; wellcome department of cognitive neurology , london , uk ) according to the methods outlined by friston . functional images were realigned to the mean volume of each session to correct for artifacts due to subject motion , were spatially normalized into the standardized brain template ( voxel size : 3.5 mm 3.5 mm 3.5 mm ) , and were spatially smoothed with a three - dimensional isotropic gaussian kernel ( 12 mm fwhm ) to improve the signal - to - noise ratio . statistical analyses were carried out using a standard peak - detection approach and the general linear model implemented in spm5 to identify the dynamic cerebral changes associated with the processing of emotional images . first , fmri data of each participant were analyzed using a fixed - effects model to investigate individual brain activation maps and to contrast the brain activity associated with different contrasts ( i.e. , negative versus neutral and positive versus neutral ) . the fixed - effects analysis produced individual contrast images that were then used as raw data for the implementation of a random - effects model to investigate the pattern of activations during the emotional contrasts in healthy and schizophrenia women . one - sample t - tests were implemented to subtract brain activity associated with neutral from that associated with emotional stimuli ( emotional minus neutral ) for each group . considering the lack of studies investigating the neural correlates of emotion processing in schizophrenia and healthy women at different phases of the menstrual cycle we also examined any potential differences between groups using a two - sample t - test . the threshold level for the statistical significance was set at a p = 0.001 uncorrected for multiple comparisons . in addition to the whole - brain exploratory analysis , the bilateral hippocampus , amygdale , and medial prefrontal cortex were selected as a priori regions of interests ( roi ) based on existing functional neuroimaging studies of emotion processing ( for reviews see : [ 4346 ] ) . the centers for each of our a priori rois were produced using the mask for roi analyses software ( marina ) . aal uses the anatomical boundaries of each region using the mni template as a reference . a search sphere with a radius of 16 mm was applied to the centre of each roi using the small volume correction function in spm5 except for the amygdala in which a sphere of 8 mm was used . the aal tool in spm provides the anatomic labelling of each activation peak within the roi . for the priori search , a probability threshold for multiple comparisons with a corrected p < 0.05 and a z - score 1.67 was used . effects at each voxel of the brain were estimated using the general linear model and voxel values for the contrasts of interest - generated statistical parametric maps of the t statistic ( spm t ) that were subsequently transformed to the unit normal distribution ( spm z ) . potential relationships between hormone levels and brain function in healthy and schizophrenia women were investigated using the second - level regression analyses in spm5 . these were correlated first with brain function during positive emotion processing and then with the cerebral activations associated with negative emotion processing for healthy and schizophrenia women separately . to increase the statistical power , the brain function associated with the first fmri scan of all the women who participated in this study ( i.e. , 21 patients and 23 controls ) was included in the regression analysis ( regions were considered significant at p < 0.001 uncorrected for multiple comparisons ) . where significant relationships were found , data were extracted for each cluster of interest ( at the maximum voxel ) and entered into spss to plot the data and to determine correlation coefficients between the hormone scores and the degree of activation . there were no statistically significant between - group differences in the levels of estrogen or progesterone ( please refer to table 1 and table 2 for details ) . nevertheless , two statistical trends of lowered hormonal levels in the subgroup of sz - w tested during two phases of their menstrual cycle were noted : estrogen during the follicular phase ( p = 0.15 ) and progesterone during the luteal phase ( p = 0.13 ) . there were no statically significant between - group differences in subjective ratings of the stimuli ( negative , positive , or neutral ) during the follicular phase ( please refer to table 3 ) . in comparison , during the luteal phase control women rated negative ( but not positive or neutral ) stimuli as more emotional than schizophrenia women did ( p = 0.016 ) . hw exhibited overall more activations during the luteal than the follicular phase ( please refer to tables 4 and 5 , as well as figure 1 ) . during both menstrual cycle phases , hw activated significantly ( among other structures summarized in tables 4 and 5 ) bilateral middle occipital cortex , fusiform gyrus , superior frontal and inferior orbitofrontal cortex ( ofc ) , supplementary motor area ( sma ) , inferior and superior temporal cortex , cerebellum , and hippocampus . these activations were overall more intense and more extensive during the luteal than the follicular phase and the bilateral thalamus , insula and amygdale , were significantly activated during the luteal phase only . in sz - w the activations were comparable in the two phases and included bilateral middle occipital cortex , fusiform gyrus , inferior frontal cortex , sma , portions of temporal and parietal cortex , cerebellum , and thalamus ( for details refer to tables 4 and 5 and figure 1 ) . thus , the two - sample t - tests revealed no significant between - group differences during the follicular phase but did show significantly more activations in hw than in sz - w during the luteal phase in the bilateral thalamus and inferior frontal cortex , left heschl gyrus and insula , right superior , and inferior temporal cortex , as well as the hippocampus and middle ofc ( for details refer to tables 4 and 5 ) . these included bilateral occipital cortex , fusiform gyrus , inferior and middle pfc , sma , inferior temporal and superior parietal cortex , as well as thalamus , hippocampus and cerebellum ( for details refer to tables 6 and 7 , as well as figure 2 ) . sz - w also showed comparable activations during both phases of the menstrual cycle , which included bilateral occipital cortex , fusiform gyrus , thalamus , inferior and ofc , sma , portions of the parietal cortex , and cerebellum ( tables 6 and 7 , as well as figure 2 ) . the direct between - group comparisons with the two - samaple t - tests revealed significantly more activations in hw than in sz - w during the follicular phase in the bilateral cerebellum and left calcarine cortex , and during the luteal phase in the right posterior cingulate , precuneus , and left calcarine cortex . thus while overall we have observed an interaction between group and menstrual cycle phase during processing of negative emotions , the analysis of positive emotion processing data revealed a significant effect of the group but no interaction . estradiolin hw there were positive correlations with activations in the right cerebellum and negative correlations in the left superior frontal cortex , while in sz - w there were only positive correlations with the left parahippocampal gyrus ( please refer to table 8 for details ) . in hw there were positive correlations with activations in the right cerebellum and negative correlations in the left superior frontal cortex , while in sz - w there were only positive correlations with the left parahippocampal gyrus ( please refer to table 8 for details ) . estradiolin hw there were negative correlations with activations in the left amygdala , right inferior frontal , and left inferior parietal cortex , as well as the posterior cingulate . in contrast , in sz - w there were positive correlations in the right inferior parietal and right cerebellum . in hw there were negative correlations with activations in the left amygdala , right inferior frontal , and left inferior parietal cortex , as well as the posterior cingulate . in contrast , in sz - w there were positive correlations in the right inferior parietal and right cerebellum . progesterone in hw there were negative correlations in the left superior temporal and the right superior medial frontal cortex , while there were no significant correlations in sz - w ( please refer to table 8 for details ) . in hw there were negative correlations in the left superior temporal and the right superior medial frontal cortex , while there were no significant correlations in sz - w ( please refer to table 8 for details ) . this is the first study demonstrating that atypical neural activations associated with emotion processing in women diagnosed with schizophrenia depend on the menstrual cycle phase and on the affective valence of presented stimuli . during exposure to negatively charged images , we observed an interesting interaction between the diagnostic group ( schizophrenia patients versus healthy controls ) and phase of the menstrual cycle ( follicular versus luteal ) . specifically , patients showed relatively less activations than controls during the luteal phase , but no between - group differences were observed during the follicular phase . this effect was apparent due to greater activations during the luteal relative to the follicular phase in healthy women , but lack of increased reactivity to aversive information in women with schizophrenia . in contrast , exposure to positively valenced material produced no significant interaction but did reveal a main effect of group . thus , in this condition there were no within - group differences between activations during follicular or luteal phase , but schizophrenia patients exhibited less activations than healthy controls during both phases of the menstrual cycle . the relative deficit in brain activations , while viewing negatively charged images by schizophrenia patients during the luteal phase , was evident in several limbic and corticolimbic structures ( e.g. , thalamus , hippocampus , insula , and middle ofc ) previously implicated in the processing of affective stimuli [ 4346 ] . these findings are consistent with numerous neuroimaging studies , which showed diminished activations in patients relative to controls during performance of diverse emotional tasks ( e.g. , [ 912 ] ) . however , examination of brain activations in female patients during two stages of their menstrual cycle in the present study revealed that the previously reported deficit in processing of negatively valenced material by schizophrenia patients may be restricted to the luteal phase and be absent in the follicular phase . studies of negative affect ( anger , fear , and anxiety ) often focus on the medial temporal lobe structures ; amygdala and hippocampus , both of which have shown deficient activations in schizophrenia patients ( e.g. , [ 3 , 50 , 51 ] ) . indeed , in the present study we have observed that only healthy women exhibited significant activations in the bilateral amygdala and hippocampus during exposure to the unpleasant images in the luteal phase . in comparison , during the follicular phase these structures were not significantly activated in either group , emphasizing again a very specific nature of the observed deficit in female patients . the between - group differences in brain activations during the positive condition were more restricted than what we have observed during exposure to the negative stimuli in the luteal phase . thus , in the follicular phase there was less activation in the circumscribed areas of the cerebellum , superior temporal , and calcarine cortex , while in the luteal phase there was less activation in the precuneus , posterior cingulated , and calcarine cortex , in schizophrenia women relative to healthy controls . although these regions are not considered to be the primary emotional zones , they have been activated during emotional tasks . for example , despite the fact that the calcarine fissure 's main function is the processing of visual information ( this is where the primary visual cortex is concentrated ) , it has also been shown to respond to emotionally arousing stimuli more than to neutral stimuli even with matched visual complexity [ 52 , 53 ] . in a similar vein , the cerebellum has been traditionally associated with the motor coordination , but there are numerous studies implicating it in complex cognitive and emotional processing , as well as in the pathophysiology of schizophrenia [ 55 , 56 ] . because the principal difference between the two phases of the menstrual cycle is hormonal , the main variables to be taken into consideration while accounting for the observed between - group differences in the pattern of brain activations are the levels of the ovarian hormones in patients relative to controls . however , the levels of estradiol were not significantly different between the two groups of women , and the trend toward a diminished level in patients was present in the follicular ( when activations were comparable between the two groups ) and not the luteal phase . in contrast , there were significant between - group differences in the progesterone level , which showed a diminished level especially in the luteal phase . thus , lowered levels of progesterone in patients could partly explain the fmri data ( obtained during processing of negative stimuli ) , and the results are consistent with some previous studies in healthy women ( discussed further below ) . moreover , the subjective rating data are consistent with functional neuroimaging findings . specifically , while there were no significant differences in subjective ratings during the follicular phase , during the luteal phase female patients reported perceiving the negative stimuli as less emotional than healthy controls . it should be noted , however , that the subjective rating data were collected only during the first scanning session . for example , response of the ofc has been shown to vary during exposure to negatively valenced words ; the activity was increased premenstrually during the luteal phase ( when estrogen levels drop ) , and increased postmenstrually during the early follicular phase ( when estrogen levels rise ) . in a different study , in addition to changes in the ofc , significantly greater responses to negatively valenced and highly arousing stimuli were found during the early follicular compared with late follicular phase ( when estrogen levels are at its maximum during ovulation ) in the amygdala , hippocampus , anterior cingulated , and hypothalamus . similarly , a more recent study has shown greater activity in the amygdala and ofc while passively viewing blocks of faces morphing dynamically from a neutral into either angry , happy , or fearful expression , during the late luteal relative to the late follicular phase ( interestingly , stress induction had opposite effects in the two cycle phases ) . in another recent study , increased neural responses to negative images have been observed during the luteal as compared to the follicular phase , in the hippocampus and amygdala . because ( similarly to our study ) the differences between estradiol levels were not significantly different between the two phases , the effect was attributed mainly to different levels of progesterone ( higher in the luteal phase ) . overall , these studies imply that enhanced levels of estradiol are associated with the attenuated cerebral reactivity , whereas progesterone appears to enhance the neural responsiveness to negative emotional stimuli . the results obtained in healthy women in the present study are consistent and extend these previous reports . thus , we have also observed enhanced cerebral activations to negatively valenced stimuli during luteal relative to follicular stage , but interestingly during processing of positive images there were no phase - dependent differences . this suggest that the reactivity is specific for aversive material , at least in healthy premenopausal women as we did not observe this effect in female schizophrenia patients . because ovarian hormones have been implicated in brain function associated with processing of emotional material [ 24 , 3032 ] , in addition to comparing fmri results in the two phases of the menstrual cycle , we also performed correlational analyses between cerebral activations and levels of both estradiol and progesterone in all participants who completed the first scanning session . the overall findings were rather intriguing as majority of correlations were in the opposite direction in the two diagnostic groups . thus , elevated levels of estradiol in control women were associated with decreased activation in the left superior frontal cortex during processing of unpleasant stimuli , and in the right inferior frontal , left inferior parietal , and posterior cingulate cortex during positive condition . in contrast , in women with schizophrenia levels of estradiol were related to enhanced activations in the left parahippocampal gyrus during processing of negative images , and right inferior parietal and right cerebellum during positive emotions . in other words , the higher the levels of estradiol in healthy women , the less neural sensitivity to the emotional material , while in the group of patients this relationship was reversed . this finding is puzzling because there were no significant differences in the overall level of estradiol between the groups . nevertheless , it is possible that even with the comparable levels of ovarian hormones , the interaction with illness processes may alter what is considered the optimal levels for patients and how these hormones interact with brain function and emotion processing . the ovarian hormones , particularly estradiol , have been implicated in the pathophysiology of schizophrenia for the past few decades . for example , symptoms have been reported to fluctuate across the menstrual cycle in women with schizophrenia such that there is clinical deterioration during times associated with low estrogen ( e.g. , premenstrual / late luteal phase ) and amelioration during high estrogen ( late follicular / ovulatory phase ) [ 57 , 58 ] . also , during pregnancy , when estrogens ( and progesterone ) levels are high , low rates of relapse have been observed in women with schizophrenia , and there is an increase in relapse postpartum when estrogens levels drop abruptly . in terms of the altered levels of sex steroid hormones , the evidence is more equivocal . some studies , which found diminished levels of estrogens in schizophrenia women , attributed the effect to the antipsychotic - induced hyperprolactinaemia , mediated by hypothalamic - pituitary - ovarian feedback mechanisms , while others have argued that hypoestrogenism in schizophrenia women occurs independently of antipsychotic use ( e.g. , [ 33 , 34 ] ) . in the present study we did not see any significant differences , but there were some statistical trends towards a diminished level of estradiol in patients relative to controls . furthermore , they emphasize the importance of studying the sexes separately and taking the hormonal status of female patients into consideration when investigating processing of emotional material .

oxaliplatin is a 3rd - generation platinum - based chemotherapeutic , possessing the 1,2 diaminocyclohexane - containing carrier ligand , useful in treating advanced colorectal cancer . it is often used in combination regimens with 5-flourouracil , capecitabine , or 5-flourouracil / irinotecan for the treatment of colorectal cancer . oxaliplatin has also been studied in clinical trials for the treatment of other cancers but has found the most success in gastrointestinal neoplasms including gastric , esophageal , and pancreatic cancers . platinum - based chemotherapeutics have effect via cell phase nonspecific mechanisms causing the formation of cross - linking dna adducts , leading to strand breaks and inhibition of dna replication . oxaliplatin produces common side effects of cytopenias , peripheral neuropathy , diarrhea , and nausea . this paper analyzes the extent , cause and risk factors for neuropathy , and the potential preventative and therapeutic treatments for oxin . acute oxin usually begins with paresthesias and dysesthesias of the hands and feet , but may include the mouth or throat . its onset may begin during the initial infusion or up to 1 - 2 days following the administered dose and is often triggered by cold . typically , the symptoms will resolve spontaneously within days , but often return upon subsequent oxaliplatin administration . it is associated with additional symptoms of muscular hyperactivity including jaw tightness , cramps , and fasciculations . the proposed mechanism of action of acute oxin includes altering the current of voltage - gated na(+ ) channels in response to oxalate , a metabolic by - product of oxaliplatin [ 9 , 10 ] . in addition , oxalate may interact indirectly with the voltage - gated na(+ ) channels through chelation of calcium ( ca ) and magnesium ( mg ) . chronic oxin , in contrast , is thought to be caused by a dose - dependent accumulation of platinum compounds in the dorsal root ganglia , causing neuronal atrophy and apoptosis . it is primarily a sensory neuropathy affecting the extremities and develops based upon the total cumulative dose of oxaliplatin . there is a wide discrepancy in the literature on how to measure and grade oxin . the common terminology criteria for adverse events ( ctcae ) is often used by clinicians to score and monitor oxin . the grading of peripheral neuropathy from the ctcae version 4.0 is shown in table 1 . other rating systems include the total neuropathy score ( tns ) , the eastern cooperative oncology group ( ecog ) toxicity criteria , the oxaliplatin - specific scale , and criteria from individual studies or the world health organization . the ctcae and the ecog criteria have been compared to the tns with reasonable validity for all chemotherapeutics . most clinical trials studying oxaliplatin use the ctcae or the who criteria [ 2 , 16 , 17 ] . grade 2 or worse neuropathy occurs in approximately 4050% of patients receiving oxaliplatin , with grade 3 neuropathy occurring in 1020% of patients [ 1 , 1820 ] . the majority of patients have improvement of their symptoms , but there is still a significant proportion of patients left with some symptoms more than 2 years after completing therapy [ 1 , 20 , 21 ] . in the mosaic trial , 44% of patients had grade 2 or grade 3 neurotoxicity at the end of treatment , 6% one year after therapy , and 4% after 18 months . in a european trial of oxaliplatin , 26% of patients with grade 3 neurotoxicity had persistent symptoms after a median followup of 28 months . more than 10% of patients in the nsabp c-07 trial who received oxaliplatin had persistent neurological symptoms 2 years after completing therapy . in a small , 25-patient prospective study specifically evaluating the development of oxin , 56% of patients developed grade 1 - 2 oxin and 8% developed grade 3 oxin based on a modified tns scoring . an australian prospective study of oxin reported that neuropathy of any grade persisted in 79% of patients , and grade 3 neuropathy persisted in 12% of patients after a median followup of 29 months . preexistent symptomatic peripheral neuropathy has served as exclusion criteria from trials of oxaliplatin , and data on exacerbation of underlying neurotoxicity by oxaliplatin is limited . however , diabetes mellitus , which carries a high susceptibility of peripheral sensory neuropathy , does not appear to be associated with increased risk of developing oxin . age also has not been shown to be a risk factor for the development of oxin . it has been hypothesized that genetic differences in the glutathione transferase pathway may lead to higher rates of neurotoxicity , perhaps due to a decreased ability to respond to oxaliplatin - induced oxidative stress [ 2628 ] . however there is conflicting evidence whether val polymorphisms of glutathione s - transferase ( gstp1 ) leads to higher rates of oxin [ 29 , 30 ] and there are currently no clinical applications of this basic scientific work . integrin beta-3 polymorphisms have been shown to be unrelated to the development of oxin , but may be related to its severity . in a genome - wide analysis of 96 colon cancer patients , a group from south korea showed a possible connection between oxin and the ddx18 and nrp2 genes , although the putative mechanism of interaction of these in relation to oxin is uncertain . two different strategies have been advocated to prevent oxin : ( 1 ) a stop - and - go approach with intermittent oxaliplatin dosing and ( 2 ) the concurrent use of neuromodulatory agents which include antidepressants , antiepileptics , or calcium and magnesium infusions . given the reversibility of oxin in the majority of patients , two separate trials have evaluated stopping oxaliplatin after 6 cycles of therapy , and reintroducing the oxaliplatin after a predefined break while continuing the 5 fu backbone of chemotherapy [ 3335 ] . the optimox1 trial was a european trial involving 620 patients , which compared continuous folfox-4 regimen ( oxaliplatin 85 mg / m ) to an intermittent folfox-7 regimen ( oxaliplatin 130 mg / m for 6 cycles with infusional 5 fu / lv then a complete oxaliplatin treatment break ) with maintenance 5 fu / lv ( 12 cycles ) followed by reintroduction of folfox-7 ( 6 cycles ) . the incidence of neuropathy was less in the intermittent arm between cycle 7 and 18 , but the overall rate of grade 3 neurotoxicity was not significantly different ( 13% intermittent folfox-7 versus 19% folfox-4 , p = 0.12 ) . notably , although 60% of patients in the intermittent arm did not receive further oxaliplatin after their scheduled break from treatment , the response rate , progression - free survival , and overall survival were similar in both arms . the concept trial also compared intermittent to continuous oxaliplatin administration , in a 2 2 design additionally randomizing patients to either calcium / magnesium infusion or placebo . this trial used modified folfox-7 ( oxaliplatin 85 mg / m ) and bevacizumab in both arms , but with alternating blocks of 8 cycles of treatment with oxaliplatin with 8 cycles of biweekly 5 fu / lv / bevacizumab in the intermittent treatment arm . grade 3 neurotoxicity was significantly reduced in the intermittent oxaliplatin arm versus the continuous oxaliplatin arm ( 10% versus 24% ) . dose delay or dose reduction was significantly less in the intermittent arm compared to the continuous arm ( 8% versus 22% ) . additionally , the intermittent oxaliplatin arm had better time to treatment failure and progression - free survival than the continuous arm . while the improvement in neurotoxicity is appealing in the stop - and - go approach use of oxaliplatin , the continuation of the 5 fu / lv may be important to maintain efficacy relative to continuous oxaliplatin . the optimox2 trial compared chemotherapy discontinuation after 6 cycles of folfox-7 with maintenance therapy after 6 cycles of folfox-7 , as was used in optimox1 . this approach resulted in inferior duration of disease control and inferior survival , and is not recommended . however , it is important to note that this study was terminated early ( and thus analyzed as a randomized phase ii ) , and tumor size was required to surpass baseline measurements prior to reintroduction an approach which is not used commonly in the clinic . the macro trial was a large randomized phase iii study that evaluated capecitabine and oxaliplatin ( xelox ) with bevacizumab for 6 cycles , followed by maintenance xelox - bevacizumab or single - agent bevacizumab . this study reported a reduction in grade 3/4 neuropathy from 24% in the continuous xelox - bevacizumab arm to 7% in the bevacizumab alone arm , with no difference in pfs , os between the two arms . therefore stop - and - go oxaliplatin use is as efficacious as continuous oxaliplatin usage when either a 5 fu / lv or bevacizumab backbone is used as maintenance , and results in reduced neurotoxicity . the timing for reintroduction of oxaliplatin is uncertain , and the optimum oxaliplatin - based regimen to use with this approach is also unknown . calcium and magnesium infusions have been tried as prophylactic therapy for oxin , on the basis that they may bind to oxalate , and reduce its effect on voltage - gated sodium channels [ 10 , 39 ] . an encouraging retrospective review of a single institution experience with calcium and magnesium infusions , given before and after oxaliplatin administration , has led to three prospective randomized trials of the strategy . patients in the concept trial were randomized to either placebo , or ca / mg infusion ( calcium gluconate 1 g , magnesium sulfate 1 g , in 100 ml of 5% dextrose in water , half an hour prior and half an hour after oxaliplatin administration ) . interim analysis of the study data which suggested a decreased response rate in the ca / mg arm led to premature closure of the trial after enrolment of only 139 patients . however a subsequent independent review demonstrated that there was no significant difference in response rate or time to treatment failure between the groups . ca / mg infusion did not lead to a significant reduction in grade 3/4 neurotoxicity either in the continuous oxaliplatin arm ( 23% ca / mg group versus 24% placebo ) or in the intermittent arm ( 11% ca / mg group versus 8% placebo ) . there was also no difference in the groups between delays and dose reductions . a retrospective review of the effectiveness of ca / mg infusions on neuropathy from the cairo2 trial also reported no substantial benefit in the reduction of grade 3/4 neuropathy . in contrast the double blinded , placebo - controlled n04c7 trial of patients receiving continuous folfox reported significantly lower grade 2 or worse neurotoxicity in the ca / mg arm ( 22% versus 41% ) . in addition , the ca / mg group had decreased amounts of muscle cramping , but there was no difference in cold sensitivity . the trial was unfortunately closed after only 102 patients were recruited due to concerns regarding the concept trial 's early erroneous assessment of inferior response in the ca / mg arm , leading to lack of statistical power to determine if there was a difference in dose delay and dose reduction between the two groups . preliminary data , taken from an early interim analysis of 52 patients on the blinded , placebo - controlled neuroxa trial , is also supportive of ca / mg infusions . there was a significantly lower frequency of grade 3/4 neurotoxicity in the group receiving ca / mg infusions ( 5% versus 24% ) , and there was no difference in efficacy between the groups . an explanation for the possible discrepancy between the insignificant outcomes of ca / mg in concept and cairo2 compared to the benefit seen in the n04c7 and neuroxa trial is the number of initial oxaliplatin cycles patients received . half the patients in the concept trial received stop - and - go therapy with oxaliplatin , and all patients in cairo2 had at most 6 cycles of oxaliplatin . these measures by themselves have been shown to reduce neurotoxicity , as demonstrated by the optimox1 trial . in contrast the n04c7 and neuroxa trial treated patients with continuous oxaliplatin , suggesting the benefit of ca / mg infusions are limited to this treatment strategy . however despite the favorable results from the n04c7 and the neuroxa trial , there are no current data available on the effect of ca / mg infusions on longer - term oxin , and a subsequent further randomized ncctg trial has begun enrollment to answer this question . a variety of pharmacological approaches to prevent neurotoxicity have been studied , but none have shown sufficient success to use routinely . glutathione , a tripeptide amino acid , has been shown to act as an antioxidant and prevent oxaliplatin accumulation in nerves [ 54 , 55 ] . a small 52-patient randomized trial of intravenous reduced dose glutathione given with oxaliplatin reported that the rate of grade 3 neurotoxicity at 8 weeks was significantly less in the group receiving glutathione ( 0% versus 26% ) . n - acetylcysteine , which increases glutathione blood concentrations , showed promising benefit in preventing oxin in a small 14-patient study . glutamine , another amino acid , has shown promising preventative qualities in a small randomized trial of oral supplementation , but larger confirmatory trials are required . the free radical scavenger amifostine was more effective than glutamine in preventing neurotoxicity when given subcutaneously before oxaliplatin - based chemotherapy . more recently , lipoic acid , an antioxidant , was shown to be no better than placebo in preventing platinum - induced neuropathy in a randomized trial of giving the drug orally during chemotherapy . carbamazepine , a widely used antiepileptic drug that blocks sodium channels , has also been tried as a preventative measure . however , the drug has a wide range of side effects and low therapeutic index , and a randomized trial of the strategy was ineffective . oxcarbazepine , an analogue of carbamazepine , was effective in a small randomized trial in preventing oxin , but larger confirmatory trials are required . xaliproden is an oral drug that acts through the 5ht1 serotonin receptor and has neurotrophic effects . a large randomized trial of xaliproden with 649 patients receiving folfox-4 chemotherapy reported reduced grade 3 neuropathy in patients taking xaliproden ( 11% versus 17% ) , but there was no difference in the percentage of patients with complete recovery after finishing treatment with oxaliplatin ( 49 versus 47 percent ) , and the use of the drug for this indication has been abandoned . there are few comparative studies to guide clinicians regarding appropriate treatment of established acute or chronic oxin . venlafaxine , an antidepressant that is a serotonin and norepinephrine reuptake inhibitor , may be appropriate for the treatment of acute oxin [ 51 , 58 ] . a randomized trial of venlafaxine in patients with acute oxin receiving folfox chemotherapy for adjuvant or palliative treatment of colon cancer demonstrated a reduction in the proportion of patients suffering from acute oxin from 31.3% in the placebo arm versus 5.3% in the venlafaxine arm ( p = 0.03 ) . although the trial was small with 54 patients , it also demonstrated in a secondary endpoint that venlafaxine was effective in reducing the incidence of chronic oxin that was grade 3 or worse from 33% in the placebo arm to 0% in the venlafaxine arm , suggestive that venlafaxine therapy may also be useful as a preventative measure . amitriptyline use resulted in a nonsignificant trend to improvement in sensory neuropathy in a small , 44-patient randomized trial of cancer patients with chemotherapy - induced neuropathy , of whom 11 patients had oxaliplatin - induced pain . however , a negative randomised trial suggests lamotrigine is ineffective as treatment for chemotherapy - induced neuropathy . based on individual case reports and small nonrandomized series , other potential therapeutic options for patients , once large confirmatory studies are done , include gabapentin and pregabalin [ 60 , 61 ] . oxin causes significant morbidity and is often the dose - limited factor in treatment of advanced colon cancer . significant advancement has been made to understand the acute and chronic phases of oxin , but further research is necessary to develop rational therapeutic options . larger future studies are needed to further elucidate the most effective strategies of prevention and treatment of oxin .

surgical site infections ( ssis ) as one of the most common causes of nosocomial infections is accounted for 20 to 25% of all nosocomial infections worldwide ( 1,2 ) . the ssis are the most common complication following surgery , with reported rates up to 30% ( 2 , 3 ) . these infections place a substantial burden on healthcare cost as a result of increased post - operative morbidity and mortality ( 2 , 4 - 7 ) . they are responsible for 30 to 40% of the deaths in the postoperative period ( 8) . with regard to the multifactorial condition of ssi , it is important to detect these factors , to investigate the procedures that bear the highest risk and , if possible , define suitable indices that can predict the risk of ssi ( 9 ) . the present study was conducted to establish the patterns and risk factors of surgical site infections at imam reza hospital in iran between 2006 and 2011 . the ssi was identified based on the presence of icd-10-cm diagnosis code in hospital discharge records of patients admitted to general surgery ward of imam reza hospital , mashhad , iran between 2006 and 2011 . surgical site infections were defined according to the cdc ( the centers for disease control and prevention ) criteria . all patients who met inclusion criteria were enrolled into the study . by using a standardized data collection form predictor variables including patient characteristics , preoperative , intra - operative and postoperative data were obtained . study data included type of surgery , wound class , infection degree , incision site , type and duration of operation , type of prophylaxis and duration of antimicrobial therapy , use of drain , preoperative and postoperative hospital stay . data were analyzed using the spss software patients diagnosed with surgical site infections who were identifiable based on the presence of icd-10-cm diagnosis code in hospital discharge records were included . patients with more than one surgery during hospitalization and those who underwent minor surgery ( surgeries that does nt require anesthesia or respiratory aid ) were excluded from the study . descriptive statistics such as frequency table is derived for categorical variables and mean and standard deviation ( sd ) for numerical variables . descriptive statistics such as frequency table is derived for categorical variables and mean and standard deviation ( sd ) for numerical variables . retrospective review of patients medical records showed that 95 patients fulfilled the inclusion criteria . study population included 51 ( 53.7% ) males and 44 ( 46.3% ) females with age ranged 12 - 88 years . the patients were admitted for various procedures of both elective ( 62.1% ) and emergency ( 37.9% ) operations as shown in table 1 . colectomy ( 13.7% ) was the leading procedure followed by umbilical herniation ( 12.6 ) and appendix perforation ( 12.6% ) ( table 1 ) . patient history of smoking , addiction , past history of diseases ( including hypertension , diabetes and autoimmune diseases ) and medications ( corticosteroid and antibiotic therapy ) were assessed . nineteen patients ( 19.7% ) were addicted to opium 8 ( 8.3% ) with diabetes , other factors ratio was less than 8% for each . except one case ( laparoscopic gastric binding ) , all patients had undergone open surgery ( 96.8% ) . the highest incidence of infection were identified in patients with midline incision above and below the umbilicus ( 40% ) followed by midline incision below the umbilicus ( 8.4% ) and above the umbilicus ( 4.7% ) . the organ involvements were also recorded based on the information contained in the operation description sheet . in most cases ( 26% ) all abdominal viscera such as appendix , colon , small intestine , ovaries and fallopian tubes have been involved during operations . the small intestine and the colon involvement alone occurred in 21.1% of cases ( table 2 ) . the highest incidence of wound infections was observed in 44 patients ( 46.3% ) with clean - contaminated wounds . ninety nine patients ( 30.5% ) had contaminated and 20 patients ( 21.1% ) had clean wounds , and 2 cases identified as dirty wounds . the highest degree of infection ( 65.3% ) was serous secretion without wound dehiscence ( table 3 ) . the mean duration of surgery , pre - operative and post - operative hospital stays was 2.91.45 hours , 1.021.42 and 7.756.75 days respectively . the missing data included duration of surgery and pre - operative and post - operative hospital stay in 40% and 33% of patients , respectively . surgical drains were used in 44.2% of cases and in 82.1% of cases , the wound was closed initially . only for 4.2% of patients , ceftriaxone and metronidazole were administered for 2.1% of patients ; and equal proportions of patients received metronidazole and erythromycin or ceftriaxone , metronidazole and ciprofloxacine ( 1.1% ) . the most antibiotics prescribed post - operatively were the combination of ceftriaxone and metronidazole ( 51.6% ) . in this study , the mean age was 47.13 years with standard deviation of 19.60 years . there were 51 ( 53.7% ) males and 44 ( 46.3% ) females in age range of 12 - 88 years . this could be explained by multiple risk factors in males such as addiction to opium . in accordance with our study , previous studies have shown that patients suffering from pre - morbid diseases , such as diabetes mellitus and hypertension are at high risk of developing ssi ( 6 - 8 , 10 , 11 ) . cigarette smoking was significantly found to be associated with ssi in other studies ( 5 , 10 , 2 ) . in contrast , in our study only 3.2% had a history of cigarette smoking ; on the contrary 20% were addicted to drugs , which showed significant association with development of ssi . in the present study the patients were admitted for various procedures including both elective ( 62.1% ) and emergency ( 37.9% ) operations . the higher rate of ssi in elective surgeries can be explained due to the higher rate of contaminated and clean contaminated wounds in elective surgeries of our institution . colectomy ( 13.7% ) was the leading procedure followed by umbilical herniation ( 12.6% ) , and appendix perforation ( 12.6% ) ( table 1 ) . the highest incidence of infection were identified in patients with midline incision above and below the umbilicus ( 40% ) followed by midline incision below the umbilicus ( 8.4% ) and above the umbilicus ( 4.7% ) . fiorio et al meta - analysis on 3066 surgical procedures demonstrated highest incidence of ssi in small bowel ( 16.3% ) and colon surgery ( 12.5% ) ( 7 ) . our findings confirmed previous knowledge that surgeries with an increased microbial load in the operative field are associated with higher risk of ssi ( 7 , 13 ) . for a long time , surgical wound classification has been recognized as an important predictive factor in developing surgical site infections after surgery ( 6 , 7 , 14 - 16).in our study , as well as previous studies , the incidence of ssi was statistically higher in clean contaminated ( 46.3% ) and contaminated ( 30.5% ) wounds . a prolonged pre - operative hospital stay has been reported to increase the rate of surgical site infection ( 17 ) . a length of operation of more than 3 hours leads to 4 times higher risk of ssi ( 9 ) . in present study , the means for duration of surgery , pre - operative and post - operative hospital stay were 2.91.45 hours , 1.021.42 and 7.756.75 days , respectively . this is not in accordance with the literature regarding the risk of ssi determined by the duration of the surgery and pre - operative hospital stay ( 5 - 7 , 9 ) . in addition , the use of surgical drains has been reported to be associated with an increased risk of ssi which was confirmed in this study ( 28 - 30 ) . this can be explained by using routine antibiotics for every procedure , and also it necessitates the use of antibiotic policy regarding different therapeutic procedures for these patients . the most prescribed antibiotics used post - operatively were ceftriaxone -metronidazole ( 51.6% ) and metronidazole ( 8.4% ) . this investigation had limitations because of missing data including prophylactic antibiotics and icd-10-cm diagnostic code in hospital discharge records . the comparison of ssi incidence between hospitals from various locations and countries must always be attentive , due to specific characteristics for each place and patient population that make it difficult to reach valid conclusions . the ideal situation would be for each hospital to critically analyze its own data , preferably focusing on the historical series and then particularizing it for various types of surgery . surgical site infection is highly related to the type of wound , namely contaminated and clean contaminated wounds and associated with higher rate of ssi . also , it seems that there is a converse relation between length of surgical incision and the rate of ssis . in short , we found that the type of surgery considered to be the main risk factor in developing ssi .

histone deacetylases are a family of enzymes that remove acetyl groups from lysine residues present in the n - terminal extension of core histones of nucleosomes and have been found in bacteria , fungi , plants , and animals . histone acetyltransferases ( hats ) and deacetylases ( hdacs ) play an important role in chromatin structural modifications and epigenetic changes in many organisms . research on histone deacetylase inhibitors ( hdac ) began nearly 30 years ago when studies were laid out to understand why dimethyl sulfoxide ( dmso ) caused terminal differentiation of murine erythroleukemia cells . this early observation led to the development of novel pharmacological agents in the field of chromatin remodeling . hdacs catalyze deacetylation reactions , which cause chromatin to coil by removing acetyl groups from lysine residues of histones . this deacetylation increases the positive charge on n - termini of the core histones . as a result , the interaction between core histones and negatively charged dna increases which causes tight coiling of dna , which in turn blocks access to the transcriptional machinery . the balance between the actions of hdacs , hats , and transcriptional elements serves as a key regulatory mechanism for gene expression and in turn governs numerous developmental processes and disease states [ 3 , 4 ] . hdacs are known to be involved in a myriad of plant physiological and developmental activities and in epigenetic events often for transcriptional repression of genes [ 5 , 6 ] . several studies in plants have reported that there is a direct correlation of dna methylation , histone deacetylation , and gene suppression [ 5 , 7 , 8 ] . in the model plant , arabidopsis thaliana , hda6 and met1 interact directly to silence transposable elements by modifying dna methylation , histone acetylation , and histone methylation status [ 9 , 10 ] . genetic analysis in arabidopsis indicates that hda6 is a component of rddm ( rna - directed dna methylation ) pathway . even in other systems like the african clawed frog xenopus laevis , relaxation of methylated dna in oocytes by the inhibition of histone deacetylases was observed [ 8 , 11 ] . removal of acetyl groups from histones at promoter regions chiefly correlates with gene silencing and transcriptional repression . however , previous studies have also shown gene repression [ 5 , 12 ] as well as activation of some genes . hence , the specificity of hdacs for regulation of distinct gene programs depends on cell identity ( cell state identified by gene regulation programs ) and the scale of available partner proteins along with the signaling networks of the cell [ 13 , 14 ] . as an example , hda6 in arabidopsis , by interacting with different proteins , can regulate flowering time [ 15 , 16 ] , leaf development , transposon silencing , salt and aba stress , ethylene and jasmonate signaling , and freezing tolerance . gene hda19 can regulate seed maturity , flowering time , immune response , and seed dormancy by interacting with other proteins [ 24 , 25 ] . hda9 has also been reported to regulate flowering in arabidopsis by repressing the floral activator agl19 . additionally , hda6 was shown to be involved in histone modifications by increasing gene expression in arabidopsis during seed germination , salt stress , and abscisic acid treatments . hdacs also showed response to various biotic stresses . in arabidopsis , hda19 showed induced expression when plants were challenged with p. syringae and the stability of induced transcripts was shown to be dependent on the levels of salicylic acid and pathogen - related 1 ( npr-1 ) gene expression . recent phylogenetic analysis of sequences from the hdacs superfamily rpd3/hda1 from arabidopsis enabled further classification into three classes , class i , class ii , and class iii . similarly , genome analysis of rice hdacs enabled the identification of an additional class , class iv , indicating the diversity and need for further studies in other commercially important crop plants including legumes . expression analysis of hdacs from all classes and families showed differential expression during developmental stages , environmental stresses , and hormonal stimuli [ 31 , 32 ] . the long - term goal of our research is to understand epigenetic modifications in common bean during infection by the rust fungal pathogen . in this study , we report progress on our understanding of the role of hdacs during infection of common bean with the bean rust pathogen , u. appendiculatus race 53 . we focus on understanding and quantifying total hdac activity present in mock inoculated ( mi ) and inoculated ( i ) leaf tissues at 0 , 12 , and 84 hours after inoculation ( hai ) and analyses of the expression profiles of selected genes from each known plant hdac family . the common bean cultivar sierra is resistant to common bean rust race 53 ( figure 1(a ) ) and carries the rust resistant genes ur-3 and crg [ 33 , 34 ] . the genotype crg , a susceptible mutant derived from sierra which carries a mutation at the crg locus also develops rust like symptoms ( rusty - yellow or bright orange spots ) on leaves . both olathe and crg were used in this experiment as control for demonstrating successful inoculations . when plants were ten days old at the primary leaf stage , half of the seedlings from each genotype were inoculated with u. appendiculatus race 53 spores with 1% tween 20 on the adaxial and abaxial sides of the two leaves and another half of the seedlings were mock inoculated ( mi ) with only 1% tween 20 along with olathe and crg as inoculation experimental controls . after inoculation , plants were placed in a growth chamber with high humidity ( approximately 90% ) to facilitate the establishment of fungal growth . sierra leaf samples were collected at 0 , 12 , and 84 hai along with mi samples for analysis as shown in figure 2 for nuclear extraction and total rna isolation . the above time points were chosen based on our previous experiments [ 35 , 36 ] . for each sampling time , leaves were pooled from three different plants ( one leaf from each plant ) and utilized for colorimetric assays , and three leaves from another set of three plants were collected and flash frozen for gene expression analyses . symptomatic leaves were collected from susceptible mutant genotype crg ( derived from sierra ) , dehydrated with ethanol , and mounted on stubs using carbon filled adhesive . the dehydrated specimens were coated with gold palladium by sputter coater 108 auto ( cressington scientific instruments ltd . , watford , uk ) and observed with an analytical scanning electron microscope s-2600n ( hitachi high technologies america , inc . , schaumburg , il ) located in the college of agriculture & related sciences at dsu . nuclear extractions were carried out from mi and i leaf samples using the epiquik nuclear extraction kit 1 ( epigentek group inc . , approximately one gram of leaf samples ( either flash frozen or fresh ) was cut into small pieces and submerged in a 1 : 10 diluted nuclear extraction buffer 1 ( ne1 ) with 1x dithiothreitol ( dtt ) in a mortar and ground thoroughly until all the leaf samples became fine paste . samples were incubated on ice for 15 minutes and centrifuged for 10 minutes at 11,000 g to obtain a nuclear pellet . the supernatant was removed and 500 l of nuclear extraction buffer 2 ( ne2 ) containing 1x dtt was added to the nuclear pellet and incubated for another 15 minutes on ice . during this time samples were vortexed for 5 sec at three - minute intervals to increase nuclear protein concentration . samples were then centrifuged at 14,000 g for 10 minutes at 4c , and the nuclear protein was quantified with qubit fluorometer ( life technologies , grand island , ny ) and stored at 80c for further analyses . hdac activity was measured with 1.545 g of nuclear protein extract following the manufacturers protocol ( hdac assay kit , colorimetric , active motif , carlsbad , ca ) . as suggested in the protocol for samples with potentially low hdacs , we extended the initial hdac reaction time to three hours . since the kit was developed based on nuclear extracts from mammalian cells , we envisioned that extending the incubation time will help complete the deacetylation reaction . samples were measured in triplicate ( standards were measured in duplicate ) using epoch colorimetric plate reader ( biotek , winooski , vt ) at 405 nm . in the assay reaction , a short peptide substrate was added along with the nuclear extract and other reagents as per the protocol . active hdacs from the experimental samples would then bind to the added substrate by removing acetyl groups from the substrate . this reaction then yielded an hdac - deacetylated colored product , which was measured by the colorimetric plate reader . the amount of deacetylated product in the reaction is directly proportional to the amount of active hdac enzymes present in our samples . representative proteins from each hdac family or class were selected based on the previous reports . the genbank protein accession numbers aac50038 , aak0712.1 , bab10553 , np_200914 , np_200915 , and aad40129 , from rpd3 ( reduced potassium dependency)/hda1 ( histone deacetylase 1 ) family , aab70032 from hd2 family , and bab09243 from sir2 ( silent information regulator 2 ) were selected for gene expression analysis . all of these sequences were derived from arabidopsis thaliana except aak01712.1 and aac50038 , which were derived from rice ( oryza sativa ) and maize ( zea mays ) . genbank protein accession numbers were used to extract model organism protein sequences from ncbi database and these sequences were compared against the common bean predicted proteome derived from the common bean genome - sequencing project from http://www.phytozome.org/ . the best match was selected and coding sequences ( cds ) were extracted for further analysis ( supplementary file 1 ) ( see supplementary material available online at http://dx.doi.org/10.1155/2015/153243 ) . proteins aak0712.1 and aac 50038.1 matched the same common bean cds phvul.009g115300.1 and proteins np_200914 and np_200915 matched phvul.003g185200.1 and other proteins matched different common bean sequences as shown in table 1 . for convenience , we named these cds ( referenced in this study as phaseolus vulgaris hdacs ) as mentioned in column 4 of table 1 . for gene expression analysis , primers were designed with primer quest software as in table 2 and tested with common bean genomic dna ( figure 4(a ) ) . total rna was extracted using trizol reagent ( invitrogen , carlsbad , ca ) from flash frozen pooled leaf tissues ( three leaves from three plants ) as per manufacturer 's protocol and the rna was digested with the enzyme rdnase ( life technologies , grand island , ny ) to remove any contaminating dna . absence of genomic dna was confirmed with known primers that can amplify intronic regions as mentioned previously . total rna was used to synthesize cdna with protoscript m - mulv first strand cdna synthesis kit ( new england biolabs , beverly , ma ) . concentrations of cdna were equalized for all the samples under consideration and qrt - pcr analysis was carried out on applied biosystems 7500 real - time machine ( foster city , ca ) using sybr green dye . gene expression was normalized to the housekeeping gene ubiquitin - conjugating enzyme e2 ubc9 ( tc362 ) and included in each pcr run . gene expression analysis was carried out by comparative 2 method and used to calculate expression values and indicated in fold changes . we collected leaf tissue from inoculated and mock inoculated rust resistant genotype sierra ( tissue pooled from 3 leaves for each time point ) at 0 , 12 , and 84 hai , from which nuclear extracts were then isolated and processed . a standard curve was generated using the standards provided with the hdac activity kit and optical density ( od ) values of the samples from mi plants and i plants were then extrapolated . colorimetric analysis revealed that there is a reduction in the amount of active hdacs ( 37.68 nmol ) in the inoculated samples at 12 hai compared to mock inoculated plants ( 48.97 nmol ) , whereas at 84 hai the activity was approximately 37.0 nmol in both the samples . the reduction in overall hdac activity at 12 hai suggests that there may be less deacetylation reactions at this time point and more uncoiled dna was available for transcription as there will be a demand for induction of stress resistant genes at this time . however , colorimetric analysis indicates that hdacs activity changes throughout the course of rust infection in common bean plants and differs between plants challenged or not challenged with the fungal pathogen . hdac protein sequences were obtained from arabidopsis and other plant species from genbank using corresponding protein accession numbers . these protein sequences were searched against the common bean predicted protein database and common bean cds were obtained ( supplementary file 1 ) for gene expression analysis . since common bean cds were derived by bioinformatics analysis , corresponding primers were initially amplified with genomic dna of the common bean ( figure 4(a ) ) and then with cdna derived from the experimental samples ( figure 4(b ) ) , which were used for qrt - pcr analysis . all the genes tested were amplified in both the genomic dna and cdna samples . between the two genes that were studied in class 1 ( rpd3 family ) , gene pvhda6 showed increased expression at both 12 and 84 hai in the inoculated samples ( figure 5(a ) ) . in the mi samples , the pvhda6 expression was seen to be slightly increased at 12 hai and was neutral at 84 hai samples ( figure 5(b ) ) . pvrpd3 expression was seen to be slightly increased at 12 hai in the i samples . however , both the genes showed slight reduction in expression at 84 hai in the mi samples . gene pvhda18 from class ii hdacs showed increased expression at 12 hai and reduced expression at 84 hai in mi and i samples ( figures 5(c ) and 5(d ) ) . class iii gene pvhda2 showed increased expression at both 12 and 84 hai in i samples whereas its expression was neutral in mi samples at both the time points ( figures 5(e ) and 5(f ) ) . similar results as observed in this study were seen in a pseudomonas syringae resistant arabidopsis plant with rpd3/hda class gene hda19 . increased expression levels of hda19 were seen when plants were inoculated with the bacterial pathogen pstdc3000 , a virulent strain of p. syringae pv . hda19 by interacting with the transcription factors wrky38 and wrky62 was suggested to help fine - tune basal defense responses to pathogen attack in arabidopsis . contrastingly , choi et al . showed that hda19 played a negative role in basal defense response mediated by salicylic acid - dependent signaling pathway , where they have observed increased expression of pathogen defense genes in hda19 mutant plants . in our analysis , gene pvhd2 was neutral in its expression at 12 hai and showed decreased expression at 84 hai in both mi and i leaf samples ( figures 5(g ) and 5(h ) ) . in a recent report , the tobacco nthd2a and nthd2b genes showed a rapid and strong reduction in their expression after treating the tobacco cells with cryptogein , an elicitor of tobacco defense and cell death . based on earlier findings , the hd2 class is plant specific and found only in plants [ 29 , 44 ] . differential expression of the barley hd2 genes ( hvhdac2 and hvhdac2 - 2 ) was observed in different tissues and during seed development in barley and they also exhibited differential expression in barley cultivars with varying seed size . in the same study , these genes responded to plant stress hormones such as jasmonic acid ( ja ) , abscisic acid ( aba ) , and salicylic acid ( sa ) suggesting a possible role in epigenetic regulation due to biotic and abiotic stresses and during seed development . gene pvsrt1 from the sir2 family showed contrasting expression at 12 hai , for which the expression levels were decreased in i samples and increased in mi samples . however we observed that the expression levels were increased at 84 hai in both the samples as in figures 5(i ) and 5(j ) . sirt1 was reported to regulate mirna in alzheimer 's disease patients [ 45 , 46 ] . sir2 genes were found to be highly expressed in highly proliferating stages such as the seedling and developing panicle stages . in the current study , we have used 10-day - old common bean seedlings for inoculation ; hence this might be a possible reason for the presence of a higher quantity of sirt proteins overall . this may be why the trends of active hdacs and the expression profiles of sirt gene are similar . additionally , we note that in this study we were able to measure one representative gene ( pvsrt1 ) from this class , and it will be interesting to measure the second gene . pointed out that only two genes from the sirt class are currently known in plants in this class . another consideration for future quantification experiments would be to determine protein turnover changes . reduced expression of the rice sir2 family gene ossrt1 by specific rna interference increased histone h3k9 acetylation , decreased h3k9 dimethylation , and also led to the development of cell death and symptoms related to plant hypersensitive response during incompatible interaction with pathogen . interestingly , in our study , pvsirt1 showed decreased gene expression at 12 hai in leaves of inoculated plants in the bean genotype that also exhibits hypersensitive response . hdacs play an important role in plant growth , development , flowering , seed maturity , and defense / tolerance to biotic and abiotic stresses . each hdac gene has unique functions and these genes substitute or complement each other 's function . a recent observation indicated that rice hdac genes showed more divergent functions than their homologs in arabidopsis and the same study also showed that their expression is tissue / organ specific in rice . based on the available literature , hdac genes interact with histone and nonhistone proteins as well as other regulatory elements . hda6 has been reported to interact with small interfering rnas ( sirnas ) that are generated through the rddm pathway to suppress gene activity [ 50 , 51 ] . hda9 has been reported to regulate flowering in arabidopsis by repressing flower - activating gene agl 19 . hda19 by interacting with wrky 38 and wrky62 showed enhanced basal resistance to bacterial pathogen . in conclusion , reduced total hdacs activity was observed at 12 hai in rust inoculated bean plants compared to mock inoculated plants . majority of the rpd3/hda1 family of hdacs studied showed increased expression at least in one time point observed after inoculation . the pvhd2 gene of plant specific hdacs did not show differential expression with inoculation and may possibly be developmentally regulated . additionally , the pvsirt1 gene showed reduced expression at 12 hai in inoculated samples . this is one of the first attempts to try to understand hdacs gene regulation in common bean . as hdacs play important roles in chromatin modification , in normal plant developmental process , and in biotic / abiotic responses

medication error can be defined as a failure in the treatment process that leads to , or has the potential to lead to , harm to the patient ( 1 ) . adverse drug events ( ades ) , defined as a drug - related injury ( 2 ) , is a widespread patient safety concern , happening in 540 % of hospitalized patients and in 1217% of patients post - discharge ( 3 - 6 ) . beyond their human toll , these errors can extend treatment courses and hospital stays as well as necessitate therapeutic and pharmacologic intervention . cost modeling study , compiled in 2000 at the university of arizona revealed that the morbidity and mortality costs for mes were in the range of $ 177 billion among the u.s . part of ades is due to medication discrepancies , or unexplained variations in medications in hospital admission and discharge or across different sites of care ( 8 , 9 ) . medication discrepancies at the time of hospital admission are common occurring in up to 67 % of patients at admission ( 10 , 11 ) . ades associated with medication discrepancies can prolong hospital stays and , in the post - discharge period , may lead to emergency room visits , hospital readmissions , and utilization of other healthcare resources ( 12 , 13 ) . medication reconciliation , the process of identifying the most accurate list of all patient s medications is a strategy to identify many medication discrepancies and reduce potential harm ( 14 ) . up to a quarter of all prescription medications taken by patients prior to admission are not correctly documented within the medical record in hospital admission ( 15 ) . several studies have reported that high - quality medication reconciliation improves the overall care provided to hospitalized patients with decreasing drug discrepancies ( 16 - 18 ) . medication discrepancies have been categorized as omission of prescription or nonprescription medications ; differing dosage form , dose , or route recorded ; and/or therapeutic substitutions ; among others . furthermore , the impact of these discrepancies can be great , with up to 40 % of noted discrepancies having the potential to cause moderate to severe despite clear evidence , which shows the importance of pharmacy - based medication reconciliation in the ed ( 19 - 22 ) , pharmacists are rarely on site at our hospital , and medication reconciliation is almost often performed by nurses . because little is known about how medication reconciliation is currently practiced in our country , the aim of current study , as the first research in iran , is evaluation of the error rate in patients ' medication history in hospitalized patients . this study was carried out in the post ccu of a 550-bed university hospital affiliated to shahid beheshti university , tehran , iran . in this prospective single center observational study , 250 patients who admitted from september 2012 until march 2013 to the post ccu were assessed by a fifth - year pharm . d. , student of the shahid beheshti university of medical sciences , with supervision of a clinical pharmacy specialist two days a week for inclusion in this study . patients were included if they were farsi - speaking , aged 18 years or older and admitted to the hospital in the past 24 h. pharmacy students were trained by his preceptor , a clinical pharmacy faculty member with an active practice site at the institution , to conduct patient interviews and obtain detailed medication histories . medication reconciliation , for the purposes of this study , was defined as reviewing the preadmission medication list ( s ) obtained by interviewing the patient and/or family member(s ) of the patient , and checking the pre - admission documents ( if available ) to obtain the most accurate pre - admission medication list . all patients , regardless of their inclusion in this study , had standard - of - care medication reconciliation performed by the hospital nursing and medical staff as part of their admission to the institution . discrepancies were defined as any deviation between the student pharmacist - obtained admission medication list at two levels ; with list obtained by the nurses and/or physicians and with list of drugs ordered at first visit by attending physician . a discrepancy was classified as an omission if a medication was not included within the nurse or physician - obtained medication reconciliation list and/or list of prescribed medications , either in whole or in part ( e.g. omission of dosage form , route , strength , or frequency ) . continuous variables in each group of subjects were expressed as mean values standard deviation ( sd ) . during the study period , 250 patients met the criteria for recruitment in the study . all patients were visited within 24 h after hospital admission for review of their medication history by pharmacy studen t. the mean interview time by pharmacy student to document the patient s medication history was 15 min . the baseline patient characteristics are listed in table 1 . in total 998 medications , including 5988 potential for errors ( 6 error potential for each medication including missed drug , generic name , dosage form , strength , rout of administration and frequency ) , were recorded by pharmacy student . comparing pharmacy student drug history with medication lists obtained by nurses or physicians revealed 3036 discrepancies . on average , 12.14 discrepancies were identified per patient ; however , the number of discrepancies per patient ranged from 0 to 68 . only in 20 patients ( 8 % ) there was 100% agreement among medication lists obtained by pharmacist and physician / nurse . comparing the medications by list of drugs ordered by physician at first visit showed 12.1 discrepancies on average ranging 0 to 72 . the omission errors according to atc classification between drug history of pharmacy student , physician / nurse medication list and first prescriptions of physician are summarized in table 3 . description of discrepancies between pharmacy student medication list , physician or nurse medication list and physician drugs ordered at first visit . this study shows that the actual use of medications at home by patients admitted to the cardiac ward of hospital was largely discrepant with the medication history recorded by usual care . the amount of discrepant medication histories varies widely from 10% to 96% in different settings ( 10 , 23 ) . in our study , compared to usual care , the pharmacist - based procedure identified a discrepancy in medication use in 92 % of the patients resulting in a total of 3036 discrepancies ( mean 12.14 , range 068 ) . the medication reconciliation on admission to a health care system has provided many challenges with respect to the admission medication order process ( 20 ) . in several studies increased accuracy of medications per patient was demonstrated using pharmacy service to obtain medication histories ( 24 - 27 ) . winter et al ( 28 ) defined discrepancies as any difference between the pharmacist - acquired medication history and that obtained by the physician and compared medication histories obtained in the emergency department ( ed ) by pharmacists versus physicians . in this prospective study , 5963 discrepancies were identified and from 3594 medication histories , 59% revealed discrepancies . the authors concluded that medication histories are very often incomplete in the ed . in our study , the disagreement between physician and pharmacist drug histories found in 92% of patients , which could be due to poor attention of clinical team to proper medication histories and lack of structured pharmacy based service for medication reconciliation process . a similar study was conducted by prins mc et al ( 29 ) in a psychiatric clinic to determine the number of discrepancies in medication use at admission , comparing the structured history of medication use procedure with the usual procedure for taking the medication history . they identified 100 discrepancies ( median 2 per patient , range 0 - 8 ) in medication use in 50 elderly psychiatric patients ; 78 % ( n= 39 ) of the patients had at least one discrepancy . of the discrepancies , 69 % were drug omissions , and 31 % were drug additions or discrepancies in the frequency or dosage of medications . the percentage of discrepancies in our study was higher than the study by prins et al . we evaluated 6 error potentials for each medication compared to three in the study by prins . to the best of our knowledge , this is the first prospective observational study in an inpatient cardiac setting that has focused on the first step of medication reconciliation and the medication prescribed by first visiting physician . the results of this study suggest that the frequency of discrepancies in medication use recorded at admission is at least in post ccu setting as high as in other settings . another finding of this study is the high number of discrepancies in first visit , which shows more than 90 % of patients did not received at least one medication at the first day of hospital stay . a factor , could be effective on the data gathering of physician or nurses , was related to the process timing . most nurses and physicians , who are involved , spent lower time than pharmacy student because of their limitations such as work loading , level of their responsibility , and stressful conditions . the data indicated that student pharmacist more reasonable document complete medication lists as compared with nurses and physicians . in addition to the patient care benefits , institutions could persuade by using student pharmacists for medication reconciliation this study had some limitations . it was not feasible to take a reliable drug history from some patients because of the level of their consciousness or education state , so some medications were missed . the nurses and physicians in this study did not receive same training on how to obtain a medication history such as pharmacy student and did not follow a same method for interviewing patients . these differences of obtaining the medication list approach may impact on accuracy of our findings . proper medication histories at the time of hospital admission are the important points of medication safety . to make a comprehensive medication history , addition to role of patients and family members , use of systematic method for standardization of data gathering seems necessary . pharmacists attendance can be a considerable element for making an accurate medication history of patients .

many current - day health problems in individual and community are associated with lifestyle changes . adopting unhealthy lifestyle oral diseases are predominantly man - made attributing to his / her lifestyle [ 1 , 2 ] . human society is undergoing continuous transformation through the harnessing of information and knowledge from the various technologies that in turn have affected our value systems , power structures , everyday routines , and environment . mobile phone usage has increased globally ; over half of the world 's 6.5 billion people now use mobile phone services . total wireless subscription in india stands at 952.34 million ( in urban 553.45 million whereas in rural 398.89 million ) at the end of january 2015 . mobile based innovations are quickly emerging as the new frontiers in transforming health due to fast - growing penetration of mobile phones into remote areas . mobile short messaging service ( sms ) has high penetration and developed into a powerful , real - time communication medium . the innovations and increase in usage of mobile phones have prompted for rapid communication as well as utilization for accessing personal and reliable health information . health information regarding prevention and treatment of diseases can be given to people through an affordable and cost - effective medium like short messaging service . the goal of planned health education program is not only to bring about new behavior but also to reinforce and maintain healthy behavior that will promote and improve individual , group , or community health . one to one approach in oral health education is promising in improving oral hygiene , but it is time - consuming and impractical from community perspective . available health education models have their own limitations to adopt healthy lifestyles such as slow feedback mechanism , expensive apparatus and training required limited access in rural areas , and an inability of customization for the target population . substitution of personal instruction by other means of communication has been investigated , such as the use of self - educational manuals and audio - visual aids . reinforcement is one of the most important principles of health education which helps to adopt healthy behavior and lifestyles . text messaging is able to elicit healthier behaviors , such as adherence to treatment guidelines , smoking cessation , dietary advice , and exercise regimes that can prevent the development of certain behavior - related diseases [ 12 , 13 ] . sparse data is available regarding the use of mobile to deliver dental health across the globe and in india . moreover , there is limited literature evidence regarding the effect of reinforcement of oral health education message through mobile phones on oral health . hence , an attempt has been made to assess the effectiveness of reinforcement of oral health education message through short messaging service ( sms ) using mobile phones among 1820-year - old bsw ( bachelors in social work ) students of north maharashtra university , jalgaon , maharashtra , india . the present study was a quasi - experimental controlled trial conducted on 400 subjects from two different social work colleges ( p. j. nehru college of social work , amalner , jalgaon , and dr . these two colleges were randomly selected from 5 social work colleges situated in north maharashtra region using lottery method . both colleges were well apart from each other ( almost 45 kms ) so that students of both colleges could not interact with each other and they were unaware about randomization and mode of intervention in the research . ethical clearance was obtained from institutional review board of acpm dental college , dhule , and informed written consent was obtained from all the participants prior to the study . subjects in the age group of 1820 years possessing personal mobile phones with sms facility and agreement to comply with the study visits were included in the study . subjects who did not have personal mobile phones and medically compromised subjects were excluded . based on the data obtained from pilot study , keeping at 5% ( p < 0.05 ) , power at 80% , considering cohen 's medium effect size 0.5 , sample size for each group was determined to be 200 . since we had two groups ( intervention and control ) , final sample size was determined to be 400 . aim and objectives of the research were explicitly explained to both institutional higher authorities and permission to conduct the research was obtained from them . thereafter details of the research were explained to all the students of both institutions and 200 subjects from each college satisfying the eligibility criteria were randomly selected . baseline data using a specially prepared and pretested proforma was obtained from the 200 study subjects from each college which included demographic details . intraoral examination was done to assess oral health using oral hygiene index ( given by john c. green and jack r. vermillion in 1960 ) and gingival index ( developed by loe h. and silness j. in 1963 ) . after the collection of baseline data , oral health education was provided to all the subjects of both colleges using common risk factor approach ( oral hygiene practices , diet , habits such as smoking and alcohol use , stress , and trauma ) . as these causes are common to a number of other chronic diseases , adopting a collaborative approach oral health education was given through powerpoint presentation ( 20-minute presentation by investigator himself ) ; demonstration of proper brushing technique on brushing model using modified bass technique and the benefit of using such audio - visual aids is that sound and sight can be combined together to create a better presentation in terms of better understanding on the part of the subjects . subjects were then allowed to ask their doubts and comprehensive explanation was given to clarify their doubts . later intervention group and control group ( college ) were selected randomly by using lottery method by a person who was not known to the examiner . oral health message included the information regarding proper oral hygiene practices , effects of harmful habits , and importance of proper intake of diet . the message was reinforced through short messaging service ( sms ) from mobile phones for the subjects belonging to the intervention group . no other oral health information was provided to the intervention group during the study period . the messages were sent by the person who was unknown to the examiner and examiner was kept blind to the group receiving the message . health related messages both in english and in local language ( marathi ) were sent . each of the two messages in both languages was sent to the intervention group twice a week for the period of 3 months . no such oral health message or any such kind of health education was given to the participants belonging to the control group after randomization . the message was as follows : hi , brush your teeth twice daily with toothbrush and toothpaste to avoid dental diseases . stay away from tobacco as it is the main cause of cancer , gum diseases , lung diseases and heart diseases . after the initial framing of the oral health education message , it was sent randomly to 30 subjects to assess their opinion regarding the comprehension , relevance , and practicality of following the message . all the oral examinations as well as oral health education presentations were performed by a single trained and calibrated examiner . the kappa coefficient value for intraexaminer reliability was 0.87 which is interpreted as very good . a set of instruments , namely , mouth mirror , explorer numbers 5 and 23 , and periodontal probe , were used for each individual patient separately . intraoral examination was done using oral hygiene index and gingival index to collect the clinical data after the 1st , 2nd , and 3rd months from both groups . after the 3rd month , sms to reinforce health education were ceased to be sent to the intervention group . from the 3rd month to the 6th month , no other oral health education was imparted to study participants . then intraoral examination of both groups was done using oral hygiene index and gingival index after six months of baseline data collection . compiled data was analyzed using statistical package for social science ( spss ) version 16 statistical software . the intervention and control groups were compared according to age and gender using nonparametric pearson 's chi square test . mean ohi and mean gi at different intervals were compared in between all subjects of intervention and control group by unpaired t - test . overall changes in mean ohi and mean gi scores within intervention and control groups were compared using anova test followed by post hoc analysis . table 1 shows gender - wise distribution of study participants between the two groups . chi square test showed no significant difference ( p > 0.05 ) in the gender - wise distribution between intervention and control groups indicating a good match ( table 1 ) . comparison of mean ohi score at different intervals between intervention and control groups showed no significant difference in mean ohi score at baseline ( p = 0.283 ) and after 1st month ( p = 0.580 ) in between intervention and control groups . however , mean ohi score in intervention group was significantly less than that of control group after the 2nd , 3rd , and 6th month ( p < 0.01 ) ( table 2 ) . when mean gi scores between intervention and control group were compared at different intervals , it was found that there was statistically no significant difference in mean gi score at baseline ( p = 0.394 ) and after 1st month ( p = 0.849 ) in between intervention and control groups . but mean gi score in intervention group was less than that of control group after the 2nd , 3rd , and 6th month ( p < 0.01 ) ( table 3 ) . the mean ohi scores ( table 4 ) and the mean gi scores ( table 6 ) of the subjects in intervention and control groups were highly significantly different ( p < 0.001 ) across baseline and after 1 month , 2 months , 3 months , and 6 months . the result of the post hoc test ( with least significant difference ) shows difference of mean ohi scores between each of the two time intervals to be highly significant ( p < 0.001 ) in both intervention and control groups except between baseline and 3 months scores in control group ( p = 0172 ) ( table 5 ) . similarly , difference of mean gi scores between each of the two time intervals was highly significant ( p < 0.001 ) in both intervention and control groups except between 2 months and 6 months ( p = 0.06 ) in the intervention group and between baseline and 3 months ( p = 0.271 ) which was not statistically significant ( table 7 ) . from the results , it can be seen that , in intervention group , baseline mean ohi and gi scores linearly decreased up to the 3rd month and gradually increased thereafter till the 6th month , but it was less than baseline mean ohi score and gi score . in control group , mean ohi and gi scores showed reduction after one month but thereafter linearly increased up to the 6th month . after the 6th month , mean ohi and gi scores were more than those of baseline scores . in the present study , there was an improvement in oral health from baseline to the 1st month in both intervention and control groups which was not statistically significant ( p = 0.58 ) . it may be due to the fact that oral health education provided before the start of the study might have the positive effect on the oral health behavior up till one month . hence , there was an improvement in mean ohi and gi scores of all the participants . after baseline data collection , oral health message was reinforced through short messaging service ( sms ) from mobile phones for only the subjects belonging to the intervention group , which might have kept them aware of the importance of oral health and motivated them to maintain proper oral health . study participants in intervention group might have been more aware , concerned about their health , and more positive towards health , thus taking proper measures like proper brushing and flossing to maintain good oral health . it indicates that reinforcement at regular intervals through sms helps to adopt healthier practices and improve oral health . these findings are in agreement with study conducted by sharma et al . which shows that text messaging was more effective than pamphlets in improving knowledge , attitude , and practices of mothers . mean ohi and mean gi scores in intervention group were less than those of control group after the 2nd , 3rd , and 6th month . these results are in concordance with the studies conducted by eppright et al . in orthodontic patients . limited literature is available on effect of reinforcement on oral health through sms , but sms reminders are effective in smoking cessation , treatment guidelines , behavior change , and so forth . the results are similar to the studies conducted by shetty et al . showing significant improvements in the health outcomes of diabetic patients . similar results obtained in the study conducted by koshy et al . on attendance reminders for ophthalmic patients showed that attendance rates were increased in patients who received sms reminder compared to patients who did not receive sms reminder . similarly huang et al . indicated that sms intervention enabled patients to consume their medication on time . apart from sms intervention , other studies have been conducted using different health interventions showing positive effect on health [ 19 , 20 ] . approaches for oral health education other than personal approach have also been used which have resulted in improvement in oral health following intervention . one such study was conducted by harnacke et al . who employed computer based training to teach either fones technique or modified bass technique . computer presentation resulted in improvement of oral hygiene skills and gingivitis using fones technique when compared to control groups . similar results were obtained in a population based survey conducted by gholami et al . using mass media campaign through tv channels . the study demonstrated a significant impact of the mass media campaign on iranian adults ' knowledge regarding periodontal health and disease . evaluated the influence of a mobile application - based approach for domestic oral hygiene maintenance in improving oral hygiene compliance and oral health in a group of orthodontic patients . the study showed positive results in improving oral hygiene compliance of adolescent patients and in improving their oral health . the present study showed that maintenance of improved oral health over longer time periods requires prolonged , repeated instructions , as explained in a study conducted by ivanovic and lekic . apart from the reinforcement of health education through sms to intervention group , regular visits of the investigator to collect the data , visit to dentist to seek care might have contributed to better oral health . however , in control group , there was no improvement in oral health ; probably lack of reinforcement might have resulted in poor oral health . investigator 's visit to both colleges for collecting data at every month during study period might have motivated maintaining good health behavior casting only for few days . impact of dental examination alone might have been responsible for improvement of oral health but for limited time period . after cessation of intervention from the 3rd month to the 6th month in intervention group , there was an increase in mean ohi and gi scores similar to control group . lack of oral health education through sms within this period and lack of investigator 's visits to participants might have resulted in relapse of ohi and gi scores towards baseline . hence , they might have been suffering from stress due to which their oral health might have deteriorated . these results are confirmatory with studies conducted by schou showing the immediate effects on the dental health status , but these effects disappeared or decreased from the 3rd month to the 6th month after dental health education program . the findings of the present study show that oral health is improved after provision of health education ; reinforcing health education helped motivate maintaining good oral health . still the study had an inherent limitation in terms of the design of the study , that is , quasi - experimental design which lacks a true randomization . beholding the internal validity limitations of the study , generalization of the results seems to be questionable . hence , we propose future research with the most valid study design among various population groups to assess the efficacy of this intervention . sms through mobile phone emerging as a new tool helps elicit healthier behaviors and reinforcement of oral health education message through short messaging service ( sms ) using mobile phones can be effective media to improve oral health . as a public health dentist , we should motivate the policy - makers to recommend telecom sector companies as a social responsibility to send free of cost oral health educational sms at community level for taking the society to pinnacles of glory .

providing comprehensive education to caregivers for the promotion of good oral health in their children is now termed as anticipatory guidance . anticipatory guidance , as defined by nowak and casamassimo , is the process of providing information about children to their parents by alerting them to impending changes , teaching them their role in maximizing their children 's developmental potential , and identifying their children 's special needs . traditional preventive strategies have been implemented after deleterious habits have progressed , and these strategies have shown to be limited in their success rate over long periods of time [ 2 , 3 ] . the timely manner in which this information is given to caregivers is a crucial point in this education strategy . anticipatory guidance has been used in the medical community in its campaign to encourage each patient to have a medical home . a medical home is an approach to providing comprehensive primary health care that is easily accessible , culturally sensitive , and family centred in a compassionate manner . studies of the medical home have shown that having a regular source of medical care has decreased the utilization of hospital emergency facilities . the literature has also shown that having a preventive dental visit by the age of one increases future preventive visits and decreases future restorative and emergency room visits . traditional preventive strategies have shown an increase in knowledge and attitudes with dental education , but this has not translated into changed behaviour patterns in the long term . for example , a randomized controlled study of the effects of a pedagogical device targeted to prevent hypoglycemia proved to be a cost - effective educational tool . the development of an anticipatory guidance model via a comprehensive audio - visual aid was achieved by alsada et al . . the aim of the study was to determine the long - term effectiveness of our anticipatory guidance model . specifically , we determined and compared three outcomes : ( 1 ) the long - term retention of knowledge ; ( 2 ) access of dental care by the caregivers for their children ; ( 3 ) the incidence of preventable oral diseases such as dental caries . the two - cohort study was approved by the research and ethics committees of the university of toronto . the anticipatory guidance model used in the study was an interactive presentation that included use of a dvd termed baby oral health in a community - oriented setting . this dvd was designed as a tool to provide comprehensive education regarding infant oral health in high - risk populations . unlike existing education materials , this aid provides a comprehensive , self - directed , and evidence - based approach to the promotion of infant oral health . the topics of the video included the role of a healthy pregnancy , stages of tooth development , early childhood caries ( baby bottle tooth decay ) , trauma , nutrition , oral hygiene , fluoride , oral bacteria , nightly feeding habits , oral habits , the first dental visit , and regular dental visits . this video was developed and previously tested in a pilot project for its effectiveness in infant oral health education . the presentations were performed by one dentist at city - operated child care centres or ontario early years centres in toronto . to assess infant oral health knowledge , there were two types of data collected to assess the effectiveness that the intervention had on preventable oral diseases . the first was the dental screening performed by one dentist in the knee - to - knee position . the second was the completion of an assessment form to review any high - risk behaviour . topics covered in the assessment form included demographics , birth history , diet and nutrition , fluoride , oral habits , injury prevention / trauma , oral development , oral hygiene , and dental visits . in order to determine the model 's effect on dental utilization , each caregiver caregivers voluntarily participated in the study based on information provided by the directors of participating centers to various parent groups . any and all parents with appropriate age children or expectant parents who consented to participation in the study were enrolled . there were no exclusion criteria as this program is intended for all parents with young children . the schematic below outlines the study design ; arrows denote the statistical comparisons made : study group : enrolled atbaseline ( n=161 ; sgb)study group followupat 18 mo ( sgfu)000000000comparison groupenrolled at followup(cg ) study group : enrolled atbaseline ( n=161 ; sgb)study group followupat 18 mo ( sgfu)000000000comparison groupenrolled at followup(cg ) for the study group at baseline ( sbg ; n = 161 ) , the assessment forms were completed by the caregiver prior to the start of the presentation . the caregivers completed the questionnaire immediately after the presentation , and then their children participated in the dental screening . the study group was followed up after an 18-month time period ( sgfu ; n = 161 ) . the follow up consisted of the caregiver completing the identical assessment form and questionnaire that they had originally filled . as well the comparison group ( cg ; n = 181 ) was enrolled from the same centres used to enroll the study sample population but did not receive the anticipatory guidance presentation before data collection . the multiple choice questionnaire was completed at the beginning of the presentation session in order to determine the level of dental knowledge prior to any anticipatory guidance given by the researcher . the presentation was provided at the end of the visit in order to provide anticipatory guidance without biasing the results of the data . summary statistics were computed using sas version 9.2 for the study and comparison groups from the questionnaire and assessment form data . chi - square tests and fischer 's exact tests were used to analyze data between the study group and the comparison group with regard to knowledge retention , presence of caries , and utilization of dental services . this cohort completed the study and was designated ( sgfu ; n = 161 ) . the children 's ages ranged from 0 to 31 months , the mean age being 17.6 months . nine children included in the study group were not born at the time of the initial data collection . the mean age for the sgfu was 35.7 months , with an age range from 16 to 49 months . the comparison group ( cg ) was enrolled based on an age range that would be approximately comparable to the sgfu and consisted of 181 children . the mean age for the cg was 34.2 months , with an age range from 12 to 54 months . as a measure of dental knowledge , a multiple choice questionnaire was administered to the caregivers of the study group at baseline ( sgb ) , at followup ( sgfu ) , and to the comparison group ( cg ) . using chi - square and fischer 's exact test , each question on the multiple choice questionnaire was analyzed comparing the sgb with the cg as well as the sgb with the sgfu . the questionnaire responses revealed that in 20 of 23 questions , the sgb had a higher percentage of correct answers than the cg . the questions that showed a significant differences between the sgb and the cg pertained to the following topics : timing and frequency of oral hygiene practices , all questions related to fluoride , transmission of bacteria , breastfeeding , providing a safe home environment , and timing of the first dental visit . knowledge retention level of the study group at the follow - up period compared to their knowledge retention at baseline revealed a general trend for some loss of knowledge retention over the 18-month period . there was no significant loss of knowledge in the sgfu at a 5% significance level over the 18-month study period for 15 out of the 24 questions . the questions which showed a significant loss of knowledge over the 18-month time period were related to the following topics : timing of the first tooth , time required for toothbrushing , swallowing toothpaste , fluoridated water , transmission of bacteria , the role of breastfeeding in causing tooth decay , and timing of the first dental visit . to determine the effectiveness of the anticipatory guidance model on preventable oral diseases , two methods of data collection were used . the first measure was the dental screening which included a record of visible caries , nonnutritive sucking habits , and trauma . statistical analysis could only be performed for caries since the incidences were too low for other preventable oral conditions , such as trauma or nonnutritive sucking habits ( nnsh ) . there was a significant difference on caries between the sgfu and the cg ( p = 0.0001 ; table 1 ) . the control group at the end of the follow - up period had a caries prevalence of 6% as compared to 24% in the comparison group . the second determinant for the effectiveness of this anticipatory guidance model on preventable oral disease was the assessment of high - risk behaviours . dental visits by caregivers : there was a higher percentage of caregivers in the sgfu ( 56.3% ) that had themselves seen a dentist in comparison to the caregivers in the sgb ( 34.8% ) and the cg ( 46.5% ) . night time feeding practices : there was a dramatic decrease in the percentage of participants in the sgfu ( 16.1% ) who allowed night time feeding for their children as compared to the sgb ( 40.4% ) and the cg ( 47.8% ) . the third objective of the study was to determine if anticipatory guidance had an effect on utilization of dental services . chi - square analysis was performed on data gathered from the assessment form , in particular , the question related to having seen a dentist in the past . the follow - up answers of the study group compared to those of the comparison group are shown in table 3 . the results showed that there was a significantly higher degree of utilization of dental services by the study group participants as compared to those in the comparison group ( p = 0.02 ) . of the 89% of the study group at baseline that had not utilized dental services , many responses were given as to the reason . the most frequent response given for the caregivers of the study group and the comparison group was that they were advised by a health care professional to go at a later age of their child ( 27.4% and 31.8% , resp . ) . an exhaustive review of the literature determined that there are no other audio - visual aids which discuss the full realm of anticipatory guidance topics for infant oral health . the unscripted , interactive aspect of the presentation was also beneficial to targeting the specific concerns of each group of caregivers and kept them engaged in the presentation . for example , presentations that included infants less than 12 months of age emphasized timing of tooth eruption . for groups with toddlers , proper home and car safety standards were emphasized . previous studies have shown that tailored preventive education may have a longer impact than methods that are uniform . the study group baseline ( sg ) approximately 10% of caregivers that were contacted were not interested in continuing with the study because their child was already under the care of a dentist consequent to our initial presentation . while this was a negative aspect to data collection , it was a positive note for the ultimate goal of the study , that is , to increase dental utilization . the results of this investigation showed that being advised by a health care professional was the most popular reason for not taking their child to a dentist . this highlights the issue that nondental health professionals need to be educated about the timing of the first dental visits for infants , so that the public receives a uniform message from all health professionals . considering the limited number of paediatric dentists , it is important for the general dentist to provide access to this young patient population . general dentists should , at least , be comfortable screening children of this age group to assess their risk and determine their need for care by a paediatric dentist . education programs have begun to address this issue at the undergraduate level and reinforce the importance of first dental visit at or before the child 's first birthday . we saw a significant reduction in the incidence of caries in children whose parents were exposed to anticipatory guidance only once . the caries prevalence of 24% in the comparison group is approximately similar to those reported by public health , establishing this population as one at risk for dental disease . an even greater degree of reduction would have been noted with an intermediate recall at a 69 month time point . this observation is based on a much larger ongoing study which shows that children who are caries free are seen to be less likely to be brought back by their parents for routine follow - up visits at these free clinics . reduction in the incidence of preventable oral disease is the ultimate goal of our model of anticipatory guidance and true test of its effectiveness . the results showed that the sgb generally had more knowledge than the cg and that the sgfu generally had some loss of retention of that knowledge . it is interesting to note that the questions that showed a significant difference between the sgb and the cg were very similar to the questions that showed a significant loss of knowledge when comparing the sgb to the sgfu . although it must be acknowledged that parents and caregivers who consented to volunteering in the study could be reasonably be assumed to be more motivated than those who did not , the loss of knowledge retention over the fairly long study period of 18 months demonstrates the attrition in recall of information and highlights areas of the presentation that need further clarity and reinforcement . additionally , the loss in knowledge also demonstrated the reliability and validity of the questionnaire instrument used in this study and our previous study . some of these multiple choice questions showed no significant difference between the sgb and the cg and/or the sgfu , and some questions showed no loss of retention when comparing the sgb to the sgfu ( p = 1.00 ) which suggests that caregivers may be receiving information about these topics from other sources . the gold - standard for caries detection would have been a complete intraoral examination with mirror , explorer , overhead lighting , and radiographic examination if deemed necessary . however , beltrn et al . found in their evaluation of two methods for assessing oral health status that visual screenings gave data comparable to that produced from visual - tactile examinations . screenings are used to seek out high - caries risk children and direct them to a dentist for further care ; its purpose is not to replace a comprehensive oral examination . the use of existing community - oriented programs is a cost effective method of delivering anticipatory guidance to caregivers of infants and increasing access to dental care . this model of delivering anticipatory guidance is more cost - effective than one - on - one counseling initiatives which are the most costly in terms of manpower , time , and financial resources , given the relatively few individuals that can be counseled . the model presented here may be a gateway program to allow parents to receive knowledge and learn whether their child is considered high risk and is in need for a dental visit or for the modification of daily hygiene routines . the model of anticipatory guidance used in this study is cost effective as very few personnel and personnel hours are required to deliver the program . the widespread use of this model can be achieved and leads to an increase in access to care for certain under serviced populations . it is important to note that populations that can not communicate fluently in english are likely the same populations that find it difficult to access care in the dental community . the audio - visual aid used in this study has been translated in to french , spanish , and arabic . it is recommended that the audio - visual aid be translated into many other languages and that multilingual personnel be trained to present this anticipatory guidance model . a second limitation to the study is access to dental services . it may have been helpful to give caregivers a list of private and public dental offices that are in their community . there is some attrition in the retention of oral health knowledge over an 18-month time period suggesting that repeated reinforcement of the same principles and concepts might be advisable over a shorter time span . a one - time exposure to anticipatory guidance has a positive effect on dental utilization . this underscores the importance of this model as a gateway into the dental system.one time exposure to anticipatory guidance has an effect on caries incidence which underlines the importance of the timing of the model . this model , ideally , should be presented to the caregivers when the child is predentate.this model of anticipatory guidance can provide long - term effectiveness in promoting the oral health of young children . there is some attrition in the retention of oral health knowledge over an 18-month time period suggesting that repeated reinforcement of the same principles and concepts might be advisable over a shorter time span . a one - time exposure to anticipatory guidance has a positive effect on dental utilization . one time exposure to anticipatory guidance has an effect on caries incidence which underlines the importance of the timing of the model . this model , ideally , should be presented to the caregivers when the child is predentate . this model of anticipatory guidance can provide long - term effectiveness in promoting the oral health of young children .

childhood obesity is increasingly being observed with the changing lifestyle of families with increased purchasing power , increasing hours of inactivity due to television , video games , and computers , which are replacing outdoor games and other social activities . the world health organization has described obesity as one of today 's most neglected public health problems . following the increase in adult obesity , the proportion of children and adolescents who are overweight and obese have also been increasing . the magnitude of overweight ranges from 9% to 27.5% and obesity ranges from 1% to 12.9% among indian children.[39 ] the most important consequence of childhood obesity is its persistence into adulthood with all its health risks . the health risks include cardiovascular diseases , diabetes , osteoarthritis , gallbladder disease , and some sex hormone sensitive cancers . it is more likely to persist when its onset is in late childhood or adolescence . although several studies have been conducted in metropolitan cities in india on overweight and obesity among children , a very few studies have been conducted in lucknow . the present study was undertaken to study the magnitude of overweight / obesity and its determinants among school - going children in lucknow city . firstly , we went to the office of basic shiksha adhikari and procured the list of government and private schools in lucknow district . probability proportionate to size of the population ( pps ) technique was used to select the number of children from each class and section . the study population includes 407 students of class 5th to 12th of coeducational school from both government and private sector . the schools are located in different areas of lucknow , where students of different socioeconomic status study . before undertaking this study , due approval was taken from institutional ( era 's lucknow medical college and hospital ) ethical committee . a predesigned and pretested questionnaire was used to interview the study participants to elicit the information on family characteristics like residence , type of school , religion , type of family , education and occupation of parents . information on individual characteristics like age , sex , eating habits , and time spent on television viewing and outdoor games were also collected . in our study , we took fast food consumption yes if the child was taking fast food more than three times a week . child was interviewed about the father 's occupation in the presence of school teacher . help of school teacher was also taken if the child had any problem in explaining father 's occupation . during data collection , body weight was measured ( to the nearest 0.5 kg ) with the subject standing motionless on the weighing scale with feet 15 cm apart , and weight equally distributed on each leg . height was measured ( to the nearest 0.5 cm ) with the subject standing in an erect position against a vertical scale and with the head positioned so that the top of the external auditory meatus was in level with the inferior margin of the bony orbit . body mass index ( bmi ) was calculated as weight in kilograms/(height in meter ) . children with bmi of 25 and above were considered overweight and children with bmi more than 30 were considered obese . a total of 407 children of 5th to 12th standard participated in the study . of them , only 141 ( 34.64% ) were normal , 246 ( 60.44% ) were undernourished , 17 ( 4.17% ) were overweight , and 3 ( 0.73% ) were obese . on applying odds ratio , risk of overweight / obesity was significantly higher in children who played outdoor games for < 30 min ( or13.97 , 95% ci=1.962.83 ) and those who consumed fast foods ( or 9.17 , 95% ci=1.281.86 ) . other factors like mother 's education , mother 's occupation , religion , residence , type of school , type of family , and type of food were not found to be statistically significant [ tables 1 and 2 ] . risk factors of overweight / obesity : family characteristics risk factors of overweight / obesity individual characteristics table 3 shows that father 's education , father 's occupation , and class were significantly associated with overweight and obesity among the study population by chi square test . obesity among children in india has become a public health problem ( prevalence > 5% ) . in the present study , overweight and obesity was found to be 4.17% and 0.73% , respectively , together constituting 4.91% for overweight / obesity . reported prevalence of overweight and obesity to be 3.1% and 1.2% , respectively , together constituting 4.3% . reported prevalence of overweight / obesity to be 2.2% in rural area of wardha district . in the present study , the important determinants of the overweight / obesity were father 's education , father 's occupation , class > 8th standard , and outdoor playing < 30 min . overweight and obesity are more prevalent in children of higher classes and belonging to affluent and higher socioeconomic group families . thus , it is recommended to start school - based programs where : there should be regular class hours on healthy food habits , nutritive values of different food items , lifestyle , and behavioral modification;teachers should be motivated to explain the health - related problems through nonconventional ways like short play , video clips , games , etc.,every student should take part in outdoor games and sports , irrespective of gender , andparents should be advised about obesity problems not only for their children but also for themselves . there should be regular class hours on healthy food habits , nutritive values of different food items , lifestyle , and behavioral modification ; teachers should be motivated to explain the health - related problems through nonconventional ways like short play , video clips , games , etc . , every student should take part in outdoor games and sports , irrespective of gender , and parents should be advised about obesity problems not only for their children but also for themselves . the findings of the study are based on interview of students of class 5th to 12th . although the full information about the study was explained to the students , there are chances that the interpretation made by them may be different because of their age and perception . the limitation of this interview - based study is that the findings are based on recall of the interviewee . children of higher classes ( above 8th standard ) belonging to higher socioeconomic group with less outdoor activities and consuming fast foods were more predisposed to overweight / obesity . as a preventive strategy , there is a need to apply health and nutritional education programs for inculcating healthy lifestyles , and incorporating more outdoor activities in physical education department of school curriculum .

systemic lupus erythematosus ( sle ) is a chronic autoimmune disease associated with considerable morbidity , increased mortality and poor health - related quality of life ( hrqol).1 2 belimumab is a human immunoglobulin ( ig)-g1 monoclonal antibody that inhibits the biological activity of soluble b lymphocyte stimulator , an immunomodulatory cytokine involved in b cell selection and survival that is overexpressed in sle.3 in two placebo - controlled trials conducted in patients with active , autoantibody - positive sle ( study of belimumab in subjects with sle ( bliss)-52 and bliss-76 ) , belimumab plus standard sle therapy resulted in significantly higher sle responder index ( sri ) response rates at 1 year compared with standard therapy ( placebo ) , indicating greater reductions in sle disease activity with treatment4 5 and improvements in hrqol measures.6 the sri is a novel composite end point that requires improvement in sle disease activity without worsening in specific organ domains or global disease activity7 consistent with us food and drug administration guidance for development of products for treatment of sle.8 a sri response requires clinically meaningful improvement in the safety of estrogens in lupus erythematosus national assessment - sle disease activity index ( selena - sledai ) and no worsening of disease , as measured by british isles lupus assessment group ( bilag ) organ domain score and physician 's global assessment ( pga ) . the present post hoc analysis examined the association of sri response at week 52 , irrespective of treatment assignment , with individual clinical and laboratory measures , and patient - reported hrqol and fatigue among sri responders and non - responders . patients with sle ( n=1684 ) who were autoantibody - positive ( antinuclear antibody titre 1:80 and/or antidouble - stranded dna ( anti - dsdna ) 30 iu / ml ) with a selena - sledai score 6 received placebo , belimumab 1 mg / kg or 10 mg / kg in addition to standard sle therapy for 52 weeks ( bliss 52 ; nct00424476 ) or 76 weeks ( bliss 76 ; nct00410384).4 5 doses of standard therapy were required to be stable for 30 days prior to enrolment . patients could not have severe active lupus nephritis or severe active central nervous system sle . progressive restrictions on immunosuppressives and antimalarials began at treatment week 16 , and restrictions on corticosteroids began at treatment week 24 . patients were stratified at screening by selena - sledai score ( 69 vs 10 ) , proteinuria ( < 2 g/24 h vs 2 g/24 h ) , and race ( african descent or indigenous american vs other ) . sri response rate at week 52 was the primary end point , defined as a decrease of 4 points in selena - sledai score , no new bilag a score and 1 new b score , and no worsening ( < 0.3-point increase ) in pga score . patients were considered non - responders if they did not meet sri response criteria , withdrew before week 52 or received protocol - prohibited medications . the bliss trials were conducted according to the principles of the declaration of helsinki and the appropriate ethical approvals were obtained.4 5 fifty - two - week data from bliss-52 and bliss-76 were pooled.4 5 of the 1684 patients enrolled , 761 were sri responders and 923 were non - responders at week 52 . clinical variables examined included the individual components of sri response , the numbers of bilag and selena - sledai organ domains with improvement,9 the proportions of patients with flares and severe flares based on the modified sle flare index ( sfi),1012 and changes in corticosteroid dose . laboratory values consisted of changes in anti - dsdna , complement ( c3 and c4 ) , and circulating b ( cd20 ) cells . hrqol , medical outcomes survey short form version 2 ( sf-36 v2 ) , and fatigue ( functional assessment of chronic illness therapy ( facit)-fatigue questionnaire ) were examined . comparisons between responders and non - responders for selena - sledai and bilag scores , changes in corticosteroid dose , and normalisation of anti - dsdna , c3 , and c4 biomarkers were performed using the likelihood ratio test . the two - sample t test was used to compare the improved selena - sledai or bilag organ domains and per cent changes in pga . changes in facit - fatigue score and sf-36 physical component summary ( pcs ) and mental component summary ( mcs ) and domain scores were analysed using an analysis of covariance model adjusted for baseline scores . comparisons of per cent changes from baseline in anti - dsdna , c3 , c4 and cd20 b cells used the wilcoxon test . no multiple test adjustments were made for the above analyses , as they were considered exploratory . the analyses were performed using sas software v.9.2 or higher and r statistical software v.1.9.1 . to examine the robustness of the univariate analysis , baseline covariate adjusted analyses were performed to assess baseline differences and the association with responder status at week 52 . for clinical and serological measures , sri responses in patients receiving placebo , and belimumab 1 and 10 mg / kg plus standard therapy were 38.8% , 46.2% ( p=0.006 ) and 50.6% ( p<0.001 ) , respectively , at week 52 . baseline characteristics were balanced across treatment groups ( table 1 ) and were generally similar between sri responders and non - responders . responders were more likely to have higher disease activity , less serological activity ( based on anti - dsdna titre ( p<0.001 ) and percentage of patients with c3 or c4 levels less than the lower limits of normal ( p<0.001 and p<0.0001 , respectively ) ) , and were more likely to have received a corticosteroid dose > 7.5 mg / d ( p<0.01 ) , but not an immunosuppressant ( p<0.0001 ) . at baseline , there were no statistically significant differences in b cell subsets or plasma cell subsets ( data not shown).13 baseline characteristics of bliss-52 and bliss-76 sri responders and non - responders * p<0.05 ; p<0.01 ; p<0.001 ; p<0.0001 ( note : p values represent comparison between responders and non - responders from the likelihood ratio test for categorical data and from the t - test for continuous variables ) . ana , antinuclear antibody ; anti - dsdna , antidouble - stranded dna ; bilag , british isles lupus assessment group ; c , complement ; facit , functional assessment of chronic illness therapy ; hrqol , health - related quality of life ; igg , immunoglobulin - g ; mcs , mental component summary ; na , not applicable ; pcs , physical component summary ; pga , physician 's global assessment ; selena - sledai , safety of estrogens in lupus erythematosus national assessment - sle disease activity index ; sf-36 , medical outcomes survey short form ; sle , systemic lupus erythematosus ; slicc , systemic lupus international collaborating clinics ; sri , sle responder index . clinical and laboratory measures of disease activity at week 52 are shown in table 2 . changes in clinical and serological measures from baseline in sri responders versus non - responders at week 52 * the analysis was adjusted for the baseline value for each listed parameter using the following methods of analysis . improved from british isles lupus assessment group ( bilag ) a to b score or better , or from b to c score or better ; dropout = failure . * * analysis of covariance test . . based on modified systemic lupus erythematosus ( sle ) responder index ( sri ) analysis that excluded anti - dsdna and complement items from determination of 4-point decrease in safety of estrogens in lupus erythematosus national assessment - sle disease activity index ( selena - sledai ) component of sri ; includes patients with data available at week 52/primary visit . anti - dsdna , antidouble - stranded dna ; c , complement ; pga , physician 's global assessment ; q , quartile ; sfi , sle flare index . more responders than non - responders achieved a 4-point reduction in selena - sledai score , with only 3.8% of non - responders meeting this sri criterion versus 100% of responders ( p<0.001 ) ( table 2 ) . a reduction of 7 in selena - sledai score occurred in 40.3% of responders versus 1.3% of non - responders ( p<0.001 ) at week 52 . mean numbers of improved organ domains per patient were higher among responders as assessed by selena - sledai and bilag ( all p<0.001 ) . mean improvements in pga scores in all patients as well as those with no worsening of pga scores at week 52 were greater among responders versus non - responders ( both p<0.001 ; 49.3% of non - responders had no worsening at week 52 ) . responders had greater improvements in pga than non - responders as early as week 4 and this continued through week 52 ( figure 1a ) . ( a ) mean % change in pga score , ( b ) risk for flare by sfi , and ( c ) corticosteroid use over 52 weeks . * p<0.05 ; p<0.01 ; p<0.001 . pga , physician 's global assessment ; sfi , sle flare index ; sri , systemic lupus erythematosus ( sle ) responder index . to evaluate whether a sri response at week 52 predicted improvement in bilag items present at baseline , an analysis was performed that required a responder to have met sri response criteria and to have had 1 bilag b score present at week 52 . at baseline 64.8% of sri responders and 57.5% of non - responders had > 1 bilag b or 1 a score . at week 52 91.9% of sri responders had 1 bilag b score ( table 2 ) compared with 35.9% of sri non - responders ( p<0.001 ) . the risks of any flare and severe flare were lower in sri responders ( 42% ( hr 0.58 ; 95% ci 0.52 to 0.65 ; p<0.001 ) and 87% ( 0.13 ; 0.09 to 0.17 ; p<0.001 ) , respectively : figure 1b ) . approximately 62% of sri responders and 55% of non - responders received a prednisone ( or equivalent ) dose > 7.5 mg / d at baseline ( table 1 ) . of these patients , more responders than non - responders had dose reductions 25% to < 7.5 mg / d at week 52 ( 25.5% vs 16.4% ; p<0.001 ) , and fewer responders who received prednisone 7.5 mg / d at baseline had dose increases to > 7.5 mg / d at week 52 ( 4.1% vs 21.3% ) . over time , fewer sri responders than non - responders had increases in prednisone dose > 7.5 mg / d , with a difference beginning at week 12 ( figure 1c ) . the proportion of non - responders with increases in corticosteroid doses rose continually over the study period , whereas the proportion of responders did not rise after week 4 . in all , 913 patients were anti - dsdna - positive , 585 had low c3 levels ( < 90 mg / dl ) and 740 had low c4 levels ( < 16 mg / dl ) at baseline . median anti - dsdna antibody levels were lower in sri responders than in non - responders at week 52 ( 34.2% vs 26.1% ) . of patients with hypocomplementaemia at baseline , median per cent increases from baseline c3 and c4 levels were greater in responders than non - responders ( c3 : 14.5% vs 9.0% ; c4 : 40.0% vs 28.6% ) . more responders than non - responders exhibited normalisation of anti - dsdna levels ( 14.4% vs 10.8% ) . similarly , normalisation of low complement levels occurred more often in responders than in non - responders ( c3 : 30.5% vs 25.3% ; c4 : 37.1% vs 29.6% ) . of 542 patients with measurements of circulating cd20 b cell subsets and plasma cell subsets at baseline and week 52 in bliss-76 , the per cent reductions in these cell types at week 52 were numerically greater in responders ( data not shown ) . this finding was driven primarily by the sri responders in the belimumab treatment groups who experienced greater reductions in b cell and plasma cell subsets than patients treated with standard therapy alone.13 overall results from the baseline adjusted analysis were similar to the univariate analysis . there was a greater response in sri responders compared with non - responders , with similar p values for all the disease activity measures , including selena - sledai , bilag , pga and sfi flare , as well as reduced corticosteroid use and improvement in complement levels . the % median change and % normalisation of anti - dsdna were not significantly different between sri responders and non - responders ; the normalisation of low c3 was significantly greater for sri responders . sri responders were more likely to have higher baseline pcs scores than non - responders ( p<0.05 ; table 1 ) . thresholds for minimum clinically important differences ( mcids ) from baseline are 2.5 points for the sf-36 pcs and mcs scores , and are generally considered 5 points for each of the eight domain scores.14 at week 52 , mean improvements in sf-36 pcs and mcs scores were greater in sri responders versus non - responders ( 4.9 vs 2.6 and 4.4 vs 1.7 , respectively ; p<0.001 ) and exceeded mcid . a higher percentage of responders reported improvements mcid than non - responders in pcs ( 59% vs 49% ) and mcs ( 56% vs 44% ) . similarly , improvements in individual domain scores were greater in sri responders and exceeded mcid ( all p<0.001 ) . improvements in non - responders exceeded mcid for pcs and role - physical , bodily pain and vitality domain scores . mean improvements were two - fold greater in responders versus non - responders in six of eight domains ( figure 2 and see online supplementary figure s1 ) ; a consistently higher percentage of responders reported changes mcid than non - responders in all domain scores ( ranging from 54% vs 42% , respectively , for the role - emotional domain to 65% vs 53% , respectively , for the general health domain ) . at week 52 , more than twice as many responders versus non - responders reported feeling somewhat better ( 76.1% vs 33.5% ) and much better ( 33.8% vs 14.6% ) than 1 year ago . mcid , minimum clinically important difference ; mcs , mental component summary ; pcs , physical component summary ; sf-36 , medical outcomes survey short form . mean improvements in facit - fatigue scores were higher in sri responders than non - responders at week 52 ( 5.2 vs 3.0 ) . improvement in the responder group exceeded mcids of 4 points as defined in patients with rheumatoid arthritis.15 greater improvements in facit - fatigue scores were observed by week 8 in responders and were sustained through 52 weeks ( figure 3 ) . these findings are supported by improvements reported by responders in the sf-36 vitality domain ( 10.4 vs 6.5 ) . comparison of sri responders and non - responders for change in facit - fatigue score over 52 weeks . facit , functional assessment of chronic illness therapy ; mcid , minimum clinically important difference ; sri , systemic lupus erythematosus ( sle ) responder index . clinical and laboratory measures of disease activity at week 52 are shown in table 2 . changes in clinical and serological measures from baseline in sri responders versus non - responders at week 52 * the analysis was adjusted for the baseline value for each listed parameter using the following methods of analysis . improved from british isles lupus assessment group ( bilag ) a to b score or better , or from b to c score or better ; dropout = failure . * * analysis of covariance test . last observation carried forward . based on modified systemic lupus erythematosus ( sle ) responder index ( sri ) analysis that excluded anti - dsdna and complement items from determination of 4-point decrease in safety of estrogens in lupus erythematosus national assessment - sle disease activity index ( selena - sledai ) component of sri ; includes patients with data available at week 52/primary visit . anti - dsdna , antidouble - stranded dna ; c , complement ; pga , physician 's global assessment ; q , quartile ; sfi , sle flare index . more responders than non - responders achieved a 4-point reduction in selena - sledai score , with only 3.8% of non - responders meeting this sri criterion versus 100% of responders ( p<0.001 ) ( table 2 ) . a reduction of 7 in selena - sledai score occurred in 40.3% of responders versus 1.3% of non - responders ( p<0.001 ) at week 52 . mean numbers of improved organ domains per patient were higher among responders as assessed by selena - sledai and bilag ( all p<0.001 ) . mean improvements in pga scores in all patients as well as those with no worsening of pga scores at week 52 were greater among responders versus non - responders ( both p<0.001 ; 49.3% of non - responders had no worsening at week 52 ) . responders had greater improvements in pga than non - responders as early as week 4 and this continued through week 52 ( figure 1a ) . ( a ) mean % change in pga score , ( b ) risk for flare by sfi , and ( c ) corticosteroid use over 52 weeks . * p<0.05 ; p<0.01 ; p<0.001 . pga , physician 's global assessment ; sfi , sle flare index ; sri , systemic lupus erythematosus ( sle ) responder index . to evaluate whether a sri response at week 52 predicted improvement in bilag items present at baseline , an analysis was performed that required a responder to have met sri response criteria and to have had 1 bilag b score present at week 52 . at baseline 64.8% of sri responders and 57.5% of non - responders had > 1 bilag b or 1 a score . at week 52 91.9% of sri responders had 1 bilag b score ( table 2 ) compared with 35.9% of sri non - responders ( p<0.001 ) . the risks of any flare and severe flare were lower in sri responders ( 42% ( hr 0.58 ; 95% ci 0.52 to 0.65 ; p<0.001 ) and 87% ( 0.13 ; 0.09 to 0.17 ; p<0.001 ) , respectively : figure 1b ) . approximately 62% of sri responders and 55% of non - responders received a prednisone ( or equivalent ) dose > 7.5 mg / d at baseline ( table 1 ) . of these patients , more responders than non - responders had dose reductions 25% to < 7.5 mg / d at week 52 ( 25.5% vs 16.4% ; p<0.001 ) , and fewer responders who received prednisone 7.5 mg / d at baseline had dose increases to > 7.5 mg / d at week 52 ( 4.1% vs 21.3% ) . over time , fewer sri responders than non - responders had increases in prednisone dose > 7.5 mg / d , with a difference beginning at week 12 ( figure 1c ) . the proportion of non - responders with increases in corticosteroid doses rose continually over the study period , whereas the proportion of responders did not rise after week 4 . in all , 913 patients were anti - dsdna - positive , 585 had low c3 levels ( < 90 mg / dl ) and 740 had low c4 levels ( < 16 mg / dl ) at baseline . median anti - dsdna antibody levels were lower in sri responders than in non - responders at week 52 ( 34.2% vs 26.1% ) . of patients with hypocomplementaemia at baseline , median per cent increases from baseline c3 and c4 levels were greater in responders than non - responders ( c3 : 14.5% vs 9.0% ; c4 : 40.0% vs 28.6% ) . more responders than non - responders exhibited normalisation of anti - dsdna levels ( 14.4% vs 10.8% ) . similarly , normalisation of low complement levels occurred more often in responders than in non - responders ( c3 : 30.5% vs 25.3% ; c4 : 37.1% vs 29.6% ) . of 542 patients with measurements of circulating cd20 b cell subsets and plasma cell subsets at baseline and week 52 in bliss-76 , the per cent reductions in these cell types at week 52 were numerically greater in responders ( data not shown ) . this finding was driven primarily by the sri responders in the belimumab treatment groups who experienced greater reductions in b cell and plasma cell subsets than patients treated with standard therapy alone.13 there was a greater response in sri responders compared with non - responders , with similar p values for all the disease activity measures , including selena - sledai , bilag , pga and sfi flare , as well as reduced corticosteroid use and improvement in complement levels . the % median change and % normalisation of anti - dsdna were not significantly different between sri responders and non - responders ; the normalisation of low c3 was significantly greater for sri responders . sri responders were more likely to have higher baseline pcs scores than non - responders ( p<0.05 ; table 1 ) . thresholds for minimum clinically important differences ( mcids ) from baseline are 2.5 points for the sf-36 pcs and mcs scores , and are generally considered 5 points for each of the eight domain scores.14 at week 52 , mean improvements in sf-36 pcs and mcs scores were greater in sri responders versus non - responders ( 4.9 vs 2.6 and 4.4 vs 1.7 , respectively ; p<0.001 ) and exceeded mcid . a higher percentage of responders reported improvements mcid than non - responders in pcs ( 59% vs 49% ) and mcs ( 56% vs 44% ) . similarly , improvements in individual domain scores were greater in sri responders and exceeded mcid ( all p<0.001 ) . improvements in non - responders exceeded mcid for pcs and role - physical , bodily pain and vitality domain scores . mean improvements were two - fold greater in responders versus non - responders in six of eight domains ( figure 2 and see online supplementary figure s1 ) ; a consistently higher percentage of responders reported changes mcid than non - responders in all domain scores ( ranging from 54% vs 42% , respectively , for the role - emotional domain to 65% vs 53% , respectively , for the general health domain ) . at week 52 , more than twice as many responders versus non - responders reported feeling somewhat better ( 76.1% vs 33.5% ) and much better ( 33.8% vs 14.6% ) than 1 year ago . mcid , minimum clinically important difference ; mcs , mental component summary ; pcs , physical component summary ; sf-36 , medical outcomes survey short form . mean improvements in facit - fatigue scores were higher in sri responders than non - responders at week 52 ( 5.2 vs 3.0 ) . improvement in the responder group exceeded mcids of 4 points as defined in patients with rheumatoid arthritis.15 greater improvements in facit - fatigue scores were observed by week 8 in responders and were sustained through 52 weeks ( figure 3 ) . these findings are supported by improvements reported by responders in the sf-36 vitality domain ( 10.4 vs 6.5 ) . comparison of sri responders and non - responders for change in facit - fatigue score over 52 weeks . facit , functional assessment of chronic illness therapy ; mcid , minimum clinically important difference ; sri , systemic lupus erythematosus ( sle ) responder index . although the lupus research community has become comfortable with selena - sledai , bilag and pga as efficacy measures , the same level of understanding does not exist for the sri . therefore , we examined the clinical meaningfulness of sri response in patients with active , autoantibody - positive sle , irrespective of therapy . improvements in a variety of clinical , serological and clinically meaningful changes in patient - reported outcome measures indicated that a sri response was associated with global benefit beyond that measured by the components of the sri . overall , reductions in severe flares and corticosteroid use as well as clinically meaningful and statistically significant improvement in patient - reported outcomes correlated with sri responder status . while sri responders would be expected to more frequently meet the sri criteria ( 4-point improvement ) for selena - sledai than non - responders , 40% of responders in this analysis had improvement of 7 points on the selena - sledai compared with 1% of non - responders . the improvement in pga score in responders was greater than that achieved in non - responders , as well as in the subgroup of non - responders with no worsening in pga scores , suggesting that sri response is associated with a marked improvement in overall health . this finding is supported by clinically meaningful improvements in patient - reported hrqol and fatigue , including pcs , mcs and all domain scores of sf-36 , facit - fatigue scores and the sf-36 transition question . in addition , sri response was correlated with higher mean numbers of organ domains with improvement on selena - sledai ( 2.00 vs 0.39 ) and bilag ( 1.45 vs 0.40 ) , as well as greater reductions in risk of any flare ( 42% ) and severe flare ( 87% ) over 52 weeks compared with non - responders . sri response was also associated with lower overall corticosteroid use ; nearly twice as many responders ( 25.5% vs 14% ) with initial prednisone doses > 7.5 mg / d were able to reduce corticosteroid doses , and 4% of responders versus 21% of non - responders with initial doses 7.5 mg / d had a dose increase at week 52 . these clinical benefits were observed in sri responders as early as 812 weeks on study medications and this improvement generally increased over time . sri response appeared to be predictive of bilag response : 92% of patients , irrespective of sle therapy , who achieved a sri response also had 1 bilag b organ score after 1 year of treatment . numerous studies have indicated that anti - dsdna antibodies and low complement levels are associated with more severe disease and reduced hrqol,1622 and the american college of rheumatology and european league against rheumatism recommend monitoring serum c3/c4 and anti - dsdna.23 24 a sri response was associated with a decrease in anti - dsdna antibodies , and increases in c3 and c4 levels in patients with low complement levels at baseline compared with non - responders and more responders had normalisation of these markers . improvements in these serological markers have been associated with reduced risk of severe flare and greater likelihood of achieving a sri response , irrespective of therapy.13 baseline values of b cell and plasma cell subsets were similar between sri responders and non - responders . although sri responders generally had greater reductions in b cell and plasma cell subsets than non - responders , this was driven by a greater proportion of sri responders receiving belimumab treatment , since belimumab treatment resulted in greater reductions in b cells and plasma cells than did standard therapy alone . other analyses of the bliss trials have shown that the benefit of belimumab plus standard therapy over standard therapy was augmented in patients with higher disease activity , as defined by baseline selena - sledai 10 , low complement levels , anti - dsdna - positivity , and corticosteroid treatment , and in patients with anti - dsdna - positivity and low complement levels.13 25 overall , sri responders were more likely to have baseline high disease activity similar to the predictors of a belimumab sri response . however , corticosteroid treatment was not predictive of a sri response , whereas patients receiving prednisone > 7.5 mg at baseline were more likely to have achieved a sri response . baseline serological activity was not associated with an overall greater likelihood of a sri response , irrespective of therapy . this differential response can be partially explained by patients in the placebo and belimumab 1 mg / kg groups with high serological activity having lower rates of sri response ( 31.7% and 41.5% , respectively ) than the overall placebo and 1 mg / kg groups ( 38.8% and 46.2% , respectively ) , whereas the sri responses in the 10 mg / kg group were similar in serologically active ( 51.5% ) and all patients in that treatment group ( 50.6%).25 the hrqol benefits in sri responders support the association of a sri response with broad improvements in sle disease activity . the impact of sle on hrqol is comparable with or worse than other chronic diseases ( eg , aids , rheumatoid arthritis , diabetes , congestive heart failure).1 21 22 26 baseline sf-36 pcs and mcs scores in the bliss trials reflected this high impact of sle on hrqol : compared with mean normative values of 50 in sf-36 summary scores , mean baseline scores were 39.1 for pcs and 40.8 for mcs . at week 52 , improvements from baseline in sf-36 pcs , mcs and all domain scores were greater in sri responders and exceeded mcid for all scores . fatigue is one of the most common clinical manifestations of sle and is associated with poor physical and mental functioning.27 mean improvements in facit - fatigue scores reported by sri responders were greater than in non - responders , with changes from baseline exceeding mcid from weeks 12 to 52 in those achieving a sri response at week 52 . however , it should be noted that a mcid of 4 , while valid in patients with rheumatoid arthritis , has not yet been validated in patients with sle . however the mcids for sf-36 summary and domain scores were independently validated in sle and correspond closely to those determined in rheumatoid arthritis . reductions in fatigue were confirmed by a greater increase in sf-36 vitality domain score , consistent with other published data indicating a high correlation between these measurements.15 28 finally , three - quarters of responders indicated that they felt somewhat or much better than 1 year before compared with a third of non - responders . interpretation of these study results is limited by the post hoc nature of the analyses . in addition , examining a clinical trial population based on achievement of the primary end point ( sri at week 52 ) eliminates the randomised balance of baseline characteristics in the treatment groups . baseline characteristics were , however , generally similar between sri responders and non - responders . further , there was a greater magnitude of difference in clinical and patient reported outcomes between these groups than in the baseline characteristics . although some individual variables examined were response criteria that , by definition , were met by responders , analysis of results for these criteria in non - responders remains instructive . our results indicate that sri responses , irrespective of therapy , are associated with global , clinically meaningful benefits in patients with active , autoantibody - positive sle .

basaloid squamous cell carcinoma ( bscc ) is an uncommon variant of squamous cell carcinoma . bscc is included as a distinct entity in the revised classification of tumors of head and neck by who in 1991 . generally , it has a predilection for head and neck region , particularly the upper aerodigestive tract , i.e. larynx hypo pharynx . in the oral cavity , bscc has a predilection for the tongue , though it has been described in other locations such as floor of the mouth , palate , retromolar trigone and gingival mucosa . the aggressive biological behavior of bscc has been commonly associated with early recurrence , cervical lymph node involvement and distant metastasis with spread to the lungs and liver . most bscc 's are diagnosed at advanced clinical stages and have an unfavorable prognosis because of poor overall patient survival rate . clinically , patients with bscc present features similar to those of the patients with squamous cell carcinoma and have the same etiological risk factors , e.g. tobacco and alcohol consumption . the recommended treatment for bscc is surgery followed by radiotherapy and chemotherapy . a 65 year - old male patient reported with a chief complaint of pain in right lower back tooth region of 6 months duration . he had a history of beedi smoking for a period of 50 years , with a frequency of 12/day . extra oral clinical examination revealed palpable , mobile submandibular and upper jugular lymph nodes on the right side , measuring approximately 11 cm and they were firm and non - tender to palpation . on intra - oral examination , an ulceroproliferative lesion involving the right side of the retromolar trigone region , measuring about 22 cm . the ulcer was tender on palpation , exhibiting irregular margins with ill - defined borders and white slough surrounded by erythematous area . after obtaining written consent from the patient , an incisional biopsy was performed under local anesthesia and sent for histopathologic examination . an orthopantomogram [ figure 2 ] was taken which showed irregular radiolucency distal to 3 molar with erosion of the ascending border of the ramus of the mandible , measuring 2 2 cm . the report showed metastatic changes in submandibular and level ii group of lymph nodes . the histopathologic report [ figure 3 ] was poorly differentiated squamous cell carcinoma . radiograph showing erosion of the bone on the right side ascending border of ramus incisional biopsy histological section ( h and e stain , 40 ) the treatment planned was hemimandibulectomy with supraomohyoid neck dissection under general anesthesia , followed by radiotherapy . an apron incision extending from the midline of the chin along the second crease of the neck extending to the mastoid process was made . anteriorly , the incision was continued around the chin to split the lower lip in the midline . the nodes in the level i and level ii regions of the neck were surgically removed . invasion of the lesion in the lymph nodes ( h and e stain , 4 ) the patient has undergone radiotherapy with 5000 cgy fractionated over 6 weeks . barium meal test , chest x - ray and endoscopic evaluation of the upper aerodigestive tract were done , which showed no evidence of lesion or metastasis . the patient has been reviewed regularly for the last 3 months and is disease free . bscc has been defined in the 2005 who blue book as an aggressive high grade variant of squamous cell carcinoma of both basaloid and squamous components . till date approximately 45 cases of bscc involving the oral cavity have been reported in literature , with a strong predilection for base of the tongue ( 61% ) and floor of the mouth ( 30% ) . it is suggested that the tumor originates from the totipotential cells in the basal layer of squamous epithelium . when we review the etiology , it is seen that the possible relationship of bscc and viruses is a matter of debate and has been reported in some locations like nasopharynx and penis . the obtained data are controversial , while kleist et al . and el mofty et al . have very recently detected a high frequency of hpv and hsv in basaloid tumors than in conventional squamous cell carcinoma in the head and neck , others have found no difference . the various sites of origin of bscc reported by authors were gingiva ( eiji hirai et al . ) , oral mucosa and maxillary tuberosity ( c. wedenberg et al . ) , floor of the mouth ( kunal sah et al . ) , retromolar trigone ( marcia sampaio campose et al . ) , nasal cavity ( joong seob lee et al . ) , hypopharynx with extensive spindle cell component ( tokuhiro kimura et al . ) , conjunctiva and paranasal sinus ( pooja vasudev ) , urinary bladder ( funda vakar lopez et al . ) and uterine cervix ( yong soon kwon ) [ table 1 ] . studied the clinicopathological and immunohistochemical features of eight bscc of floor of the mouth which reveals high recurrence , worse prognosis , metastasis , mortality and shorter survival than squamous cell carcinoma . grossly , most of the previously reported bsccs are flat or slightly elevated tumors , often with a central ulceration similar to our case . jung yeon kin et al . reported that all bscc showed positivity for high molecular weight cytokeratin ( hmw ck ) with heterogenous or diffuse staining pattern , but lacked activity for neuroendocrine markers and bcl-2 oncoprotein . pinar atasoy et al . reported a case of bscc of lungs whose cells showed a high mitotic rate and peripheral palisading . grazia salerno et al . reported that low levels of p27 expression significantly correlated with poor prognosis , biological aggressiveness and consequent shortened survival . the supposed higher clinical aggressiveness of bscc compared with the conventional squamous cell carcinoma remains a continuous matter of debate . did not find significant differences in behaviour between these two neoplasms in different anatomical sites while others did . cosmo e et al . discussed the clinicopathological and follow - up study of 40 cases and review of literature and concluded that solid nest with typical cell population , basaloid at the periphery [ figure 5 ] and squamous at the centre , are the most common growth patterns of bscc , which was the histopathologic picture seen in our case . bscc is a rare and aggressive variant of squamous cell carcinoma which is reported to occur predominantly in men between 60 to 70 years of age . the most frequent site to be affected by bscc is the upper aerodigestive tract with strong predilection for the base of the tongue , supraglottic larynx and hypopharynx , but it is also found in the anus , thymus and uterine cervix . clinically , it is an aggressive tumor with high rates of nodal ( 64% ) and distant metastases ( 44% ) . results of a case control study by soriano et al found a 6 times higher risk of distant metastases compared to the usual type of squamous cell carcinoma . some authors recommend a chest ct and fdg- pet in all cases to rule out early distant metastases . treatment of choice is complete surgical excision of the lesion with neck dissection supplemented by radiotherapy or adjuvant chemotherapy . our case report was a bscc in the retromolar trigone which is an unusual site of occurrence . has also reported an atypical presentation of oral bscc in the retromolar trigone in a 39 year old man , in which he described the immunohistochemical characteristics of the lesion . due to its biological and morphologic features , it may be confused with adenoid cystic carcinoma of the solid subtype , small cell neuroendocrine carcinoma undifferentiated carcinoma , basal cell adenocarcinoma and squamous or adenosquamous carcinoma . in the large majority of cases , the distinction between these tumors is readily made on the basis of standard h&e morphology . our case mimics squamous cell carcinoma clinically and was reported on incisional biopsy as poorly differentiated squamous cell carcinoma . on examining the deeper sections of the excisional biopsy , the histologic picture showed nests and cords of closely packed pleomorphic basaloid cells with nuclear palisading along the periphery of the neoplastic nests surrounded by a fibrous stroma with prominent areas of comedo necrosis . the features included : predilection for head and neck region in men in their 60s or 70s.an ulcerated or exophytic mass with submucosal soft tissue infiltration.solid basaloid appearing dysplastic island with biphasic pattern showing comedo type necrosis [ figure 6 ] and pseudo - glandular pattern.abrupt foci of squamous differentiation with or without keratin pearls , and surface mucosal epithelium showing dysplastic features . solid basaloid appearing dysplastic island with biphasic pattern showing comedo type necrosis [ figure 6 ] and pseudo - glandular pattern . abrupt foci of squamous differentiation with or without keratin pearls , and surface mucosal epithelium showing dysplastic features . histopathological picture on high power magnification showing comedo necrosis ( h and e stain , 10 ) quite recently , however coletta et al . have demonstrated the importance of cytokeratins 1 , 7 and 14 in the diagnosis of scc and have shown significantly higher agnor and pcna positivity in bscc when compared with squamous cell carcinoma . the glandular carcinomas can be excluded as glandular lesion present immunoreactivity for ck7 unlike bscc . expression of mmp-1 , mmp-2 and mmp-9 were reported higher in cells of bscc than in cells of squamous cell carcinoma , suggesting of aggressive behaviour . emanuel et al . have stressed the value of p63 in making the distinction between bscc and adenoid cystic carcinoma of head and neck . though in a comparative study of oral bscc and squamous cell carcinoma done by ferrada c grizza et al . they concluded that their study supported the opinion that the prognosis of bssc does not differ from that of conventional squamous cell carcinoma of the oral cavity when matched for clinical classification . regional nodal metastasis was seen in 75% and distant nodal metastasis was seen in 35 - 50% of the cases in wizenburg et al series , lung is reported to be the main target for distant metastasis in bscc . in our case , finding a second primary tumor is a common clinical situation in the head and neck . cosmo e et al . have observed in 17.5% of their patients , a second primary tumor and therefore support thompson 's advice for keeping in mind the possibility of finding a second primary tumor in any sites either synchronic or metachronic , when diagnosing a bscc in the head and neck bscc , a distinct clinicopathological entity with aggressive clinical behavior , which usually is reported to occur in the upper aerodigestive tract , has been reported by us occurring in the retromolar trigone area which is an uncommon site for an uncommon lesion such as bscc .

while adrenal adenomas are quite frequent in the general population , adrenal cortical carcinomas ( accs ) are rare malignancies affecting only 2 per million per year in the world population . a bimodal age distribution has been recognized , with first peak in childhood ( < 5 years of age ) and second peak in 4 - 5 t decade of life . most of the accs are sporadic ; however they may also arise within the context of familial or hereditary diseases . although different studies have been undertaken in order to elucidate the molecular pathogenesis of sporadic accs , to date none of them has been proven to be completely exhaustive . in particular , the genetic alterations characteristic of the adenoma - carcinoma sequence has not clearly established . to date , modification in three main molecular patterns has been identified : insulin - like growth factor ( igf)-2 , the metabolic pathway wnt/-catenin and tp53 . accs can be either functioning ( i.e. producing hormones of the adrenal cortex ) or non - functioning . adrenal malignancies are more likely to be functioning with respect to adenomas and can have different clinical presentations , depending on the type of hormonal secretion . in fact , patients may have symptoms of hypercortisolism , hyperaldosteronism , or sex hormone excess ( either virilization or feminization ) , although mixed clinical features can be observed . feminizing accs , i.e. malignant adrenal tumors causing features of estrogen excess , are an extremely rare cause of abdominal mass . we herein present a case of a young man having a giant feminizing acc with distant metastases , which posed serious management problems principally related to the burden of the primary tumor . a 41-year - old gentleman presented complaining of a 6-month history of bilateral gynecomastia without galactorrhea , and a significant weight loss ( 8 kg in the last three months ) . in the last two months he also noticed abdominal and back pain , dyspepsia , and some episodes of vomiting , bloating and difficult passage of stools . the rest of his medical history was not relevant and he was on no medications . his family history revealed the presence of a 37-year - old sister with an established diagnosis of men1 ( primary hyperparathyroidism , pancreatic gastrinoma , and prolactinoma ) with germline mutation 894 - 9 ga , and a 50-year - old brother affected by an adrenocorticotropic hormone ( acth)-secreting pituitary adenoma causing cushing s disease . as the patient was in good health before the appearance of gynecomastia , he had previously refused to undergo laboratory and genetic testing . laboratory tests showed : i ) extremely high estradiol plasma levels ( e2>500 ) , together with undetectable gonadotropin levels and low testosterone ; ii ) the likely coexistence of cushing s disease ( hypercortisolemia with increased acth levels ) ( table 1 ) . in relation to the latter , an overnight dexamethasone suppression test ( 1 mg at 11.00 p.m. ) was carried out , which failed to show serum cortisol suppression ( 19 g / dl at 08.00 a.m. ) . on abdominal examination , a large , firm mass adherent to the deep layers was palpable in the left quadrants . the total body computed tomographic ( ct ) scan showed a large tumor of the left adrenal ( 25 cm in its maximum diameter ) with colliquative areas infiltrating the left kidney ( figure 1 ) , left renal vein thrombosis , and multiple liver and lung metastases . pelvic ct scan revealed no abnormality , while breast and testicular ultrasound confirmed the presence of gynecomastia and reduced testicular volume . after a multidisciplinary discussion , it was decided to operate on the patient to remove the adrenal tumor , in an attempt to reduce the symptoms related to the mass , principally pain and recurrent subocclusive episodes . before the operation , a medication with ketoconazole was started for hypercortisolemia ; however , replacement of ketaconazole with metyrapone was necessary to normalize the cortisol levels . the patient underwent an exploratory celiotomy where a large encapsulated mass was encountered , displacing the kidney inferiorly and the distal pancreas and spleen superiorly . the right adrenal also appeared increased in size , thus a bilateral adrenalectomy was performed . on gross pathology examination , the left adrenal tumor ( which included approximately half of the left kidney , with diffusely infiltrated areas ) weighed 3.3 kg , and measured 271712 cm ( figure 2 ) . the histological examination revealed an acc with oncocytic changes with a weiss score of 8 ( figure 3 ) . based on the histological finding , the laboratory tests , and the presence of distant metastases , we made the diagnosis of feminizing adrenocortical carcinoma at stage iv according to the american joint committee on cancer ( ajcc ) classification . the postoperative course was uneventful , and the patient was discharged on postoperative day 12 . he was given replacement therapy with hydrocortisone , fludrocortisone , and mitotane therapy without any important side effects . six months after surgery , laboratory tests showed a normalization of cortisol and estrogens levels . gynecomastia was markedly improved , and a mild reduction of the size of liver and lung metastasis was observed . he went on to live 12 months with an acceptable quality of life before dying from metastatic disease . feminizing accs are very rare , accounting for only 1 - 2% of all malignant neoplasms of adrenal cortex . in 1994 , zayed reported fewer than 100 cases described in male sex . clinical features related to steroid excess occur in about 60% of accs , while feminizing symptoms due to estrogen secretion are extremely uncommon . the main presenting signs and symptoms of feminizing acc are gynecomastia , testicular atrophy , decreased libido and impotence . in line with other reported cases , gynecomastia was the presenting symptom of acc in our patient . gynecomastia recognizes various causes , being most commonly due to medications , imbalance between testosterone / estrogen levels , kidney failure , cirrhosis , and much more rarely to pituitary and adrenal tumors . it has been suggested that feminizing symptoms can be linked to aberrant aromatization of adrenal androgens in estrogens , principally e1 . feminization is often combined with hypercortisolism , as we observed in our patient . while aacs are for the most part sporadic , they can occur as part of various familial tumor syndromes , such as men1 , li - fraumeni syndrome , beckwith - wiedemann syndrome , and carney complex . the familiar history of our patient denoted the presence of well - defined endocrine pathologies ( men1 and acth - producing pituitary adenoma ) . adrenal neoplasms are not commonly included in men1 ; however , non - functioning macronodular hyperplasia is observed in up to 40% of patients with men1 and , even rarely , accs can occur . as a consequence , we investigated the existence of men1 syndrome in our patient , but the laboratory and imaging studies prevented us from confirming this hypothesis . in the described case , however , differentiation between benign tumors ( adenomas ) of the adrenal cortex and accs is not always simple to achieve . the likelihood of being malignant is very high for adrenal tumors producing sex hormones ; in particular , feminization is quite consistent with malignancy more often than virilization . as for the histological criteria , the model proposed by weiss in 1984 still remains the most accurate and the one that best correlates with the prognosis of accs . this model includes nine histological features distinguishing benign from malignant tumors . in our patient , one cause for this finding is certainly the fact that many accs are detected at advanced stage . according to the literature , only 25 - 30% of patients present with stage i - ii ( disease confined to the adrenal ) , whereas about 70% present with stage iii - iv ( disease extending beyond the adrenal ) . the two most important prognostic factors have been identified in stage at diagnosis and surgical radicality , though age , mitotic count , and ki67 expression have also been considered . historically , overall 5-year survival rates in patients with accs range between15% and 20% . in a study from the memorial sloan kettering cancer center , patients with stage i - ii disease and with iii - iv disease had 5-year survival of 60% and 10% , respectively . complete surgical resection is the only potentially curative treatment , and nowadays remains the mainstay in the treatment of localized accs ( stage i - iii ) , even in patients with extension to adjacent organs and positive local lymph nodes . even patients who undergo radical surgery bear high risk ( about 80% ) of local relapse and metastases , which typically occur within 2 years . when surgical removal is not achieved with radical intent , although the use of laparoscopic adrenalectomy for malignant aacs has been reported to be safe by some authors , there is a common agreement that open surgery should be preferred in this setting . our patient had stage iv disease because of distant metastases ( lungs and liver ) and invasion of adjacent tissues . in stage surgical resection of metastatic acc remains controversial , although surgery may have a role in metastatic accs providing that greater than 90% of the tumor and metastases can be removed , according to the national comprehensive cancer national guidelines . in the case described herein , the management decision was quite complex , as a result of the patient s young age , the presence of widespread disease and the local extension of the primary tumor . following a multi - disciplinary approach , we decided to operate on the patient because of the local effects of tumor . the intention was obviously not to cure the patient with surgery , but to ameliorate his quality of life , and thus obtain a good palliation for mass effects and hormonal symptoms . to note , he lived with an acceptable quality of life for 12 months after the surgical removal of the adrenal mass . we can reasonably speculate that , in this case , surgery might have prolonged the overall patient survival . mitotane , alone or in combination with other chemiotherapics has historically been the main medicament used in the adjuvant setting following radical surgery as well as in metastatic disease . its use is limited by significant , dose - dependent gastrointestinal and neurologic toxicity ; in our case the mitotane therapy was quite well tolerated . survival rates from acc have not substantially changed over the last 20 years , and systemic treatment , to date , is unsatisfactory . the phase iii firm - act trial comparing two different regimens in metastatic accs ( etoposide , doxorubicin , cisplatin , and mitotane versus streptozotocin and mitotane ) showed no difference between the two regimens in terms of overall survival ( 14.8 vs 12.0 months ; p=0.07 ) . as knowledge of the molecular mechanisms of accs continues to improve , it is likely that targeted therapies will improve survival outcomes in the near future . numerous trials are investigating targeted agents such as epidermal growth factor inhibitors , antiangiogenic agents , tyrosine kinase inhibitors , igf-1 inhibitors , and fibroblast growth factor receptor inhibitors . in our opinion , this report contains two principal points of interest . first , we emphasize the fact that feminizing acc should be taken into account as a possible diagnosis in patients presenting with gynecomastia , especially when the common causes for this disorder have been ruled out . second , surgery may have a role even in patients with functioning accs at stage iv , with the aim of improving quality of life by relieving symptoms of hormonal excess and mass effects .

a 27-year - old man was admitted to our hospital with a 10-day history of headache and vomiting . he was born with multiple pigmented areas on his skin ; specifically , there were multiple confluent hairy nevi on his extremities , back and most of the anterior trunk . the nonenhanced ct scanning showed multiple intracranial lesions ; these included a hyperattenuated mass with an adjacent cyst in the left temporal lobe , an irregular fatty mass containing marginal calcific foci within the temporal horn of the right lateral ventricle and a midline posterior fossa cyst with hypogenesis of the cerebellar vermis ( fig . the unenhanced mr scans showed a left temporal lobe mass with high signal intensity on the t1-weighted images and mixed low signal intensity on the t2-weighted images . in addition , there was a right intraventricular mass with bright signal intensity on the t1-weighted images and heterogeneous signal intensity on the t2-weighted images , relative to the cerebral cortex ( figs . further , there was hypoplasia of the inferior cerebellar vermis , dilatation of the inferior fourth ventricle and an enlarged posterior fossa , which confirmed the presence of a dandy - walker variant ( fig . the gd - dtpa enhanced t1-weighted images showed minimal marginal enhancement of the cystic lesion of the left temporal lobe mass with associated diffuse leptomeningeal enhancement ( fig . 1f ) . an excisional biopsy of the proximal hairy melanocytic nevus of the left ankle was done ; the diagnosis proved to be a melanocytic congenital nevus . the histologic sections of the temporal lobe mass revealed a melanocytoma , and the cyst wall showed a reactive gliosis . our patient had congenital giant hairy melanotic nevi of the skin as well as cns melanocytoma , and these findings fulfilled the diagnostic criteria of ncm as suggested by kadonaga and frieden ( 5 ) the pathogenesis of ncm is not yet sufficiently clear ; it is thought to come about by an error that occurs in the embryonic neuroectoderm during morphogenesis , and particularly in the neural crest ( 5 ) . melanocytes that originate from the neural crest are normally found within the basal layer of the epidermis , the pia mater , the reticular formation of the medulla and the substantia nigra . in ncm , there is a marked increase of the concentration of melanotic cells in their normal location with concomitant cell infiltration into the perivascular space . the melanocytes within the pia mater are responsible for the development of the leptomeningeal melanosis ( 4 ) . parenchymal melanosis is less commonly seen than leptomeningeal melanosis , and the former is thought to be caused by either the primary migration of the melanotic cells early in development or it is caused by their subsequent secondary spread via the virchow - robin spaces ( 1 ) . the anterior temporal lobes , and particularly the amygdala , seem to be the most frequent locations for the parenchymal melanocytic accumulation ( 3 , 4 ) , as was seen in the present case . the clue for the diagnosis of leptomeningeal melanosis or parenchymal melanin deposits is t1 shortening on the mr imaging . the cause of this effect is still controversial ; it might be the result of the presence of stable free radicals in the melanin in which the unpaired electrons interact with the water protons via an electron dipole - dipole interaction , and this causes the subsequent shortening of both the t1 and t2 relaxation times ( 6 ) . minimal diffuse enhancement of the leptomeninges without evidence of t1 shortening also indicated the presence of leptomeningeal melanosis ( 2 ) . ( 2 ) , the hyperintensity on the t1-weighted mr images depends upon the number and maturity of the melanocytes . the overall incidence of malignancy within the involved meninges is estimated to be on the order of 50% ( 1 ) . it is not possible to distinguish benign accumulations of melanocytes from malignant ones just based on the mr images . ( 3 ) suggested that only necrotic or hemorrhagic intracranial masses or the masses eliciting vasogenic edema in the patients with ncm can confidently be identified as malignant melanomas ; those masses without hemorrhage , edema or necrosis can not be classified unless they show growth on the subsequent scans . in our case , peritumoral cyst has been described in a case of intracranial metastatic melanoma reported by ogawa et al . ( 7 ) the cyst might have been formed either directly by the tumor itself or by pooling of the cerebrospinal fluid ( csf ) that was caused by blockage of csf flow by the tumor . neurocutaneous melanosis may be associated with other neurocutaneous syndromes such as sturge - weber or von recklinghausen 's disease . associations were also reported with dandy - walker complex , spinal lipoma and arachnoid cyst ( 8 , 9 ) . however , to the best of our knowledge , we are not aware of any previous description of the concurrence of intraventricular dermoid in a patient with ncm . a review of the relevant embryological data is helpful for considering the pathogenesis of such concurrent lesions in relation to the neural crest . intracranial dermoids originate from ectodermal inclusions of the primitive pleuripotential cells , and this is due to defects of the neural tube closure at around 3 - 5 weeks of gestation ( 10 ) . they are often located at the cranial midline within the posterior cranial fossa , the suprasellar cistern and the subfrontal areas . dermoids have characteristic ct and mri appearances ; they appear round or lobulated on ct with attenuation values from -150 to 0 hu , and they usually show a slight mass effect and foci of calcification without evidence of enhancement or surrounding edema after contrast . they have high signal intensity on t1-weighted mr images due to their lipid content , with a heterogeneous signal on the t2-weighted mr images due to the mixed composition of the tumor . fat - suppression techniques can be used to definitely demonstrate the presence of lipid within these lesions ( 10 ) . although pathologic correlation of the intraventricular dermoid in this case was not available since the patient 's family refused further surgical intervention , we feel quite confident of the diagnosis of dermoid because of the characteristic ct and mr imaging findings . the dandy - walker malformation is a rare developmental abnormality of the cns ; it is characterized by hypoplasia or aplasia of the cerebellar vermis , cystic dilation of the posterior fossa and a cystic dilatation of the fourth ventricle that communicates with the broad posterior fossa . the dandy - walker variant is a less severe form of the dandy - walker complex in which there is a better development of the vermis and the fourth ventricle posterior fossa cyst is smaller . ( 8) have proposed that the concurrent development of the dandy - walker malformation and the ncm , as seen in our patient , is not an incidental finding . dandy - walker complex may result from an insult to the development of both the cerebellar hemisphere and the fourth ventricle . any failure of incorporation between the choroid plexus and the roof of the fourth ventricle or the delayed opening of the foramen magendie may form the fourth ventricle - cisterna magna cyst . the meningeal cells , the melanin - containing abnormal leptomeninges may disrupt the development of both the cerebellum and the fourth ventricle ( 8) . in summary , we report here on a case of ncm that manifested as a temporal lobe melanocytoma and leptomeningeal melanosis with coexistent intraventricular dermoid cyst and dandy - walker malformation . this type of unusual presentation should be added to the spectrum of imaging abnormalities of ncm .

this commentary discusses the findings of udy and colleagues about the augmented renal clearance in septic and traumatized patients . antibiotic therapy is a key element of the treatment of patients with severe sepsis and septic shock , and success of antibiotic therapy is determined by multiple factors . susceptibility of the microorganism to the antibiotic is crucial , but reaching an adequate concentration of the drug is equally relevant . in recent years several studies found that plasma concentrations of antibiotics - with beta - lactam antibiotics investigated most intensely - are highly variable in critically ill patients , with a considerable number of patients not reaching adequate concentrations . several factors , such as increased volume of distribution , hypoalbuminemia and increased elimination from the circulation , have been found to be involved in this phenomenon . as most of these antibiotics are primarily cleared via the kidneys , renal elimination has been studied most extensively , and the state of increased elimination of drugs via the kidney has been coined ' augmented renal clearance ' ( arc ) , which is defined as a creatinine clearance of 130 ml / minute/1.73 mor higher . the incidence of arc has mainly been studied in smaller studies , and depending on the cutoff used for its definition and on the studied population , the incidence varied from 30% to 85% [ 3 - 5 ] . in their article , udy and colleagues confirm the high incidence of arc in a trauma and sepsis population , and found that age , trauma as admission diagnosis and a sequential organ failure assessment ( sofa ) score of 4 or less were independently associated with arc . although they provided us with new insights in the characteristics of patients at risk of arc , they could not translate their results into easy to use clinical guidance . identifying patients who present with arc is obviously a desirable goal , in order to appropriately treat patients with severe infections . however , evaluation of kidney function in the icu is notoriously difficult , and conventional biomarkers such as serum creatinine or estimates of glomerular filtration rate , such as cockcroft - gault or modification of diet in renal disease ( mdrd ) formulas , are not reliable , which is also the case in patients with arc . at present , creatinine clearance can most conveniently be measured using 8 or 24 h calculated creatinine clearance . when these data are not available , the results of the current study can serve as a guide to screen for arc . in the study by udy and colleagues , cardiac index correlated with creatinine clearance - albeit poorly - and was not retained in the multivariate analysis . although the methodology for cardiac index measurement used could also have influenced this finding , it may also demonstrate that renal clearance is determined by multiple factors and the search for surrogate markers may be elusive . the incidence of arc in more diverse and other icu populations has been incompletely explored . better methods for the early recognition of patients with arc are necessary - potentially novel biomarkers such as neutrophil gelatinase - associated lipocalin ( ngal ; currently mainly investigated in the setting of acute kidney failure ) are worth exploring . finally , diagnostic accuracy of different methods to diagnose arc , including measured creatinine clearance , needs urgently to be determined . other than focusing on factors associated with the determinants of changed pharmacokinetics of antibiotics , performing actual therapeutic drug monitoring ( tdm ) may be an alternative approach . this is currently standard for drugs that have a narrow therapeutic index such as aminoglycosides or glycopeptides , but for other - safer - drugs , tdm may offer a solution to monitor efficacy rather than toxicity . however , tdm of beta - lactams is rarely used in routine practice because the potential benefits of tdm have not yet been established , and because of long turn - around time and high costs .

kidney transplantation is the optimum replacement therapy for patients with established renal failure ( erf ) , as it offers better quality of life and improved survival . the demand for renal transplantation has increased due to the growing prevalence of erf and extension of the criteria for accepting patients onto the waiting list . in response to the increasing need for organs , deceased donor programs ( donation after circulatory death ( dcd ) and donation after brain death ( dbd ) ) are being optimized , and living kidney donation expanded in several countries to include both related and unrelated donation . further developments include abo ( blood group ) incompatible transplantation , legalised altruistic nondirected living donation , and adoption of paired or more complex exchange of living donor programs . in the last decade in the uk , there has been significant growth in living donor kidney transplantation with 485 transplants in 2005 , increasing to 1,055 in 2012 . all these have not succeeded in meeting the demand for renal transplantation , and efforts to provide more donors have included the use of marginal living donors , particularly elderly living donors . the use of grafts from elderly deceased donors ( dd ) is associated with less than ideal graft function and graft survival in recipients [ 3 , 4 ] . this is attributed to reduced nephron mass , senescence , greater susceptibility to ischemic injury , and acute rejection episodes being more prevalent in the elderly . whereas donor age is a strong determinant of death censored graft survival among recipients of deceased donor kidneys , the relationship between living donor age and graft survival is less clear [ 6 , 7 ] . kidneys from deceased older donors are more likely to be transplanted into older recipients , and this may potentially confound the impact of donor age on outcome . acceptance of elderly living donors remains controversial due to the higher incidence of comorbidity and greater risk of postoperative complications . such a risk to a donor whose benefit is at best psychological may be difficult to justify , as it may stretch the first do no harm principle [ 58 ] . as waiting times for transplantation increase , older candidates are more disadvantaged by their rapidly deteriorating health , often resulting in death or removal from the waiting list before transplantation . uk transplant statistics for 2011/2012 show that 18% of patients on the transplant waiting list have either died or been removed from the waiting list by five years of listing . furthermore , only 65% of patients listed would have received a transplant by five years . this is a review of published evidence about renal transplantation from elderly living donors to determine trends in elderly living donation , the effect of donation on elderly donors , and outcomes of such transplantation . english language publications on living donor renal transplantation from 2000 to 2013 were obtained from electronic databases such as medline , the cochrane central register of controlled trials , embase ( 20002012 ) , the database of abstracts of reviews of effects ( dare ) , ovid system , health technology assessment ( hta ) database , google scholar , and elsevier 's scientific search engine ( scirus ) ; pertinent journals were searched to identify relevant studies including randomizes trial , meta - analysis , and case series . the search strategy included but not limited to the terms kidney , renal , transplantation , older , living , donor , outcomes , graft survival , and patient survival . all related referenced articles in the english language literature between 2000 and 2013 were reviewed , and studies involving combined renal and other solid organ transplantation were excluded . all potential and relevant citations were retrieved in full text for detailed evaluation . when the same group of donors was studied in multiple publications , all were reviewed , and the strongest evidence - based article with long - term followup was cited . the number of glomeruli per kidney and the mean glomerular volume negatively correlate with age beyond 60 years but positively correlate with kidney weight . the number of sclerosed glomeruli per kidney also increased to 3050% after the age of 60 [ 11 , 12 ] . humans may lose renal reserve as they age because of nephron loss , possibly secondary to glomerulosclerosis and/or renal microvascular disease . in healthy nonnephrectomised individuals , induced renal hyperfiltration varies between 20 , and 35% of basal nonstimulated glomerular filtration rate ( gfr ) . although the postnephrectomy gfr may not be affected by age , the post nephrectomy reserve capacity of the remaining kidney as assessed by amino acid - induced hyperfiltration was significantly impaired in older and heavier donors . kidneys from elderly donors have a lower functional nephron mass at the time of transplantation , compared with allografts from younger donors . following kidney donation however , there is no accelerated loss of kidney function . the short - term kidney function in the donor recovers to 70% of predonation estimated gfr . the incidence of erf among donors is similar to that of the general population and ranges from 0.2 to 0.6% [ 18 , 19 ] . the process of aging results in complex alterations in how the kidney copes with normal homeostasis as well as acute and chronic injury . distinct processes that can explain why the kidney from an elderly donor regenerates less satisfactorily than the kidney from a younger donor include a diminished proliferative reserve , which might in part depend on altered progenitor cell function , an increased tendency for apoptosis , alterations in growth factor profiles , and important changes in immune responses [ 20 , 21 ] . according to the 2006 united states renal data system report , the risk of dgf in transplants from living donors above the age of 65 years is double that of transplants from younger donors . another report , a retrospective cohort study of 49,589 recipients between 2000 and 2009 in the united states , showed that for every 10-year increase in living donor age , the odds of dgf increased by 15% . these observations can be partly explained by the impaired ability for repair in kidneys from elderly donors . the goal of living donor assessment is to ensure the suitability of the donor and minimisation of risk of complications [ 25 , 26 ] . a key component of donor selection is a comprehensive medical assessment according to the criteria set by the amsterdam forum . this is in line with the procedure advocated by the british transplantation society ( bts ) . during assessment , the donor should be considered to be a patient just like the transplant recipient and should get the same level of care and protection against risks . elderly donors require even more rigorous assessment due to the higher prevalence of co - morbid conditions . living kidney donors with normal renal function prior to donation are at no greater risk of developing erf after unilateral nephrectomy than individuals in the general population [ 18 , 31 ] . a direct measurement of gfr using iodinated or radioactive isotopes is ideal for assessing renal function in a potential donor . however , most transplant centres determine gfr by measuring creatinine clearance using a 24-hour urine collection . several studies recommend that living donors should have a gfr of 80 ml / min or , alternatively , a normal kidney function level within two standard deviation , ( sd ) for age and gender [ 26 , 33 , 34 ] . however , some centres utilise a gfr of 80 ml / min per 1.73 m to be the lower limit for donation [ 3436 ] . there are relatively few published data on kidney function in normal populations stratified by age . the use of a single cutoff value does not take into account the decline in gfr with aging [ 37 , 38 ] . therefore , to identify potential donors at increased risk of developing erf , cutoffs should vary based on age on the premise that an individual would not develop clinically significant renal impairment as a result of unilateral nephrectomy . most of the centres that utilise a cut - off gfr do so on what the likely gfr would be at the age of 80 . whether this means that , after 80 , a donor might be accepted with an even lower gfr is debateable . had shown that transplants from living donors with a gfr of 80 ml / min ( not adjusted for body surface area ) had graft survival worse than those from donors with higher gfr . this highlights the fact that the evaluation of kidney function is important not only to protect the health of a donor , but also to ensure adequate function for the recipient . measurement of egfr in living donors has not been validated to predict the risk of long - term kidney disease and should not be used in this context . the bts guideline recommends that a prospective donor should not be considered for donation if the corrected gfr is predicted to fall below a satisfactory level of kidney function within the lifetime of the donor . for example , a predicted gfr of at least 37.5 ml / min/1.73 m at the age of 80 is recommended as a minimum standard . there is a lack of evidence to guide acceptable levels of kidney function for donors over 60 years of age . brindel et al . conducted a population - based study of noninstitutionalised individuals aged 65 years and reported that 62% of 9090 people were hypertensive with 81% of these on antihypertensive drugs . this finding is similar to another study using the database of the national health and nutrition examination survey , which showed the prevalence of hypertension ( defined as 140/90 mmhg or taking antihypertensive medications ) to be 7.3 0.9% , 32.6 2.0% , and 66.3 1.8% in the 18 to 39 , 40 to 59 , and 60 age groups , respectively . the framingham study showed that hypertension was more common in the elderly , and the overall risk for a cardiovascular events and deaths due to cardiovascular disease was two to three times higher in subjects with definite hypertension compared with normotensives for all age and sex groups considered . mild to moderate hypertension that is controlled with single or double antihypertensive agents is not a contraindication to kidney donation providing significant end - organ damage has been excluded . the presence of hypertensive end organ damage , poorly controlled hypertension , or hypertension that requires more than two drugs to achieve adequate control are relative contraindications to living donation . evaluating the potential medical risks to individual donors presents a complex problem , particularly in the elderly , where age - associated conditions such as the decline in gfr , hypertension , impaired glucose tolerance , and weight gain assume increased significance after nephrectomy . in assessing people for kidney donation , postnephrectomy risks should be evaluated in terms of exposure over the duration of remaining lifetime . the mayo kidney / pancreas transplant program stratifies medical criteria according to age , allowing more liberal criteria for older donors based on their belief that many of the long - term results of kidney donation are likely to hinge upon future behaviour , including smoking , weight management , and medical follow - up care . older donors are more likely to have established behavioural patterns , an element that makes them better candidates in many respects . such an approach requires careful follow up in order to determine the impact of donor nephrectomy in the current evolving environment . studies addressing the issue of accelerated kidney damage in patients with single kidneys who develop diabetes have produced conflicting results . it is thought that hyperfiltration of the remaining kidney would cause donors who develop diabetic nephropathy to accelerate to erf more rapidly than patients with two kidneys , but there is no evidence in donors who develop diabetes to prove this hypothesis . however , those who develop diabetes after kidney donation do have more proteinuria and hypertension . many guidelines , such as the one by the amsterdam forum , while recommending that individuals with diabetes should be excluded from donating , they do not address donors in the prediabetes state . on the basis of cohort studies , erf in living donors , a rare event , occurs in a median of 20 years after donation ; therefore , the younger the potential donor with impaired fasting glucose , the greater the cumulative risk for developing diabetes and its resultant complications . the observation by vigneault and coworkers that younger patients with prediabetes have more time to develop diabetes and its complications would imply that exclusion of older donors with impaired glucose tolerance could be relaxed , as the interval to developing diabetic nephropathy would make it irrelevant , as most elderly donors would have died from other causes . data from 2006 indicate that the greatest number of living donor kidney transplants were performed in the united states ( 6,435 ) , united kingdom ( 2,020 ) , brazil ( 1,768 ) , iran ( 1,615 ) , mexico ( 1,459 ) , and japan ( 939 ) . during the last decade , 62% of the countries reported at least a 50% increase in the number of living kidney donor transplants . also , the number of living donor kidney transplants performed in the us and canada doubled and represented about 40% of all donor kidneys . reported that about 27,000 related and unrelated living donor kidney transplants were performed worldwide in 2006 , representing 39% of all kidney transplants . this growing trend is also reflected by the increase in the number of living donors > 60 years during the last decade . since 2007 , there have been more living donor transplants performed in the uk than deceased donor transplants . whereas 34% of deceased donors in the uk were aged over 60 years , the proportion of living donors aged over 60 in 2011/2012 was 14% ( nhs blood and transplant personal communication ) . furthermore , the characteristics of the living donors have changed , particularly with the acceptance of progressively older living donors . friedman et al . reviewed 6320 cases of living donors and reported a complication rate of 18.4% with no mortality . independent predictors of donor complications were older age ( odds ratio ( or ) , 1.01 ) , male sex ( or , 1.19 ) , charlson comorbidity index of at least 1 ( or , 1.49 ) , obesity ( or , 1.76 ) , medium - size hospitals ( or , 1.88 ) , and low - volume hospitals ( or , 1.37 ) . other factors affecting donor risk of chronic kidney disease ( ckd ) include baseline renal function , older age , and duration after kidney donation . the clinical course and risk factors for developing erf after kidney donation have not been properly investigated . donor nephrectomy represents a sudden loss of approximately 50% of the nephron mass with an immediate and corresponding decrease in gfr ; however , the remaining contralateral healthy renal parenchyma has the ability to recover a significant percentage of lost function within a relatively short period as early as one month . . showed that as early as one week after nephrectomy , renal function has recovered to levels slightly lower than those achieved at six months after nephrectomy . others have shown that the gfr at one year after donation was essentially similar to the value achieved at one week after donation [ 55 , 56 ] , suggesting little recovery of function after the initial period . it has been observed that donors with a decreased renal mass may have a higher risk of developing proteinuria , hypertension , and chronic renal disease during long - term followup . saxena et al . also examined the magnitude of adaptive hyperfiltration in the remaining kidney of 16 older ( > 57 yr ) and 16 younger ( < 55 yr ) individuals who had undergone a contralateral nephrectomy and concluded that the magnitude of adaptive hyperfiltration is similar in the elderly to that in young subjects with single kidneys , albeit at a lower absolute gfr level . velosa et al . evaluated 140 donors ( 105 were < 35 years and 35 were > 55 years old ) in whom the predonation gfr in the younger group of 113 ml / min was compared to 88 ml / min in the older group and showed that postnephrectomy percentage change of gfr was 68 8 and 65 8 in younger and older groups , respectively . this is similar to the finding in a larger prospective study ( 19942006 ) of 539 consecutive recipients of kidneys from 422 living donors < 60 years , compared to 117 living donors > 60 years , in which elderly donors had lower gfr before donation ( 80 versus 96 ml / min resp . , the maximum decline in egfr was 38% 9% and the percentage maximum decline was not different in both groups . on long - term followup , significantly more elderly donors had an egfr < 60 ml / min ( 131 ( 80% ) versus 94 ( 31% ) , p < 0.001 ) [ 59 , 60 ] . a study by poggio et al . of 1015 donors showed that the decline in gfr was approximately 4 ml / min per 1.73 m per decade of life for donors who were younger than 45 years , compared to 8 ml / min per 1.73 m in those older than 45 years . several investigators hypothesized that kidneys from older donors would have a decreased renal reserve capacity that would manifest as impaired kidney function after donation . examined the effect of donor nephrectomy on gfr at 3 months and the occurrence of stage 3 ckd using i - iothalamate gfr ( igfr ) , modification of diet in renal disease ( mdrd ) estimated gfr , cockcroft - gault estimated creatinine clearance , and endogenous 24-hr creatinine clearance and found that the prevalence of stage 3 ckd was greater in the elderly . the long - term impact of stage 3 ckd after uninephrectomy is poorly understood and may not have the same implications as stage 3 ckd brought on by other causes . living donors have long - term risks that may not be apparent in the short term . the japanese study of eight donors who developed erf and were compared with a control population of 24 donors matched for age , sex , and follow - up time since donation showed that , apart from one donor who developed erf caused by a traffic accident , none developed progressive renal dysfunction immediately after donation . however , after 10 years , the development of persistent proteinuria , cardiovascular event , or major infection heralded ckd . the organ procurement and transplantation network ( optn ) examined its database , cross - checking it with renal waiting list history files to identify previous living donors subsequently listed for cadaveric kidney transplantation . they identified a total of 56 such people43 having received transplants and two candidates had died while waiting . a survey by optn in conjunction with the centre for medicare and medicaid services identified 126 cases of erf among 56458 living kidney donors ( 0.22% ) , who donated during 19872003 . the overall erf risk was 0.134 per 1000 years at risk with an average duration of followup of 9.8 years . erf rates for living donors overall and for black , white , male , and female donors were compared favourably to the erf incidence in the general population . the erf rate in living donors was nearly five times higher for blacks than for whites and two times higher for males than females . in another report , which focused on african americans , optn data revealed that , although african americans comprised 14% of living kidney donors , they constituted 43% of former donors who were listed for transplantation . however , these ethnic and gender - related differences were similar to those previously reported for erf in the general population and support the current practice of living kidney donation . only three of 84 donors in a cohort of 464 living donors died with / from kidney failure . conducted a retrospective analysis of 146 living - related donors > 50 years old from 1976 to 2005 and reported that the rate of diabetes and hypertension was similar to that of an age matched general population . other reports including a larger swedish study of 402 live donors found the age - adjusted prevalence of hypertension among donors to be similar to that in the general population . however , a retrospective norwegian study of 908 donors ( 19972007 ) , showed a progressive increase in hypertension rate after kidney donation . this increase in hypertension risk was also the conclusion of a meta - analysis concluded by the donor nephrectomy outcome research network , revealing that kidney donors may have a 5 mmhg increase in blood pressure within 5 to 10 years after donation over that anticipated with normal aging . the type of nephrectomy may exert important influence on the quality of life after donation . laparoscopic nephrectomy is associated with less postoperative pain and better quality of life compared to open donor nephrectomy . kok and coworkers have reported that , one year after donation , patients who underwent laparoscopic nephrectomy had less physical fatigue and better level of physical function than those who had open donor nephrectomy . minnee and coworkers conducted a prospective study of postoperative complications and quality of life in 105 consecutive living donors who underwent a laparoscopic donor nephrectomy between 2002 and 2006 , comparing donors over 55 years with younger donors . they found no significant differences in intra- and postoperative complication rates even though elderly donors ( n = 34 ) had both a significantly lower postoperative pain on day one ( p = 0.019 ) and a lower total pain score in the analysis for the whole follow - up period ( p = 0.002 ) . although their cut - off age for the elderly was relatively low at 55 years , their study gives support to the use of elderly donors in screening programs for transplantation . also showed that the effect of donation on quality of life is not related to donor age or gender . o'brien et al . performed a cross sectional study on 383 living donors stratified into groups according to age ( < 65 years , > 65 years ) and bmi in a single centre with followup of over 5 years and showed no significant differences in operative parameters such as operative time and estimated blood loss between groups . although rates of early postoperative complications were not significantly different , subgroup analysis showed a higher incidence of respiratory complications at the extremes of obesity ( body mass index 40 kg / m ) . they concluded that nephrectomy in selected donors who may otherwise have been precluded on account of their age or weight resulted in perioperative or longer term outcomes comparable with their younger counterparts . in a study comparing 115 recipients of kidneys from living donors > 60 years with 158 from donors < 60 years , the frequency of acute rejection ( ar ) episodes was found to be similar in both groups , but delayed graft function occurred more frequently in the former group . the frequency of chronic renal allograft dysfunction in the first posttransplant year was significantly higher in transplants from older donors . ferrari et al . assessed the impact of donor - recipient age difference on living donor kidney transplant outcomes by using a multivariate competing risks cox model and showed that donor - recipient age difference was neither associated with increased risk of acute rejection within the first six months , nor with increased patient death , death - censored graft failure , or serum creatinine at five or 10 years . however , the european senior program ( esp ) has shown that allocating elderly donor organs to elderly recipients while resulting in shorter cold ischemia times and reduced dgf rates , it is associated with a 510% higher rejection rate . graft and patient survival were not negatively affected by the esp allocation policy when compared to standard allocation rules . in a series of 147 dcd recipients , akoh and rana demonstrated a significantly higher acute rejection rate in younger recipients of older dcd grafts in spite of better hla mismatch profile . with the increasing use of elderly donors , it would be interesting to see what effect this has on transplant outcomes of younger recipients of grafts from older living donors . the outcome of transplantation of kidneys from living donors has been shown to be superior to those from deceased donors with regards to early graft function and patient survival , irrespective of the degree of mismatch [ 6 , 8083 ] . kerr and coworkers conducted a univariate analysis of 1,126 consecutive transplants ( 19851995 ) and demonstrated that the graft survival of kidneys from older living donors is better than deceased donor kidneys from older donors and is comparable to deceased kidneys from younger donors . with living donor kidneys , delayed graft function ( dgf ) is unusual , and long - term results are equivalent for both related and unrelated donor transplantation . a systematic review of transplant outcomes for recipients of living donor kidneys from 1980 to june 2008 showed that recipients of kidneys from older living donors ( > 60 years of age ) have poorer 5-year patient and graft survival than recipients of kidneys from younger donors . this meta - analysis included studies with varying levels of evidence , but recent studies have showed no difference in graft survival in transplantation from older versus younger donor kidneys [ 78 , 85 ] . chang et al . showed that with the exception of recipients aged 1839 years , who had the best outcomes with donors aged 1839 years , living donor age between 18 and 64 years had minimal effect on allograft half - life ( difference of 1 - 2 years with no graded association ) . this study however , had a relatively small number of living donors > 60 years and did not account for death as a competing risk . a more recent retrospective cohort study demonstrated significant differences in overall survival , and death censored survival and death with graft function in recipients stratified according to living donor age categories . they reported a 53% increase in the hazard for total graft failure in recipients of kidneys from donors > 60 years compared to donors between 18 and 29.9 years . in a large retrospective series of 73,073 first kidney - only transplant recipients in the united states between 1995 and 2003 , it was reported that the risk of graft loss with living donors of 5564 years was similar to that with deceased donors < 55 years . however , recipients from living donors over 65 years had a higher relative risk of graft loss ( hazard ratio ( hr ) = 1.3 , 95% ci : 1.11.7 ) and > 70 years ( hr = 1.7 , 95% ci : 1.12.6 ) . in a select group of recipients from 219 living donors over 70 years in the us , graft loss was significantly higher than matched 50- to 59-year - old live donor allografts ( subhazard ratio 1.62 , 95% confidence interval 1.162.28 , p = 0.005 ) but similar to matched nonextended criteria 50- to 59-year - old deceased donor allografts ( subhazard ratio 1.19 , 95% confidence interval 0.871.63 , p = 0.3 ) . gill and coworkers evaluated 23,754 kidney transplantations performed in recipients 60 years and older of which 7,006 were living donors ( 1,133 were > 55 years , and 5,873 were 55 years ) showing that outcomes were best in younger living donor transplantations , followed by standard criteria deceased donor and older living donor transplantations . kidneys from older donors provide a statistically poorer outcome in transplant recipients [ 49 , 89 ] . a multivariate analysis of 1,063 recipients revealed that the age of a living donor is an important determinant of long - term graft survival , particularly in younger recipients . analysed 1,632 recipients who underwent ld kidney transplantation between 1990 and 2009 in the us . donor age more than 65 years , five to six hla mismatches , dgf , and acute rejection were independent predictors of decreased patient and graft survival . even after controlling for recipient age , donor age of more than 65 years remained a risk factor for a worse outcome . this is in agreement with a large uk retrospective study of 3,142 first adult kidney transplants from living donors . however , a retrospective cohort study showed no significant difference in total graft loss when transplants from older living donors ( > 60 years ) were compared with younger donors ien and coworkers performed a cox regression analysis to estimate the association between different risk factors including donor age , hla - dr mismatch , female gender , graft survival , and acute rejection episodes in a prospective cohort study of 739 first time living donor transplantation . their multivariate analysis further showed donor age > 65 years was a risk factor for graft loss in all time periods after transplantation . however , graft survival was not affected by donor age above 50 years if recipients did not experience an early acute rejection episode . in their series , the incidence of acute rejection increased in recipients of grafts from donors > 65 years ( p = 0.009)similar to the finding by akoh and rana in a series of recipients of dcd kidneys from elderly donors . a study from korea evaluated the outcomes from 269 living donors , in which 64 were expanded criteria living donors and 205 were standard criteria donors . their definition of an expanded criteria living donor included at least one of five criteria ( age greater than 60 years , body mass index > 30 kg / m , history of hypertension , estimated gfr < 80 ml / min , and proteinuria or microscopic hematuria ) . the recipients of organs from the expanded criteria living donor group showed a lower estimated gfr at one year after transplantation than the standard criteria group ( 66.9 16.0 versus 58.3 11.2 ; p < 0.001 ) although graft survival was not different ( p = 0.518 ) . most studies showed that kidneys from older donors had relatively lower graft function , increased rejection episodes and poor long - term graft survival compared to kidneys from younger donors . the prevalence of hypertension , established renal failure , and quality of life in donors is comparable to that of the general population . a multicentred , long - term , and prospective database , which is specifically aimed to address the outcomes of kidneys from elderly living donors is recommended . this review demonstrates that there is no clear definition or agreement on who should be regarded as an elderly living donor . many of the studies cited have used different age cutoffs50 , 55 , 60 , and 65 years . age stratification may be required in any future study to properly elucidate the effects of aging on kidney function and living donation . this review has uncovered two areas of urgent need of study in relation to elderly living kidney donors . firstly , there is a dearth of robustly conducted studies on quality of life of elderly donors . given the rising trend of living kidney donation , particularly in the elderly , and the increasing complexity of dependency needs of an aging population , such a study will provide much needed information . secondly , there is a clear need to analyse the outcome of parents to offspring living donor transplantations , particularly from donors over 65 years . such transplantation involves donation of a relatively lower nephron dose , and it is therefore important to determine whether they perform better long - term than deceased organs from younger donors .

in middle age people , total cholesterol levels have been established as a risk factor for a cardiovascular disease ( cvd ) risk marker . in finland data have been shown that mortality rate from cvd among people high plasma total cholesterol level people ( > 300 mg / dl ) is fivefold higher than other factors and reducing plasma total cholesterol level by 10% can reduce the mortality due to cvd up 30% . according to a survey in the usa , 50% of adults showed cholesterol level higher than 200 mg / dl , while 37 million people had levels higher than 240 mg / dl . showed that the prevalence of mortality and morbidity due to cvd can be estimated by the determination of plasma cholesterol levels in young and adult people . several other studies have shown that many factors such as lifestyle , diet , smoking , bmi , gender , physical activity , and age are correlated with mean plasma cholesterol level . of 1.6 million people in golestan province ( northern iran and south east of caspian sea ) , 66.39% are 25 - 65 years old , whereas 43.9% and 56.1% are living in urban and rural area , respectively . the main objective of this study was to determine the plasma total cholesterol status and some associated factors in people of urban and rural areas in northern iran . this study may suggest the ways to decline or prevent the risk of cvd in this area . this was a population - based cross - sectional study conducted in golastan province ( northern iran ) . regarding the previous study and 95% confidence interval , 1995 subjects ( 997 males and 998 females ) 100 clusters of 20 cases were randomly selected by family code in primary health centers in rural areas and postal code in urban areas with equal proportions of genders . from each district , one team was trained to complete the questionnaire and measure anthropometric indexes . the questionnaire included demographic characteristics , residential area , educational level , and physical activity . all family members in blocks who were in 25 - 65 years were included in the clusters . pregnant women and those who were unwilling to participate in this study were excluded from the study . weight was measured with light clothing without shoes and height was measured with standing up and head , back , and buttock on the vertical land of the height gauge . body mass index ( bmi ) was calculated as weight ( kg)/height ( m ) and using world health organization classification . bmi of 25.0 - 29.9 kg / m was classified as overweight , bmi of 30.0 - 39.9 kg / m was classified as obese , and bmi 40 kg / m was classified as pathologic obese . waist circumference higher than the normal range ( men > 102 and women > 88 cm ) was determined as abdominal obesity . physical activity was categorized into five categories based on daily work and activity including no physical activities ( without moving from one place to another ) , low physical activity ( activity that requires extension of the muscular skeletal system and moving from one place to another ) , moderate physical activity ( activity that requires sometimes increased respiratory rate like cleanliness , gardening , building painting , etc . ) , high physical activity ( activity that requires highly increased reparatory rate like manual labor , building labor , etc . ) , and very high activity ( a combination of above activities ) . for measuring plasma total cholesterol level , plasma total cholesterol was measured with commercial kits ( pars azmoon , karaj , iran ) by an auto - analyzer . the plasma cholesterol level spss 16.0 software was used for the statistical analysis , the chi - square test was used for comparing frequencies and the t - test and anova were used for comparing the means . logistic regression analysis was applied in order to estimate the odds ratio ( or ) of hc considering the socio - demographic factors at 95% significant level . the reliability was assessed using cronbach 's alpha coefficient and was found to be 0.86 . this study was approved by ethical research committee and consent was received from all participants . the mean and standard deviation of age was 44.2 11.3 years . of the 1995 subjects , 50% , 46.7% , 29% , and 43.3% were men , urban residence , general , and abdominal obese , respectively [ table 1 ] . characteristics of study subjects ( n=1995 ) the mean and standard deviation of plasma total cholesterol levels were 203.6 40.7 mg / dl and it was 12.7 mg / dl higher in women than in men . there was a positive significant correlation between age and plasma total cholesterol level ( p = 0.001 ) . plasma total cholesterol level decreased with physical activity ; the mean of plasma total cholesterol level in the low active group ( 205.1 mg / dl ) was 14.9 mg / dl higher than in the very active group ( 190.2 mg / dl ) which was statistically significant ( p = 0.019 ) . the plasma total cholesterol level had a positive correlation with bmi p = 0.001 ) , and in the obese group ( bmi 40 kg / m ) ( 255.5 mg / dl ) it was 71.4 mg / dl higher than in the thin group ( bmi < 18.5 kg / m ) ( 184.1 mg / dl ) [ table 2 ] . the mean and standard deviation of plasma total cholesterol levels based on some related factors the overall prevalence of hc was 50.9% and it was up to 12.3% higher in women ( 57% ) than in men ( 44.7% ) ( p = 0.001 ) . the prevalence of hc was 25.1% among 55 - 65 years age participants ( 61.5% ) higher than among that observed in the 25 - 35 years age group ( 36.4% ) . hc was significantly common in abdominal obese subjects ( 63.2% ) higher than in normal subjects ( 41% ) ( p = 0.001 ) and in the urban area ( 53.1% ) it was 4.1% higher than in the rural area ( 49% ) without statistically significant difference [ table 3 ] . the prevalence of hypercholesterolemia based on some related factors multiple logistic regressions were used to identify variables that contribute to hc . the risk of hc was found to be 1.64 [ 95% ci : 1.31 - 1.99 ] in female compared to male ; 2.79 [ 95% ci : 2.15 - 3.631 ] in 55 - 65 years subjects compared to 25 - 35 years ; 10.00 [ 95% ci : 3.75 - 26.67 ] in bmi 40 compared to bmi 18.5 and 2.47 [ 95% ci : 2.06 - 2.98 ] in abdominal obesity compare to normal people . no significant differences were found among residential area and physical activities [ table 4 ] . the results of this study show that half of adult population living in northern iran is hypercholestrolomic . the prevalence of hc has been reported to be in romania ( 70% ) , northwest mexico ( 52.6% ) , indian rural ( 22.3% ) , spain ( 24% ) , western samoa ( 36% ) , koki ( 25% ) , and saudi arabia ( 54% ) . hc prevalence in tehran ( capital of iran ) and in arak ( a capital city in central iran ) has been reported up to be 40.4% and 26.7% , respectively . as like as mentioned studies the prevalence of hc in the north of iran is high and should be consider as the most common health problem in this area . in our study , the prevalence of hc was seen to be higher in urban than in rural and higher in women than in men . there was a positive association between age , waist circumference , and bmi with plasma total cholesterol level . increasing hc in an urban population in the worldwide has been shown in some studies . after menopause , estrogen has a positive role in serum cholesterol level , therefore , estrogen therapy has been recommended for the control of cvd . in our study , half of the women were over 45 years , which may be used as a interfering factor for increased plasma total cholesterol level . the correlation between plasma total cholesterol level and age , waist circumference , and bmi in our study is similar to the earlier reports . the influence of physical activity on the serum cholesterol level was not similar in all studies . although the role of physical activity in decreasing plasma total cholesterol level has been shown in many studies , there was not any correlation between them in another . physical activities decreased the plasma lipid profile with statistical significant differences in hdlc and apoa1 . we do nt know whether there is any signification between physical activity and hc , but it seems that other related factors which are not included in our study such as ethnicity and food behavior do have influence on the plasma total cholesterol level . our study showed that hc is a health problem in northern iran and it is common in half the adult population . socio - economic status , general , and abdominal obesity are predispose factors for hc . further studies are necessary to examine the related factors such as life style , food behavior , ethnic differences , and awareness with hc .

hair transplantation is an accepted technique of restoring hairs on bald scalp in pattern baldness , by different techniques such as follicular unit transplantation , follicular unit extraction ( fue ) , etc . , however , one of the limiting factors in achieving successful outcome is the limited donor hair on occipital scalp , particularly in extensive baldness . body hair has been shown to grow longer when transplanted to scalp , as a result of the influence of scalp dermis on the transplanted hair , referred to as recipient influence . however , proper studies of successful body hair transplantation are yet to be documented and pubmed search revealed only few published case reportsr.[35 ] we report the outcome of transplantation of hairs from different areas such as chest , abdomen , arms , back , and thighs in a patient . further , he gave history of previous punch transplantation 18 years back , which had failed to give satisfactory results . occipital scalp examination showed multiple punch scars and poor density , of only 41 follicular units / sq cm . he was counselled about the poor donor area on scalp and was advised against a transplantation . he was determined to have a transplantation and requested for body hair transplantation , as he had good body hair and had read about the procedure on internet . detailed counselling was done about body hair transplantation , detailed history taken , general examination performed to rule out any contraindications for the surgery . investigations including blood counts , sugar , liver and renal function tests , electrocardiogram were within normal limits . a test graft session by fue technique was performed to determine its feasibility with 400 units from chest [ figure 1 ] . the hairs showed satisfactory growth after 5 months and after detailed counselling , larger sessions were planned . immediate postoperative photograph of test grafting with body hair in frontal area with 400 grafts initially , the procedure was planned under local cream anaesthesia , using eutectic mixture of lignocaine and prilocaine . however , this proved inadequate in relieving intraoperative pain and it was therefore decided to use tumescent anaesthesia , which was administered as per standard guidelines . sessions were performed from different areas , such as chest and abdomen [ figure 2 ] , arms , and thighs on different dates as shown in table 1 . table 2 shows characters of body hair , such as density and length from different areas . chest and abdomen immediately after extraction the dates of surgery and number of grafts transplanted hair unit density ( no of units per sq cm ) over different areas transection rates and yield after transplantation from different areas extracted grafts were kept in ringer lactate solution [ figure 3 ] and then transplanted in to the frontal , parietal , and vertex scalp [ figure 4 ] . the grafts started growing on recipient area from fourth month onwards and reached a length of 34 cms by 8 months [ figure 6 ] . table 2 shows length of the grafted hairs after transplantation in to scalp from different areas . yield ( ratio of hairs growing after transplantation and the number of grafted hairs ) after grafting from different body areas is shown in table 3 . extracted hairs in a petri dish with ringer lactate solution extraction sites over thigh healed 2 days after extraction frontal area immediately after implantation satisfactory result after 8 months patient was satisfied with the results . the patient has been followed up for a period of 24 months after the first session and 8 months after the last session . the patient s appearance has altered so much that he has had to change his passport ! the scientific basis for the procedure is provided by the concept of recipient influence , which suggests that dermis in the recipient area may exert influence on the growth pattern of transplanted hair and hence when body hair is transplanted on to scalp , it would grow longer , thicker . the rationale has been confirmed in a few published reports.[35 ] further , since the procedure is performed by extraction method which produces very small scars , and body is a cosmetically less significant area , body hairs can help to provide an alternative source of donor hair in selected patients . this is particularly so in indian patients , who do not expose body in public as sunbathing so common in the west , is not popular in indians . our report represents additional proof that body hair indeed can be a valuable alternative source in patients who lack scalp hair . however , the technique has limitations and the concerns that are yet to be addressed fully : the procedure is possible only in patients who possess good body hair , as in our patient . his chest hair density was particularly good , with density of 22 units per sq cm ( roughly half the occipital area density).routine methods of anaesthesia such as infiltration and blocks are not useful and hence the procedure can be painful . this was indeed so in our patient and hence tumescent anaesthesia had to be followed , which provided pain - free surgery.only the fue method is suitable for harvesting body hair . this makes the procedure slow and painstaking.the procedure is slow and needs high degree of motivation for the patientbody hairs are usually found as 1 and 2 hair units , and hence the density and thereby results achieved may not be as impressive as with scalp hair [ table 2].body hairs are shorter in length , are thinner and hence their extraction needs special technique and expertise . on an average the roots were located at a depth of only 34 mm . [ figure 3 ] . the transection rates varied in our patient [ table 3 ] from 13% to 32% . chest hair showed the least transaction rate , while back , arms , and abdomen showed much higher transection rate . however , since the patient was not bothered about loss of body hair , the cosmetic impact of transection was not relevant to the patient . the procedure is possible only in patients who possess good body hair , as in our patient . his chest hair density was particularly good , with density of 22 units per sq cm ( roughly half the occipital area density ) . routine methods of anaesthesia such as infiltration and blocks are not useful and hence the procedure can be painful . this was indeed so in our patient and hence tumescent anaesthesia had to be followed , which provided pain - free surgery . the procedure is slow and needs high degree of motivation for the patient body hairs are usually found as 1 and 2 hair units , and hence the density and thereby results achieved may not be as impressive as with scalp hair [ table 2 ] . body hairs are shorter in length , are thinner and hence their extraction needs special technique and expertise . on an average the roots were located at a depth of only 34 mm . [ figure 3 ] . the transection rates varied in our patient [ table 3 ] from 13% to 32% . chest hair showed the least transaction rate , while back , arms , and abdomen showed much higher transection rate . however , since the patient was not bothered about loss of body hair , the cosmetic impact of transection was not relevant to the patient . lastly , strong evidence for the procedure is lacking ; published reports are few and represent isolated case reports only . hence the procedure should be performed only in selected patients , in those who are well motivated , and after proper counselling and consent . this was indeed done in our patient , with a test grafting being done initially and separate special consent obtained . our case showed variable yield in grafts from different areas , the least yield was from back ( 29% ) , while the best yield was from chest [ table 3 ] . however , even the chest hair yield was much below that would be expected from scalp hair . the length of hairs after transplantation of body hair is a matter of debate , though it is thought that hairs grow longer on scalp . this is borne out to some extent in our patient , though the growth was only marginally higher than the length over the body hair . chest hair grew best and longest , by about 2 cms longer than the growth on chest . analysis of data showed that chest hair is the best of the different body hairs . it has best density , least transection , highest yield and longest growth after transplantation . we used tumescent anaesthesia in our patient - this is very helpful as the method helps to provide anaesthesia over a larger area , as it lasts longer , and is safer than using large doses of lignocaine by local infiltration . the method of extraction is similar to the one on scalp , but since the body hair is thinner and is located more superficially , the extraction has to be done more carefully . the long - term of outcome of body hair is yet to be determined , as the procedure is only a few years old . in our patient , it is only 24 months after first session and 8 months from last session and hence this is a limitation . it is also not known whether the method would work consistently in all patients and hence proper case selection and counselling of the patient is very important . in summary , body hair can be an alternative source in highly selected patients , with poor donor scalp and good body hair . chest hair is possibly the best among the different body sources , but even this chest hair growth and yield is moderate . the method needs to be established properly with more data to define its role and hence the author strongly feels that the current trend of publicizing it on internet websites is unjustified and should be discouraged .

it was first described in 1959 by bernstein and lichtenstein as a distinct variant of chs . this affects females more frequently than it does males ( f / m = 1.4/1 ) . these neoplasms are characterized by sheets or clusters of highly undifferentiated , small , ovoid cells that alternate with small zones of neoplastic cartilage . this type of neoplasm shows aggressive local behavior as well as a high metastatic potential . in this report , we present a case of mchs primarily involving the maxilla . a 40-year - old female patient reported with a chief complaint of swelling in right side of face since 3 months . the swelling was initially pea - sized in upper right posterior region of jaw which gradually increased extending extraorally to a present size of 4 4.5 cm . patient gave history of paresthesia with right upper lip and right ala of nose since 2 months . patient underwent biopsy from right maxillary posterior region 2 months back and histopathologic report was suggestive of a inflammatory lesion . patient also gave a history of cataract of the right eye for which she had visited ophthalmologist 6 months back . extraorally , facial asymmetry was noted due to diffuse swelling on right side of face extending supero - inferiorly from right infra - orbital region to lower border of mandible [ figure 1 ] . antero- posteriorly , the swelling extended from midline causing obliteration of right nasolabial fold to 2 cm anterior to tragus of right ear . the swelling was round to oval , same color as that of surrounding mucosa , smooth and tensed with well - defined margins and measured 6 4.5 cm in size approximately . the temperature was raised , it was soft to firm in consistency and non - tender . lower right submandibular lymph node was palpable and enlarged to the size of 0.51 cm ; was tender , hard in consistency , mobile and single in number . extraoral profile of the patient showing a large swelling on the right side of the face . intraoral examination revealed expansile fleshy swelling on right maxillary posterior region of size 4 4.5 cm in relation with 13 to 18 involving right buccal mucosa , extending upto upper right gingivo - buccal sulcus involving the attached gingiva extending to the palatal gingiva and hard palate upto the midline . obliteration of upper gingivo - buccal sulcus and labial vestibule was noticed on right side [ figure 2 ] . the swelling was yellowish white with red inflammatory marks representing the indentation of lower anterior teeth over labial and palatal mucosa . the swelling was dome - shaped over the palate with smooth surface , ill - defined margins and was soft to firm in consistency . intraoral photograph of the patient showing the lesion involving the right posterior gingivobuccal sulcus and the palate with displacement of teeth . radiological examination by orthopantomogram showed severe inter - dental bone resorption with 11 to 16 , cloudy and hazy radio - opacity over right maxillary antrum with loss of demarcation of posterior wall and anterior tuberosity [ figure 3 ] . on the basis of clinical and radiological evidence orthopantomogram of the patient showing hazy radiopacity in the right maxillary posterior and maxillary antrum area . under all aseptic conditions , on gross examination , specimen consisted of multiple bits of tissue , soft to firm in consistency that were creamish in color . microscopic examination revealed connective tissue stroma showing a bimorphic pattern consisting of sheets of small round or spindle - shaped mesenchymal cells , interspersed with islands of hyaline cartilage . the chondrocytes showed features of malignancy like nuclear hyperchromatism , pleomorphism , altered nuclear - cytoplasmic ratio and abnormal mitotic figures [ figures 4 - 9 ] . also , areas of mineralization was seen which was confirmed by performing goldner 's stain [ figure 10 ] . from the above histopathological features , the diagnosis of mchs of maxilla photomicrograph showing cellular areas along with chondroid tissue ( h&e stain , 40 ) photomicrograph showing areas of chondroid tissue ( h&e stain , 200 ) photomicrograph showing a bimorphic pattern composed of lobules of cartilage and sheets of mesenchymal tissue ( h&e stain , 200 ) photomicrograph showing connective tissue stroma with malignant chondrocytes and areas of highly cellular tissue composed of small round or spindle - shaped cells along with areas of calcification ( h&e stain , 200 ) photomicrograph showing malignant chondrocytes ( h&e stain , 400 ) photomicrograph showing small round or spindle - shaped cells ( h&e stain , 400 ) photomicrograph showing areas of mineralization ( goldner stain , 100 ) the lesion was treated by radical surgery and follow - up of the patient was done periodically every 6 months . mchs is a rare highly malignant tumor that arises in bone but can occur in extraskeletal sites and is characterized by highly cellular areas composed of undifferentiated small round or spindle cells admixed with lobules of mature hyaline cartilage . chs can be classified based on the topographical location of the tumor , the histological characteristics of the cancerous cartilage cells and the make - up of the surrounding matrix material associated with the tumor as : dedifferentiated chs . a small minority of secondary chss occur in patients with maffucci syndrome and ollier disease . the pathogenesis and biologic behavior of the chondrogenic tumors is not fully understood , but it is evident that these lesions represent a spectrum from benign chondroma to the malignant chs , through all degrees of intermediate type . it may be induced by irradiation , arise from pre - existing paget 's disease of bone or in association with fibrous dysplasia and the solitary bone cyst or it may arise from the vestigial cartilaginous rests . in premaxilla and maxilla , these cell rests are quite possible because of the proximity of chondrocranium throughout the fetal development . in mandible , the lesion may arise from the coronoid or condyloid process , from mental symphysis or from remnants of meckel 's cartilage . whereas , some believe that chs can arise de novo from osseous tissues without the presence of cartilaginous rests . some authors believe that mandible is more common a site for chs than maxilla . while terezhelmy suggested that the tumor is found in equal frequency in both arches and huvos the most common clinical finding of mesenchymal chs is painless swelling , expansion of buccal and lingual plates , premature eruption or exfoliation of teeth . the mass is usually rapidly growing and covered with mucosa which can ulcerate and there can be pain at later stages . it can also cause nasal obstruction , nasal discharge or epistaxis , facial paralysis and bleeding from the lesion . histologically , chs continues to be defined as a malignant tumor composed of fully developed cartilage without tumor osteoid , being directly formed from a sarcomatous stroma . evan 's and co - workers have attempted to associate the histologic grade ( grade i to iii ) of chs with the ultimate biologic behavior of the tumor , depending on the cellularity , nuclei size , presence of mitotic figures , multinucleation , spindle cell formation and mineralization in the form of osseous development at the edge of the cartilagenous lobules . in head and neck , the largest percentage of chss has been reported as grade i. mchs has a characteristic histological feature showing presence of highly cellular , undifferentiated zones with islands of chondroid differentiation as seen in this case . other findings are opacification of air spaces , a densely calcified bone mass and root resorption . cortical destruction occurs late in the course of disease and periosteal bone formation is often limited . some authors have reported a uniform widening of periodontal membrane space . in late stage disease , the primary lesion may penetrate the cortical plate and extend into adjacent soft tissues , resulting in a fuzzy soft tissue , peripheral shadow radiologically . the immunohistochemical markers that can aid in the diagnosis of chondroid tumors are epithelial membrane antigen ema , panck , d2 - 40 , s-100 and glial fibrillary acidic protein ( gfap ) . chondroma typically shows positivity for ema and panck and negative for d2 - 40 and gfap , whereas chs revealed positivity for d2 - 40 , s-100 and is negative for ema , panck and gfap . the mchss of maxilla are classically treated by radical surgery with radiotherapy being used as an adjunct or a form of palliative treatment for recurrent lesions . these lesions are radio - resistant and therefore radiotherapy is not generally recommended as a primary modality . the prognosis of mchs of jaws is disappointingly poor as compared to that of long bones . the cause of death is usually by direct extension into the base of skull and also through distant metastasis , chiefly to lungs and bones .

compared with traditional laparotomy , laparoscopic choledochotomy with t - tube drainage has dramatically decreased surgical trauma for patients . in this case , the problems caused by placing a t - tube for example , the inconvenience to the patient caused by the t tube and drainage bag , bile - induced peritonitis after removal of the t tube , and accidental slippage of the t tube from the common bile duct ( cbd)gradually aroused the concern of surgeons . with the use of laparoscopic choledochotomy and extraction of stones with primary closure , we have improved the traditional technique used for laparoscopic choledochotomy in china , which was laparoscopic transcystic choledochotomy and extraction of stones with primary closure . by making use of the natural tube of the cystic duct , this procedure minimizes the size of the incision into the common bile duct ( cbd ) , and the application of separate laparoscopic double - layer suture of the mucous and seromuscular layers dramatically decreases the postoperative incidence of bile leakage . furthermore , the novel procedure can shorten the postoperative stay , reduce hospitalization costs , and speed the patient 's recovery . from october 1 , 2009 , through april 30 , 2012 , 194 laparoscopic transcystic choledochotomies with extraction of stones and primary closure were performed . the group of 194 patients consisted of 69 men and 125 women aged 29 to 93 years ( sd 57.2 18.1 years ) . all of them had a medical history of intermittent right upper quadrant pain for 3 months to 10 years . on admission , 37 patients ( 19.1% ) had acute onset of abdominal pain with concurrent jaundice and fever for 3 to 5 days . different degrees of jaundice were found in 128 patients ( 81.5% ) , with total bilirubin ranging from 41 to 310 twenty - three patients had concurrent fever , with body temperature from 37 to 38.5c ( sd 37.6 1.2c ) . diagnostic blood biochemical tests , b - type ultrasound , computed tomography ( ct ) , and magnetic resonance cholangiopancreatography ( mrcp ) were completed in each patient ( table 1 ) . normal level for alt ( alanine aminotransferase ) , 740 u / l ( to convert to the si unit kat / l , multiply by 0.0167 ) ; ast ( aspartate aminotransferase ) , 1335 u / l ; ggt ( -glutamyltransferase ) , 745 u / l ; alp ( alkaline phosphatase ) , 35135 u / l ; tbil ( total bilirubin ) , 530 mol / l ; and dbil ( direct bilirubin ) , 05mol / l . finally , cholecystolithiasis combined with concurrent cbd stones was diagnosed by mrcp in 194 patients ; all were secondary choledocholithiasis without cirrhosis . chronic cholecystitis without concurrent acute cbd inflammation was present in 112 patients ( 58% ) . inclusion criteria were cbd diameter more than 5 mm ; somewhat large cbd stones or incarcerated ampullary stones that could be treated with a lithotripter ( holmium laser lithotripter ; aikekaineng technology co , ltd , beijing , china ) ; stones completely extracted from the intrahepatic and extrahepatic ducts , confirmed by choledochoscopy after laparoscopic bile duct exploration ; and no edema or stricture in the cbd ampulla , duodenal papilla , or cbd outlet . the surgical procedure would be converted to conventional laparoscopic choledochotomy with primary closure or t - tube drainage in the following conditions : intrahepatic stones could not be extracted entirely during the operation ; if the cystic duct enters the cbd through the back wall of cbd ; and the cystic and common hepatic ducts ran in parallel for too long . two 10-mm trocars were placed below the xiphoid process and through the right margin of the umbilicus . two 5-mm trocars were then placed at the right costal margin on the collarbone midline and lower right abdomen . first , a laparoscopic cholecystectomy was performed , and the residual cystic duct diameter was retained at 1 to 1.5 cm . the cystic duct was cut longitudinally on its ventral side up to the confluence of the cystic duct and cbd , after which the a longitudinal cut of approximately 3 cm was made in the anterior wall of the cbd ( figure 1 ) . a choledochoscope ( 4.5 mm chf - v electric choledochoscope ; olympus , tokyo , japan ) was then inserted through the enlarged opening for exploration of the distal cbd and extraction of stones ( figures 2 and 3 ) , after which it was turned upward for exploration of the common hepatic duct and intrahepatic ducts , bypassing the valvular septum around the confluence ( figure 4 ) . the distal cbd was checked for a second time after the intrahepatic exploration identified no stones . if no edema or stricture was found in the duodenal papilla , the irrigation water flowed smoothly through the cbd , and the sphincter of oddi worked normally , the mucous layer was continuously sutured from the distal end of the combined incision to the cystic duct end with 5 - 0 sutures ( figure 5 ) . the muscular layer was then closed by the lembert technique , with the same absorbable suture ( figure 6 ) . the cystic duct , 0.2 cm away from the cbd , was double ligated with an endoloop . excess distal cystic duct was removed and a drainage tube was placed in the gallbladder bed . two 10-mm trocars were placed below the xiphoid process and through the right margin of the umbilicus . two 5-mm trocars were then placed at the right costal margin on the collarbone midline and lower right abdomen . first , a laparoscopic cholecystectomy was performed , and the residual cystic duct diameter was retained at 1 to 1.5 cm . the cystic duct was cut longitudinally on its ventral side up to the confluence of the cystic duct and cbd , after which the a longitudinal cut of approximately 3 cm was made in the anterior wall of the cbd ( figure 1 ) . a choledochoscope ( 4.5 mm chf - v electric choledochoscope ; olympus , tokyo , japan ) was then inserted through the enlarged opening for exploration of the distal cbd and extraction of stones ( figures 2 and 3 ) , after which it was turned upward for exploration of the common hepatic duct and intrahepatic ducts , bypassing the valvular septum around the confluence ( figure 4 ) . the distal cbd was checked for a second time after the intrahepatic exploration identified no stones . if no edema or stricture was found in the duodenal papilla , the irrigation water flowed smoothly through the cbd , and the sphincter of oddi worked normally , the mucous layer was continuously sutured from the distal end of the combined incision to the cystic duct end with 5 - 0 sutures ( figure 5 ) . the muscular layer was then closed by the lembert technique , with the same absorbable suture ( figure 6 ) . the cystic duct , 0.2 cm away from the cbd , was double ligated with an endoloop . excess distal cystic duct was removed and a drainage tube was placed in the gallbladder bed . acute cholecystitis with concurrent acute cholangitis and acute liver injury were found in 82 patients ( 42% ) and 71 patients ( 36.5% ) , respectively . acute pancreatitis occurred in 21 patients ( 10.8% ) , with elevated blood and urine amylase level of 110 to 1230 u / l ( to convert to si unit kat / l , multiply by 0.0167 ) and 460 to 9800 u / l , respectively ( normal level for blood and urine amylase , 096 twelve patients ( 6.2% ) had blood electrolyte disturbances , with serum k , 2.9 to 3.5 mol / l ; na , 129 to 130 mol / l ; cl , 89101 mol / l ; ca , 1.92.3 mol / l ( normal level for k , na , cl , and ca : 3.55.3 mmol / l , 137147 mmol / l , 99110 mmol / l , and 2.12.6 mmol / l , respectively ) one patient had a 3.5-cm abscess on the anterior right lobe of the liver , identified by ct . in the 194 patients , concurrent coronary heart disease , type 2 diabetes mellitus , hypertension , and arrhythmia were present in 18 ( 9.3% ) , 13 ( 6.7% ) , 10 ( 5.1% ) , and 11 ( 5.7% ) , respectively . one patient had a history of subtotal gastrectomy , and another 2 had a history of biliary tract surgery . laparoscopic cholecystectomy with transcystic choledochotomy and intraoperative bile duct exploration was performed in 194 patients . a comparison between preoperative and postoperative mrcp of 55 patients showed no obvious change in the cbd ( figure 7 ) . of all 194 operations , the choledochoscope was directly inserted through the cystic duct without incision in 5 ( 2.5% ) , whereas in 56 ( 28% ) , it was inserted through a mini - incision in the cystic duct . in the rest ( 133 patients ) , a 0.1- to 1.1-cm transcystic incision was made in the cbd for choledochoscopic exploration and extraction of stones . bile leakage occurred in 2 patients , without obvious abdominal pain , distention , or signs of peritoneal irritation . after the patients fasted and underwent fluid infusion , the bile leakage stopped , 9 after the operation in one and 13 days in the other . in 2 patients , postoperative abdominal pain and distension abated spontaneously after 48 hours . the average postoperative hospital stay of the 194 patients was 8.0 days ( sd 8 2.1 days ) . perioperative conditions of the patients a , preoperative and b , postoperative mrcp . owing to the low position of the valvular septum around the confluence , 92 patients ( 47% ) had unsuccessful intrahepatic duct exploration by choledochoscope . a residual hepatic duct stone was found in 1 patient , who had intermittent abdominal pain and jaundice on postoperative days 5 and 8 , respectively . mrcp of this patient showed the residual stone located in the cbd ( figure 8a ) . both the abdominal pain and jaundice ceased on postoperative day 9 after the stone was discharged spontaneously ( figure 8b ) . follow - up of 176 patients ( 90% ) showed no recurrent cbd stones or strictures . fifty patients were observed for 6 months , 60 for 6 to 12 months , and 66 for more than 12 months . a , residual stone in cbd . duodenal endoscopic extraction of stones has been reported in the treatment of common bile duct calculi , both at home and abroad . endoscopic papillary balloon dilation ( epbd ) is used for stones less than 5 mm , and endoscopic sphincterotomy ( est ) , with a net basket and holmium laser lithotripsy , is applied for stones more than 5 mm or incarcerated stones . duodenal endoscopic stone extraction has certain requirements : smaller stones , fewer stones , and location close to the duodenal papilla . , it is very important to avoid cutting the duodenal papilla and impairing the structure and function of the sphincter of oddi . therefore , we usually perform a laparoscopic choledochotomy , no matter how many stones , their size , or the presence or not of duodenal papilla lesions . even if stones can not be completely removed intraoperatively , a t tube may be placed , to remove the residual stones postoperatively by choledochoscope . disadvantages are the need for surgery , increased trauma , longer operative time , and more surgical participants compared with stone extraction by duodenal endoscopy . choledochotomy with t - tube drainage has been the classic surgical treatment for secondary bile duct stones . since 1997 , our hospital has performed laparoscopic treatment of cbd stones , and 1385 laparoscopic choledochotomies with t - tube drainage have been completed through april 2012 . compared with traditional laparotomy , laparoscopic choledochotomy with t - tube drainage has dramatically decreased surgical trauma . new problems have aroused concerns , however , such as discomfort of patients caused by the prolonged need for the t - tube , peritonitis caused by bile leakage after removal of the tube , and t - tube complications such as accidental slippage out of the cbd . est can be used as an alternative to extract stones from the cbd , but the sphincter of oddi , along with its normal function can be badly injured . for a long time , surgeons have been looking for a procedure for the removal of cbd stones in one operation , with less damage to the sphincter and fewer complications . in 1932 , mirizze introduced the technology of intraoperative cholangiography . later , the choledochoscope was widely used intraoperatively and decreased the incidence of residual biliary stones greatly . laparoscopic common bile duct exploration avoids intraoperative radiologic injury to patients and operator , allows removal of the cbd stones , and shortens the postoperative hospital stay . above all , transcystic exploration avoids cbd injury , reduces the incidence of postoperative bile leakage and cbd stricture , and shortens the operative time and postoperative hospital stay . for the patients who are inappropriate for transcystic extraction of stones , it is still necessary to convert to choledochotomy to extract the stones . if there is neither residual calculi nor edema and stricture after laparoscopic common bile duct exploration , it is safe and effective to perform primary closure of the cbd . according to reports in the literature , primary closure of the cbd is generally by full - thickness continuous suture . however , in the case of postoperative high bile duct pressure , bile leakage from a suture pinhole may occur . a preoperative endoscopic nasal biliary drain or an intraoperative bile duct duodenal drainage tube is usually placed , to prevent bile leakage . based on the current treatment of secondary cholangiolithiasis , we have described laparoscopic transcystic choledochotomy with primary double - layer suture . by making full use of the natural dilatation around the confluence between the cystic and hepatic ducts found in most patients , the surgeon can easily insert the choledochoscope into the cbd through the natural orifice in the lumen of the cystic duct or by a combined continuous incision in the cystic duct and cbd ( figure 9 ) . the use of primary double - layer suture of the mucous and seromuscular layers can avoid the placement of a t - tube drain , decrease the occurrence of bile leakage , and avoid discomfort from the presence of the t - tube . among the 194 patients in this study , postoperative bile leakage occurred only in 2 ( 1% ) . according to a report by cai et al the postoperative rate of bile leakage for laparoscopic choledochotomy with t - tube drainage or primary cbd closure was 4.50% and 4.00% , respectively . in another study on laparoscopic choledochotomy with primary cbd closure , a further two original studies on laparoscopic transcystic cbd exploration reported a rate of 7.4% ( 2/27 ) and 3.39% ( 2/59 ) , respectively . we found during the operation that the cbd at its confluence with the cystic duct had a greater diameter than other points along the duct . in this article thus , the suture of the cbd and cystic duct generally had no effect on the diameter of the cbd and made primary closure feasible , decreasing the possibility of postoperative stricture of the cbd dramatically . postoperative mrcp in 55 patients in this study showed no abnormal change in the cbd caliber , compared with that seen in the preoperative scans . in addition to the surgeon 's proficiency in performing laparoscopies , it has been reported that the cbd can be continuously sutured if neither tissue fragments nor stone sludge is directly viewed in the cbd and if the ampulla of vater is found to have no stricture . moreover , it has been recommended that a t tube should be placed if the distal bile duct has high biliary pressure , if a postoperative cholangiogram is deemed essential , or if residual stones must be extracted postoperatively . based on our laparoscopic experiences , we suggest the following indications for laparoscopic cbd exploration with primary closure : ( 1 ) definite diagnosis of cbd stones with a cbd diameter of more than 5 mm . in general , the cbd diameter should be more than 8 mm , to allow primary closure . however , by making use of the improved procedure , the incision site of the cbd is the area of natural dilatation around the confluence between the cystic duct and the cbd , along with the natural lumen of the cystic duct . thus , the length of the incision in the cbd , the postoperative incidence of cbd stricture , and bile leakage are decreased . the use of a lithotripter makes it possible to have primary cbd closure in the presence of large stones or incarcerated ampullary stones , but the gravel must be removed completely . ( 3 ) no edema or stricture of the cbd , ampulla , or duodenal papilla must be present in intraoperative inspection by choledochoscope . ( 4 ) the surgeon has proficient experience in laparoscopic choledochotomy , suture , and manipulation of the choledochoscope . it is important to minimize stimulation of the ampulla or duodenal papilla with the choledochoscope ; both are risk factors for bile leakage . one patient in our group had a 3.5 1.5-cm cylindrical stone in his 1.7-cm diameter cbd . the use of the cystic duct makes it possible to shorten the cbd incision , because the longitudinal incision of the cystic duct offers a passage for the choledochoscope to enter the cbd . the caliber of the choledochoscope routinely used was 0.45 cm , and the diameter at the confluence between the cystic duct and cbd in all the patients was 0.3 to 0.8 cm . in transductal choledochotomy , however , by laparoscopic transcystic choledochotomy , the cbd incision was 0.1 to 1.1 cm ( average , 0.5 cm ) . moreover , less injury of the cbd ensured a lower risk of bile leakage or bile duct stricture . , the incision should be made along the longitudinal axis of the cbd , with care taken to avoid opening the cbd transversely . the shorter the cbd incision , the more difficult it is to perform the choledochoscopic exploration in the direction of the hepatic duct . another factor affecting exploration in the direction of the hepatic duct is the existence of the valvular crescent septum at the junction of the cystic duct and common hepatic duct wall . for cbd exploration , it is easier for the choledochoscope to pass the septum , but it is more difficult to bypass the septum upwardly for intrahepatic exploration . in this study , intrahepatic duct choledochoscope exploration failed in 92 patients ( 47% ) , and 1 patient had postoperative residual stones . therefore , it is essential to determine the locations of stones by preoperative ct or mrcp . the caliber of the cbd was seldom narrowed by this novel procedure of cut and suture , because the cbd incision was shorter ( average , 0.5 cm ) . in addition , the cbd is capable of self - adjustment and can regulate its caliber in accordance with the bile duct pressure . moreover , we left a margin of 1.5 to 2 mm from the stitches to the edge of the incision in the cbd when closing the cbd . these efforts ensured that there would be no postoperative stricture in the cbd during follow - up . to determine whether postoperative bile duct stricture occurred , the comparative results between preoperative and postoperative mrcp in 55 patients objectively proved the effectiveness of this new procedure in avoiding postoperative cbd stricture . the process of laparoscopic primary closure of the bile duct has rarely been reported in the literature . interrupted suture is rarely reported , perhaps because of the complexity of suture by laparoscope . however , the method of primary closure is known to be very important , in that it may influence the postoperative incidence of bile leakage and bile duct stricture . in this study , first , continuous suture of the mucosa from the distal cbd to the distal cystic duct with 5 - 0 absorbable sutures was performed , to close the mucous layer tightly with good mucosal apposition . second , lembert 's seromuscular suture technique was used , with care taken to avoid penetrating the mucosal layer and causing bile leakage . if the cbd wall was thin and bile was seen leaking from a pinhole during the operation , the hepatoduodenal ligament was sutured by the lembert method . the abdominal drainage tube can be removed 3 to 5 days postoperatively if no bile is discharged from the drainage tube and if the patient has no abdominal pain , fever , or jaundice . there is evidence that t - tube placement compromises the advantage of the minimally invasive technique and increases the risk of complications and length of hospital stay . comparatively , primary closure of the cbd may shorten the hospital stay , reduce hospitalization expenses , and increase patient comfort , while avoiding the postoperative complications of bile leakage and peritonitis . a transcystic drainage tube or temporary bile duct stent can be placed intraoperatively , if necessary . ha et al . reported a study of 36 patients with choledochotomy and extraction of stones with primary closure . the study also supported that primary closure is feasible and can shorten the hospital stay and reduce medical costs . duodenal endoscopic extraction of stones has been reported in the treatment of common bile duct calculi , both at home and abroad . endoscopic papillary balloon dilation ( epbd ) is used for stones less than 5 mm , and endoscopic sphincterotomy ( est ) , with a net basket and holmium laser lithotripsy , is applied for stones more than 5 mm or incarcerated stones . duodenal endoscopic stone extraction has certain requirements : smaller stones , fewer stones , and location close to the duodenal papilla . , it is very important to avoid cutting the duodenal papilla and impairing the structure and function of the sphincter of oddi . therefore , we usually perform a laparoscopic choledochotomy , no matter how many stones , their size , or the presence or not of duodenal papilla lesions . even if stones can not be completely removed intraoperatively , a t tube may be placed , to remove the residual stones postoperatively by choledochoscope . disadvantages are the need for surgery , increased trauma , longer operative time , and more surgical participants compared with stone extraction by duodenal endoscopy . choledochotomy with t - tube drainage has been the classic surgical treatment for secondary bile duct stones . since 1997 , our hospital has performed laparoscopic treatment of cbd stones , and 1385 laparoscopic choledochotomies with t - tube drainage have been completed through april 2012 . compared with traditional laparotomy , laparoscopic choledochotomy with t - tube drainage has dramatically decreased surgical trauma . new problems have aroused concerns , however , such as discomfort of patients caused by the prolonged need for the t - tube , peritonitis caused by bile leakage after removal of the tube , and t - tube complications such as accidental slippage out of the cbd . est can be used as an alternative to extract stones from the cbd , but the sphincter of oddi , along with its normal function can be badly injured . for a long time , surgeons have been looking for a procedure for the removal of cbd stones in one operation , with less damage to the sphincter and fewer complications . in 1932 , mirizze introduced the technology of intraoperative cholangiography . later , the choledochoscope was widely used intraoperatively and decreased the incidence of residual biliary stones greatly . laparoscopic common bile duct exploration avoids intraoperative radiologic injury to patients and operator , allows removal of the cbd stones , and shortens the postoperative hospital stay . above all , transcystic exploration avoids cbd injury , reduces the incidence of postoperative bile leakage and cbd stricture , and shortens the operative time and postoperative hospital stay . for the patients who are inappropriate for transcystic extraction of stones , it is still necessary to convert to choledochotomy to extract the stones . if there is neither residual calculi nor edema and stricture after laparoscopic common bile duct exploration , it is safe and effective to perform primary closure of the cbd . according to reports in the literature , primary closure of the cbd is generally by full - thickness continuous suture . however , in the case of postoperative high bile duct pressure , bile leakage from a suture pinhole may occur . a preoperative endoscopic nasal biliary drain or an intraoperative bile duct duodenal drainage tube is usually placed , to prevent bile leakage . based on the current treatment of secondary cholangiolithiasis , we have described laparoscopic transcystic choledochotomy with primary double - layer suture . by making full use of the natural dilatation around the confluence between the cystic and hepatic ducts found in most patients , the surgeon can easily insert the choledochoscope into the cbd through the natural orifice in the lumen of the cystic duct or by a combined continuous incision in the cystic duct and cbd ( figure 9 ) . the use of primary double - layer suture of the mucous and seromuscular layers can avoid the placement of a t - tube drain , decrease the occurrence of bile leakage , and avoid discomfort from the presence of the t - tube . among the 194 patients in this study , postoperative bile leakage occurred only in 2 ( 1% ) . according to a report by cai et al the postoperative rate of bile leakage for laparoscopic choledochotomy with t - tube drainage or primary cbd closure was 4.50% and 4.00% , respectively . in another study on laparoscopic choledochotomy with primary cbd closure , a further two original studies on laparoscopic transcystic cbd exploration reported a rate of 7.4% ( 2/27 ) and 3.39% ( 2/59 ) , respectively . we found during the operation that the cbd at its confluence with the cystic duct had a greater diameter than other points along the duct . in this article thus , the suture of the cbd and cystic duct generally had no effect on the diameter of the cbd and made primary closure feasible , decreasing the possibility of postoperative stricture of the cbd dramatically . postoperative mrcp in 55 patients in this study showed no abnormal change in the cbd caliber , compared with that seen in the preoperative scans . in addition to the surgeon 's proficiency in performing laparoscopies , it has been reported that the cbd can be continuously sutured if neither tissue fragments nor stone sludge is directly viewed in the cbd and if the ampulla of vater is found to have no stricture . moreover , it has been recommended that a t tube should be placed if the distal bile duct has high biliary pressure , if a postoperative cholangiogram is deemed essential , or if residual stones must be extracted postoperatively . based on our laparoscopic experiences , we suggest the following indications for laparoscopic cbd exploration with primary closure : ( 1 ) definite diagnosis of cbd stones with a cbd diameter of more than 5 mm . in general , the cbd diameter should be more than 8 mm , to allow primary closure . however , by making use of the improved procedure , the incision site of the cbd is the area of natural dilatation around the confluence between the cystic duct and the cbd , along with the natural lumen of the cystic duct . thus , the length of the incision in the cbd , the postoperative incidence of cbd stricture , and bile leakage are decreased . the use of a lithotripter makes it possible to have primary cbd closure in the presence of large stones or incarcerated ampullary stones , but the gravel must be removed completely . ( 3 ) no edema or stricture of the cbd , ampulla , or duodenal papilla must be present in intraoperative inspection by choledochoscope . ( 4 ) the surgeon has proficient experience in laparoscopic choledochotomy , suture , and manipulation of the choledochoscope . it is important to minimize stimulation of the ampulla or duodenal papilla with the choledochoscope ; both are risk factors for bile leakage . one patient in our group had a 3.5 1.5-cm cylindrical stone in his 1.7-cm diameter cbd . the use of the cystic duct makes it possible to shorten the cbd incision , because the longitudinal incision of the cystic duct offers a passage for the choledochoscope to enter the cbd . the caliber of the choledochoscope routinely used was 0.45 cm , and the diameter at the confluence between the cystic duct and cbd in all the patients was 0.3 to 0.8 cm . in transductal choledochotomy , however , by laparoscopic transcystic choledochotomy , the cbd incision was 0.1 to 1.1 cm ( average , 0.5 cm ) . moreover , less injury of the cbd ensured a lower risk of bile leakage or bile duct stricture . , the incision should be made along the longitudinal axis of the cbd , with care taken to avoid opening the cbd transversely . the shorter the cbd incision , the more difficult it is to perform the choledochoscopic exploration in the direction of the hepatic duct . another factor affecting exploration in the direction of the hepatic duct is the existence of the valvular crescent septum at the junction of the cystic duct and common hepatic duct wall . for cbd exploration , it is easier for the choledochoscope to pass the septum , but it is more difficult to bypass the septum upwardly for intrahepatic exploration . in this study , intrahepatic duct choledochoscope exploration failed in 92 patients ( 47% ) , and 1 patient had postoperative residual stones . therefore , it is essential to determine the locations of stones by preoperative ct or mrcp . the caliber of the cbd was seldom narrowed by this novel procedure of cut and suture , because the cbd incision was shorter ( average , 0.5 cm ) . in addition , the cbd is capable of self - adjustment and can regulate its caliber in accordance with the bile duct pressure . moreover , we left a margin of 1.5 to 2 mm from the stitches to the edge of the incision in the cbd when closing the cbd . these efforts ensured that there would be no postoperative stricture in the cbd during follow - up . to determine whether postoperative bile duct stricture occurred , the comparative results between preoperative and postoperative mrcp in 55 patients objectively proved the effectiveness of this new procedure in avoiding postoperative cbd stricture . the process of laparoscopic primary closure of the bile duct has rarely been reported in the literature . interrupted suture is rarely reported , perhaps because of the complexity of suture by laparoscope . however , the method of primary closure is known to be very important , in that it may influence the postoperative incidence of bile leakage and bile duct stricture . in this study , first , continuous suture of the mucosa from the distal cbd to the distal cystic duct with 5 - 0 absorbable sutures was performed , to close the mucous layer tightly with good mucosal apposition . second , lembert 's seromuscular suture technique was used , with care taken to avoid penetrating the mucosal layer and causing bile leakage . if the cbd wall was thin and bile was seen leaking from a pinhole during the operation , the hepatoduodenal ligament was sutured by the lembert method . the abdominal drainage tube can be removed 3 to 5 days postoperatively if no bile is discharged from the drainage tube and if the patient has no abdominal pain , fever , or jaundice . there is evidence that t - tube placement compromises the advantage of the minimally invasive technique and increases the risk of complications and length of hospital stay . comparatively , primary closure of the cbd may shorten the hospital stay , reduce hospitalization expenses , and increase patient comfort , while avoiding the postoperative complications of bile leakage and peritonitis . a transcystic drainage tube or temporary bile duct stent can be placed intraoperatively , if necessary . ha et al . reported a study of 36 patients with choledochotomy and extraction of stones with primary closure . the study also supported that primary closure is feasible and can shorten the hospital stay and reduce medical costs . laparoscopic transcystic choledochotomy and extraction of stones with primary double - layer closure decreases surgical injury , incidence of bile leakage , hospital stay , and admission costs . it is essential for the surgeon to have a good mastery of both the surgical indications and the laparoscopic surgical technique .

endoscopic retrograde cholangiopancreatography ( ercp ) is a common interventional procedure used for more than 40 years in diagnosis and management of pancreaticobiliary pathologies . by using a specialized side - viewing duodenoscope , the procedure involves injection of radio - opaque contrast into the biliary ducts allowing for both visualization of the anatomy and performance of a variety of therapeutic interventions . relying heavily on the use of real - time fluoroscopy , ercp has been recognized to carry a radiation risk , not only for patients undergoing the procedure but also for physicians performing it 1 2 . guidelines have now been developed by the world gastroenterology organization ( in collaboration with the international atomic energy agency , the american society for gastrointestinal endoscopy , as well as the european society of digestive endoscopy ) , to ensure that doses of ionizing radiation received by individuals involved in the procedure are as low as reasonably achievable in order to prevent rare but harmful consequences of radiation exposure 3 4 5 . there is therefore a need to identify modifiable factors that could help reduce total radiation exposure during ercps , and allow for overall safer procedures . in the past , various factors have been shown to affect the total radiation dose received by patients and staff during ercp . these include : the type of x - ray tube used ; distance of personnel from the x - ray source ; and shielding the body using radio - protective shields . in addition , time spent during fluoroscopy has been shown to have a linear relationship to the radiation dose during ercp 6 . therefore , limiting fluoroscopy time ( ft ) seems to be a simple way of reducing radiation exposure during ercp . increased interest in this area has also recently led to benchmarks for ft in ercp being established in the united states , allowing endoscopists to monitor and improve their own procedure ft over time 7 . in fact , recent recommendations based on currently available evidence and working party expert consensus suggests that endoscopists should record the time and dose of fluoroscopy as part of their documentation for every fluoroscopic session in which they are involved 8 . while some factors affecting ft , such as fluoroscopy equipment used , procedure type , and patient characteristics , are non - modifiable ; identification of potentially modifiable factors would not only allow the endoscopist to predict prolonged fluoroscopy durations but also to possibly undertake relevant precautions accordingly . recently , the design of one of our ercp suites was modified and as a result , the endoscopy and fluoroscopy screens were placed closer together ( fig . 1 b ) for ergonomic reasons ( reducing endoscopists need to rotate their head and body to view both screens ) . this provided the opportunity to test whether this specific change would impact the total fluoroscopy time during each ercp performed . a multivariate analysis was therefore undertaken to investigate these effects , as well as to identify and validate other clinical & procedural factors associated with prolonged fluoroscopy time ( and thus radiation exposure ) . we performed a retrospective analysis from january 2012 to june 2013 of all ercps performed in 1 of 2 ercp suites at kingston general hospital which is a tertiary care center in kingston , ontario , canada . the duration spanned 9 months before to 9 months after the screen move date ( september 2012 ) . all inpatients / outpatients undergoing ercps in this fluoroscopy suite for any clinical indication were identified using a clinical endoscopy database . after obtaining appropriate ethics approval from our institution s research ethics board , various predetermined clinical , procedural and ergonomic factors as well as fluoroscopy times for each procedure were obtained via thorough chart review ( table 1 ) . all procedures performed prior to september 2012 were included in the screens away group ( endoscopy and fluoroscopy screens located away from each other ) ; while those performed october 2012 onwards were included in the screens together group . left lateral decubitus dilator ( balloon / rigid ) other ( including l / r hepatic , common hepatic & cystic ) moderately difficult ( 2 ) ercp , endoscopic retrograde cholangiopancreatography ; ft , fluoroscopy time ; cbd , common bile duct data were entered into an excel file designed for the study , and imported into ibm spss ( version 21.0 , armonk , new york , 2012 ) for statistical analysis . data were initially analyzed descriptively , and both fluoroscopy time and procedure time were plotted to assess the underlying distribution . factors associated with lengthened ft were then assessed using independent samples t - tests for 2-level variables , 1-way anovas for categorical variables with more than 2 levels , and correlations for the continuous data . promising factors from the univariate analysis including our primary study factor ( screen position ) were then included in the multivariable linear regression analysis with ft as the dependent variable ( age , sex , prior ercp , grade based on difficulty , volume , fellow involvement and screen distance ) . variables were then removed sequentially using a backward , manual approach , based on the updated p values at each iteration . final significance was established at a p value < 0.05 with factors having p values < 0.05 affecting the fluoroscopy time significantly . a total of 299 ercps were performed on 121 males ( 40.5 % ) and 178 females ( 59.5 % ) in the selected ercp suite in the chosen time frame ( table 1 ) ( representing approximately 50 % of the total ercps performed ) . the mean fluoroscopy time was 6.67 5.75 min ; and the mean procedure time was 33.6 18.19 min . both were reasonably normally distributed , with only a slight skew to the right for fluoroscopy time . most procedures were performed for therapeutic ( 91.3 % ) compared to diagnostic reasons ( 8.7 % ) . one hundred nineteen patients ( 39.8 % ) had previously had an ercp , with a previous sphincterotomy in 99 patients ( 83.2 % ) . most procedures were performed in the left semi - prone position ( 95.3 % ) . four endoscopists performed the ercps ( 3 gastroenterologists , 1 general surgeon ) . a gastroenterology fellow ( residents in their fifth or sixth year of post - graduate training in gastroenterology ) most procedures ( 74.7 % ) were considered grade 1 on a validated 3 point scale of difficulty ( 1 : least difficult , 2 : moderately difficult , and 3 : most difficult ) 22 . a total of 160 procedures ( 53.5 % ) had been performed with the screens away from each other , whereas 139 procedures ( 46.5 % ) had the screens placed next to each other . a total of 10 cases ( 3.3 % ) encountered some sort of an adverse event ( hemodynamic instability or minor to moderate bleeding requiring epinephrine injections , balloon tamponade or clip application ) . multiple instruments ( including extraction balloons , baskets , stone - crushers , balloon / rigid dilators , snares , needle knives etc . ) were identified as having been used in the procedures depending on the cases and the pathologies identified . in the univariate analysis , a strong positive correlation was found between fluoroscopy time ( ft ) and procedure time ( r = 0.693 , p < 0.001 ) . mean ft was found to be prolonged in cases with prior ercps ( 7.78 min , p = 0.008 ) ; fellow involvement ( 7.26 min , p = 0.046 ) ; use of basket ( 12.56 min , p < 0.001 ) , stone - crushers ( 14.97 min , p < 0.001 ) , balloon / rigid dilators ( 10.26 min , p < 0.001 ) , stents ( 8.26 min , p < 0.001 ) ; stones ( 7.84 min , p = 0.002 ) , and procedures with adverse event(s ) ( 9.29 min , p < 0.001 ) . large stones had a significantly greater ft than small or moderate - size stones [ f(2,125 = 13.879 , p < 0.001 ] . after controlling for confounding factors and interactions , the multivariable regression analysis demonstrated that placing the endoscopy and fluoroscopy screens next to each other was associated with a significantly lesser ft than when the screens were away ( 1.4 min , p = 0.026 ) . other significant predictors associated with a prolonged ft included those with prior ercps ( + 1.4 min , p = 0.031 ) and the grade of procedural difficulty ( + 4.2 min for each grade of difficulty , p < 0.001 ) . endoscopists performing high - volume endoscopies had shorter fluoroscopy times compared to low - volume endoscopists ( 1.82 mins , p = 0.015 ) ( table 2 ) . variables were removed sequentially using a backward , manual approach in the following order based on updated p - values with each iteration : sex , age and fellow involved . final model adjusted r = 0.232 , f = 21.6 , p < 0.001 ercp , endoscopic retrograde cholangiopancreatography in our study , various factors were identified as affecting the total fluoroscopy duration ( and therefore the total radiation received ) during ercp , validating the results from research in the past . however , based on our multivariate analysis , we also concluded that a simple ergonomic modification in the ercp suite made by reducing the distance between the endoscopy and fluoroscopy screens , significantly reduces the fluoroscopy time during the procedure . prior to making these modifications , the endoscopy and fluoroscopy screens in our ercp suite were located away from each other ( fig . 1 a ; distance between the centers of both screens = 130 cm ) . as a result , the endoscopists found themselves having to constantly move their heads in order to switch views between the 2 screens , which not only affected overall body positioning but also risked scope positioning . even though not formally tested in this study , we believe that bringing the screens together ( fig . 1 b ; new distance between the centers of both screens = 70 cm ) narrows the endoscopists field of vision leading to decreased head / body movements ; improved simultaneous visualization of both images ; and therefore better stability with the scope . this may therefore allow for more successful procedures , and based on our results , decrease fluoroscopy times and lower radiation risk to both patients and staff involved in the procedure . it may seem obvious to assume that the endoscopy and fluoroscopy screens would be placed next to each other in most endoscopy suites ; however , in the absence of any formal recommendations , we found a great deal of variability in the designs of ercp suites , specifically screen positioning , via an informal survey conducted across many north american institutions . currently , there are no formal guidelines on the ergonomics of endoscopy ; however , certain recommendations have previously been extrapolated from the laparoscopic surgery and general ergonomics literature to minimize musculoskeletal complaints and overuse injury of endoscopists 9 . specifically , recommendations to position the endoscopy and fluoroscopy monitors somewhere directly in front of the endoscopist , with monitor height just at or below eye level , as well as having the examination table at or below the elbow height , have been recommended . no recommendations with regards to exact screen positioning have been made and no ergonomic modifications have ever been shown to affect fluoroscopy time in the past . while acknowledging the need for further research in ergonomics in gastrointestinal endoscopy 10 , the asge ( american society for gastrointestinal endoscopy ) in their evaluation report highlight that methods to limit patients and staff exposure to fluoroscopy are paramount to maintaining a safe work environment . bringing the screens right next to each other , and directly in front of the endoscopists view , would not only reduce fluoroscopy times , but also allow for compliance with previous recommendations . retrospective research in the past has established various factors that directly affect the total radiation dose received by personnel during ercp and other fluoroscopic procedures . these factors include : the type of x - ray tube used ( over - couch c - arm units vs. under - couch units ) 11 ; mobile vs. stationary x - rays 12 ; distance of personnel from the x - ray source 11 12 ; use of radio - protective shields to shield the body 13 14 ; and total time spent in fluoroscopy during the procedure ( a collinear relationship)1 . in addition , fluoroscopy time during ercp has recently been shown to be affected by several factors including : type of fluoroscopy used ( pulsed vs. continuous vs. time limited ) 15 16 17 ; indication for ercp ( diagnostic vs. therapeutic ) ; physician education and experience 18 19 ; altered anatomy ; and various procedure - specific factors including stent insertion , lithotripsy , taking biopsies , and use of instruments 20 . moreover , an observational prospective study from greece identified multiple clinical factors that were found to prolong total fluoroscopy time during ercp . these clinical factors included : choledocholithiasis , multiple cbd stones , stone size > 10 mm , needle - knife papillotomy , presence of periampullary diverticulum and mechanical lithotripsy 21 . however , this study was performed by a single endoscopist , which limits the overall generalizability of the findings . in our cohort of patients , various procedural factors determined the grade of procedural difficulty such as difficult / failed cannulations , failed stone retrieval , difficult stent insertions , patient non - compliance , and distorted anatomy secondary to prior surgeries . as expected , the multivariate analysis found that the grade of procedural difficulty was associated with prolonged fluoroscopy times ( ft ) . difficulty of procedures was based on the widely used and validated ercp difficulty grading scale by cotton et . grade 3 procedures ( most difficult ) had the highest fluoroscopy time ( an additional 12.6 mins ) , followed by the moderately difficult grade 2 procedures ( additional 8.4 mins of fluoroscopy time ) . grade 1 procedures ( least difficult ) had the lowest fluoroscopy time . even though grade of difficulty as a predictor of fluoroscopy time , would be considered a non - modifiable factor , anticipating prolonged fluoroscopy times in ercps that are going to be technically difficult such as cases involving removal of larger cbd stones , intrahepatic stones , or patients with billroth ii anatomy , would not only allow for a more informed consent about potential radiation risks but would also allow the endoscopist and ercp personnel to take relevant precautions accordingly . using instruments such as baskets , stone - crushers , and balloon / rigid dilators in the common bile duct significantly increased the ft , as did presence of stones . interestingly having multiple stones did not increase the ft , primarily because most of these stones were small and easily removed with 1 to 2 basket sweeps without adding to the ft . having a prior sphincterotomy was not associated with longer fts , most likely due to easier cannulation of the papilla . patients with prior ercps were found to have longer ft than those who were ercp - nave , and this maintained significance in the final regression analysis . this was most likely because most ercps in these patients were performed for non - functioning stents , requiring removal and reinsertion of newer stents , thereby prolonging both the procedure and ft . the volume of ercps performed in the chosen endoscopy suite may not have accurately reflected the overall volume of procedures performed at our center . however , the significant decrease in fluoroscopy time / exposure achieved by simply moving the endoscopy and fluoroscopy screens together can not be ignored . in addition , even though a retrospective study has its drawbacks , we feel that performing this study prospectively may well have modified the endoscopists behavior and confounding may have occurred due to the hawthorne effect . recently , there has been a lot of interest generated by research in the field of fluoroscopy duration during ercp , due to the associated radiation risks to both patients as well as the endoscopy staff involved in the procedure 8 23 . while being aware of the clinical factors affecting fluoroscopy time during ercp such as the presence of stones or having a stricture does hold some merit , these factors are for the most part non - modifiable . therefore , one needs to search for other means to reduce radiation exposure during ercp , and a simple ergonomic change by reducing the distance between two screens in the endoscopy suite is an easily achievable alternative . currently , sample frameworks for endoscopy suite designs are available , yet there is no established standard in that regard in either canada or the united states . this is the first study to show that decreasing the distance between the fluoroscopy and endoscopy screens in the ercp suite significantly reduces ft during the procedure .

the toxicity of these preservatives against ocular surface cells , namely the cornea and conjunctiva , has been investigated extensively110 because epithelial disorders are of particular concern postoperatively , as well as in patients using eye drops over the longer term , and those with dry eye . in the present study , we examined the toxicity of ophthalmic solutions commonly used after eye surgery to control infection and inflammation in a bioassay using four established ocular surface cell lines . a further aim of the present study was to develop a scoring method of cell viability to enable the easy comparison of drugs , because many toxicity studies use multiple methods to evaluate cell damage and data evaluation consequently becomes complicated . the commercially available cell lines used in the present study were the sirc ( rabbit corneal epithelium ; atcc ccl-60 ; american type culture collection ( atcc ) , manassas , va , usa ) , bce c / d-1b ( bovine corneal epithelial cells ; jcrb-9129 ; health science research resource bank , osaka , japan ) , rc-1 ( rabbit corneal epithelium ; jcrb-0246 ; health science research resource bank ) and chang conjunctiva ( human conjunctival cells ; atcc ccl-20.2 ; atcc ) lines . all cells were cultured according to standard protocols provided by the distributors . after cells had reached 80%90% confluence and had been cultured under standard conditions for 48 h , a 100l aliquot of the culture containing approximately 2 10 cells was harvested from the culture wells . the different drugs and preservatives to be tested were diluted 10- , 20- , 100- , 1,000- , and , when necessary , 10,000-fold with phosphate buffered saline before being added to the cells in culture . cell viability was determined after 48 h incubation in the presence of the test solutions using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2h - tetrazolium bromide ( mtt ) assay ( sigma , st louis , mo , usa ) , a quantitative colorimetric measure of mitochondrial activity as an index of cell viability and proliferation . absorbance was read on a spectrophotometer ( benchmark microplate reader ; bio - rad , hercules , ca , usa ) . the mtt assay detects living cells only and the signal generated is directly proportional to the number of live cells . the viability of cells in test solutions was calculated as a percentage of cells incubated in growth medium without test solution . experiments were repeated between eight and 16 times and the results are given as the mean standard deviation ( sd ) . because the results of preliminary experiments indicated that viability increased markedly in solutions that had been diluted 20-fold or more and the differences among the drugs were evident enough in 10-fold diluted solutions , we expressed the results as a cell viability score ( cvs50 ) , a number of cell lines with viability 50% in the presence of a 10-fold dilution of the drug , to enable easy comparison of the effects of different drugs . this concept is similar to the mic50 ( ie , the minimum inhibitory concentration of a drug required to inhibit the growth of 50% of organisms ) . the antibiotic and anti - inflammatory drugs evaluated in the present study are listed in table 1 . the ophthalmic solution preservatives tested in the present study were 0.01% bak ( wako pure chemical industries , osaka , japan ) , 0.5% chlorobutanol ( wako ) , 1.0% polysorbate 80 ( tween 20 ; icn biomedicals , aurora , oh , usa ) , 0.04% methyl paraoxybenzoate ( wako ) , and 0.04% propyl paraoxybenzoate ( wako ) . for all cell lines tested , cell viability after exposure to cefmenoxime was 80% ( figure 1 ) . cell viability after exposure of cells to the fluoroquinolones was concentration dependent , with 80% viability observed for solutions diluted 100-fold or more . low toxicity was observed for betamethasone - containing drugs without bak or polysorbate as the preservative ( figure 2 ) . bak - containing anti - inflammatory drugs had less cell viability than the drugs without bak . nonsteroidal anti - inflammatory drugs had less cell viability than fluoromethorone with same concentration of bak . using cvs50 as an indicator of cytotoxicity , the order of cell viability after exposure to the antibiotic solutions was determined to be cefmenoxime tosufloxicin dibekacin levofloxacin norfloxacin = gatifloxacin = moxifloxacin ( table 1 ) , whereas that for the anti - inflammatory drugs was betamethasone betamethasone + fradiomycin > preservative - free diclofenac preservative - free bromfenac > > 0.02% fluoromethorone 0.1% fluoromethorone = diclofenac + preservative ( diclod ) = bromfenac + preservative ( bronuck ) = pranoprofen ( table 2 ) . the calculated cvs50 of the antibiotics ranged between one and four , whereas that of anti - inflammatory drugs preserved with bak and diclod was zero . these data are consistent with the results presented in figures 1 and 2 , which indicate that cell viability after exposure to antibiotics was generally higher than after exposure of cells to anti - inflammatory drugs . of the preservatives tested , bak and polysorbate were cytotoxic to all cell lines ( figure 3 ) . propyl paraoxybenzoate exhibited cytotoxicity only in the sirc and conjunctival cell lines , whereas chlorobutanol and methyl paraoxybenzoate did not exhibit any cytotoxic effects . the fluoroquinolones are broad - spectrum antibiotics that are the most commonly used drugs for the pre- and postoperative control of infection after eye surgery . in the present study , cefmenoxime , dibekacin , and tosufloxacin appeared to be the least cytotoxic . these results are partially consistent with those of a previous study performed in a single cell line11 and support previous reports that levofloxacin is less toxic than moxifloxacin12 or gatifloxacin.13 many previous studies evaluating the cytotoxicity of fluoroquinolones have used solutions preserved with bak,14,15 which had a significant effect on the results . however , in the present study we were able to evaluate the toxicity of the pharmaceutical components of the ophthalmic solutions because we used preservative - free fluoroquinolone solutions . the results of the present study indicate that antibiotics are more toxic to conjunctival cells than to corneal cells , except for cefmenoxime , which was not cytotoxic against any of the cell lines used . dose - dependent cytotoxicity was observed for all anti - inflammatory drugs preserved with bak in all cell lines . the corneal toxicity of preservative - containing solutions of diclofenac1618 and bromfenac19 has already been documented and the present study further revealed that preservative - free diclofenac and bromfenac exhibit considerable toxicity . pranoprofen preserved with bak exhibited the greatest toxicity , although this may be due to the fact that this solution contained the highest concentration of bak . of the actual preservatives evaluated in the present study , the cytotoxic effects of bak , polysorbate,8 and cholorobutanol9 were comparable to those reported previously ; both methyl paraoxybenzoate and chlorobutanol exhibited significant cytotoxic effects at high concentrations.10 on the basis of the results of the present study , it is likely that any postoperative epithelial damage may be caused primarily by anti - inflammatory drugs because the cell viability determined in the present study for the routinely used nonsteroidal anti - inflammatory drugs ( ie , diclod and bronuck ) were very low compared with values determined for the antibiotics . cvs50 was demonstrated to be a useful system for comparisons of the cytotoxicity of different ophthalmic solutions , as evidenced by comparable results for mtt assay using multiple cell lines and various drug concentrations . in addition , we calculated another number [ ( number of cell lines with viability > 80% ) ( number of cell lines with viability < 40% ) ] for each drug at a 10-fold dilution based on the cell viability of different cell lines and the order of this number for tested solution was the same as that for cvs50 . considering biological fluctuations in cellular reaction to the drugs , we have to be careful of evaluation of the value of cell viability and the method for expressing general trend such as cvs50 may be one of the solutions . the limitations of the present study include the use of bioassays only to evaluate cytotoxicity and the relatively long exposure time . however , the standardization of methods to evaluate the toxicity of ophthalmic solutions is still under development and the cytotoxic and/or ocular toxic effects of drugs clearly need to be evaluated more comprehensively . in the present study , we attempted to address these issues by evaluating the effects of a range of drug concentrations in four commercially available cell lines . we were able to compare the cytotoxic effects of the drugs easily using the cvs as well as graphically by plotting cell viability against drug concentration . with regard to exposure time , optimal exposure times for in vitro cell culture systems have not yet been established . it is common to use 48 h exposure followed by 48 h recovery in in vitro cytotoxicity assays ; however , epithelial cells proliferate rapidly and further studies in epithelial cells are needed using shorter exposure times . in conclusion , the toxicity of a wide range of bak - free ophthalmic solutions used postoperatively to control infection and inflammation was evaluated in four ocular surface cell lines . because sufficient data regarding cell viability from multiple cell lines with different drug concentrations were available , the results of the mtt assay were expressed as cvs to enable the simple comparison of drug effects . the cytotoxicity of antibiotics was found to be dependent on the pharmaceutical components of the solution , whereas the toxicity of anti - inflammatory drugs was found to be dependent on both the pharmaceutical components of the solution and the preservative used . based on the results of the present study , postoperative drug - induced epitheliopathy is most likely due to the effects of anti - inflammatory drugs .

a 63 year - old female patient was admitted to our hospital for laparoscopic splenectomy . her diagnosis was ovarian cancer with splenic metastasis and there were no specific findings in the laboratory data and physical examination . at admission , blood pressure and heart rate were 100/63 mmhg and 74 beats / min , respectively . anesthesia was induced with thiopental sodium ( 4 - 5 mg / kg ) and was maintained with sevoflurane 2 - 2.5 vol% . the patients were given rocuronium ( 40 mg ) to facilitate tracheal intubation . ventilation was mechanically controlled with o2/air mixture ( fractional inspired oxygen [ fio2 ] = 0.5 ) and adjusted to keep an end - tidal concentration of co2 between 35 and 40 mmhg throughout the operation . after the induction of anesthesia , central venous catheterization through the right internal jugular vein was attempted with two 14 g lumen indwelling catheter sets ( spectrum central venous catheter set , cook medical , usa ) . the enclosed introducer steel needle was used for puncture of the right internal jugular vein and free aspiration of blood was confirmed . the first attempt to insert the j - shaped guidewire failed with resistance . while lefting the introducer needle and guidewire , a more experienced clinician tried a second attempt and the guidewire passed through the venipuncture needle with resistance at the final step . the tissue dilator was inserted over the guidewire without any problems . after removing the dilator , was felt while attempting to withdraw the guidewire , so the guidewire was pulled out with force . the core wire was cut at the middle of the guidewire , and its surrounding cover coils detached and extended from the core wire ( fig . it was not clear whether the j - shaped end was lost or not because the outer cover coil was extended long ( fig . chest x - ray was taken , which revealed that the guidewire including the j - shaped tip was entrapped in the cvc ( fig . 2 ) . we suspected fracture of the guidewire and a radiologist was called for correct diagnosis as well as to remove the guidewire . to remove the fractured fragment , a snare - loop catheter 6 fr . ( multi - snare set 30 mm , pfm - prodokte fr die medizin ag , kln , germany ) with a loop diameter of 30 mm was introduced through the femoral vein with the help of a fluoroscopic intensifier ( series 9800 , ge oec medical systems inc . , the catheter passed into the superior vena cava adjacent to the j - shaped tip of the fractured guidewire . the removed fractured guidewire revealed a 19 cm length without detaching the outer cover coil . the guidewire curved from 3 cm proximal to the j - shaped tip by snare loop during removal of the guidewire . the total time from the start of anesthesia induction to the removal of fragmented guidewire was 60 min . we report a case of fractured j - tipped guidewire being entrapped in the cvc during internal jugular venous catheterization and its successful removal with snare - loop catheter under fluoroscopic guidance . the reported rate of mechanical complications such as arterial puncture , air embolism , nerve injury and hemo- or pneumothorax is between 5% and 19% . central venous catheterization is usually carried out by seldinger 's technique using guidewire which may cause complications such as failure to pass , loss in the vessel , kinking , knotting , breakage and fracture [ 4 - 11 ] . safe use of guidewires for central venous access requires care in handling and understanding of the physical characteristics . the guidewire consists of an inner single filament core wire and a surrounding coiled wire cover . apart from the two ends of the guidewire , there is no further point of attachment between the core and the outer wire . hence , any damage to the guidewire on its stem may lead to unwinding of the whole outer spiral . in our case , the j - shaped end results from rounding and flattening of the core which causes structural weakness and may lead to potential breakage . in our case , the j - shaped end was damaged 3 cm distal from the tip of the end . however , the etiology of this damage may be the force applied to this part while removing the guidewire with the snare - loop . this could be evidenced by the initial chest radiograph showing an intact j - shaped end of the guidewire which was entrapped in the cvc ( fig . the removed guidewire ( 19 cm length ) including the j - shaped end was fractured without the outer coil detachment . the proximal part of the fractured guidewire showed a cut core wire with a detached and stretched outer coil from the cut end ( fig . , we felt resistance mainly at the final step when the guidewire reached about 19 cm from the puncture needle tip ( 20 cm from the skin ) . at this time , initial damage to the guidewire due to the bevel of the accompanying puncture needle could have occurred . although no problem was encountered while inserting the tissue dilator , secondary damage could be added to the guidewire since the initial damage had already occurred . during the insertion of the catheter with subsequent withdrawal of the guidewire , the guidewire may have been cut completely and the distal part of the fractured guidewire remained in the cvc . further pulling of the guidewire may have caused the uncoiling of the outer cover coil of the proximal part of the guidewire . reported that if breakage occurred only in the inner part of the wire , it could exclude the effect of the needle . in our case , the inner wire and outer covering coil was fractured together and the damage to the guidewire could be from the puncture needle . however , there may be damage to the guidewire because the initial damage had already occurred to the guidewire . breakage or fracture of the guide wire can cause complications including myocardial perforation , pulmonary embolism , arrhythmias , sepsis , endocarditis and even cardiac arrest . techniques for removal of foreign bodies vary and depend on the type of fragment , its location , and the experience of the operator . the most commonly used techniques are snare - loop or basket catheter . in our case , the fractured guidewire entrapped in the cvc was removed using a snare - loop catheter ( fig . we suggest that no force should be applied during insertion or withdrawal of the guidewire if resistance is felt and care should also be exercised when passing the tissue dilator over the guidewire . furthermore , the snare - loop technique under fluoroscopic guidance could be considered a safe method for the removal of a fractured guidewire .

adult athletes competing primarily in aerobic modalities are characterized by higher levels of maximum cardiac output and aerobic power when compared with non - athletes or those who participate in predominantly anaerobic modalities.1 - 3 as the heart rate ( hr ) accelerates during exercise , there is a reduction in the duration of the cardiac cycle , especially in diastole , and consequently , ventricular filling time is also reduced . thus , it has been hypothesized that end - diastolic volume is limited in some individuals by progressively higher hr levels , potentially resulting in an increase in blunted stroke volume ( sv ) during exercise as a result of the frank - starling mechanism.4 the ratio between oxygen consumption ( vo2 ) and hr defines the oxygen pulse ( o2 pulse ) , which according to the fick equation , is numerically equal to the product of sv and arteriovenous o2 concentration difference.5 - 7 thus , because vo2 and hr tend to increase linearly but at different rates based on exercise intensity , the shape of the o2 pulse curve will reflect the relative differences in the magnitude of the incremental adjustments in these variables . because the shape of the curve representing the arteriovenous o2 difference does not vary appreciably between healthy individuals subjected to an incremental exercise protocol,5 o2 pulse reflects sv ( i.e. , the effective blood volume ejected from the left ventricle with each heart beat).8 - 11 this phenomenon occurs even more consistently in high - performance athletes.8 therefore , it is possible to analyze the behavior of sv by following the o2 pulse curve during progressively more intense exercise , e.g. , during cardiopulmonary exercise testing ( cpet ) performed with a ramp protocol . although some studies12 - 14 suggest that increases in sv may be limited at higher hr levels , there is evidence15 , 16 that this does not actually occur in the last few minutes of cpet and that the sv may continue to increase up to the end of an exercise protocol in which intensity is progressively increased . to establish a better physiological understanding of these responses , we evaluated a large group of professional soccer players from whom expired gases were sampled every ten seconds , and continuous electrocardiograms were recorded during a maximally progressive exercise test . we compared the o2 pulse curves throughout cpet between two groups of athletes with high and low maximum hr values . our hypothesis was that the o2 pulse curves would have similar slopes but different intercepts . this would suggest that sv , reflected by the o2 pulse curves , would behave similarly during progressive exercise testing , regardless of the magnitude of the maximum hr achieved . in addition , we sought to describe a normal pattern for the relative o2 pulse curves for young , healthy individuals with high aerobic fitness during a cpet performed using a treadmill ramp protocol in which only the velocity was continuously increased . we retrospectively analyzed the results of sports medicine evaluations of 180 professional soccer players from first - division clubs in brazil ( n = 151 ) and angola ( n = 29 ) that were overseen by our research team between 2005 and 2010 . we started with a total sample of 189 players and excluded those who a ) did not provide valid data for a true maximal cpet , i.e. , due to poor motivation and/or limiting muscle / joint pain ; or b ) were prescribed any medication that could affect the physiological response to exercise . the players included in this study were evaluated immediately after the holidays , which is the typical time for pre - season assessment . in the preceding week , subjects did not participate in any formal training or competition . subjects underwent a specialized medical evaluation aimed at identifying relevant diseases or clinical conditions that could affect their performance or competitive eligibility . any abnormalities in the resting electrocardiogram were identified and , when necessary , confirmed as physiological adaptations based on clinical findings and echocardiography.17 after this medical evaluation , all athletes were cleared for professional soccer training and competition . the mean age , weight and height of the players were 24 4 years , 75 8 kg and 178 6 cm ( mean standard deviation ) , respectively . all players provided informed consent explicitly authorizing the evaluation and use of the data ( excluding identifiable information ) for research and statistical purposes . all players were assessed using the same ramp protocol on an atl master treadmill ( inbrasport ; porto alegre , brazil ) programmed to achieve a maximum duration of 10 to 15 minutes . after one minute at 5.5 km / h , the velocity was rapidly increased to 8 km / h and then increased by 0.1 km / h every 7.5 s ( 0.8 km / h every minute ) . considering that the sport of soccer is played on a level field , we intentionally did not incline the treadmill . the criteria we adopted to ensure a maximal test were a ) achievement of maximum voluntary exhaustion , despite verbal encouragement , accompanied by a maximum effort sensation ( a grade of 10 on the borg scale ) ; and b ) a respiratory exchange ratio greater than 1.10 . ventilatory and hr data were collected starting at the third minute of the cpet , at a velocity of 8.8 km / h . we eliminated data collected during the first two minutes , which included the initial walking and running phases . we disregarded these initial phases , which comprise the transition between rest and exercise , because the responses during that time tended to be non - linear . hr was measured every 10 s from a continuous recording on a single derivation ( using cc5 or cm5 chest leads ) measured by a digital micromed electrocardiograph with the elite ergopc software versions 3.2.1.5 or 3.3.6.2 ( micromed ; braslia , brazil ) . later , in an effort to eliminate artifacts , the hr values were visually compared , and when there was a difference between two consecutive measurements that exceeded five beats , the values were confirmed on the electrocardiographic tracing and , if appropriate , corrected from the reading of five r - r intervals ( cardiac cycles ) . in about 3% of the readings , excess electrocardiographic tracing artifacts hindered this measurement and , as a result , the hr values were interpolated . the greatest observed hr value over a 10-s interval during the cpet was considered to be the maximum achieved hr . ventilatory expired gas was collected using a prevent pneumotachograph ( medgraphics ; saint paul , united states ) with the aid of a nose clip and was expressed every 10 s by a vo2000 metabolic analyzer ( medgraphics ; saint paul , united states ) , which was calibrated with known gas concentrations before and after the cpet . the o2 pulse values were collected every 10 s during the maximum cpet and divided by the athlete 's body weight to provide the relative o2 pulse . to facilitate reading of the data , the relative o2 pulse values were multiplied by 100 . to minimize the intrinsic variability of ventilatory measurements , the maximum relative vo2 and the maximum relative o2 pulse were defined as the highest mean values obtained from a 10-s interval during the maximum cpet . the cpet data were analyzed at intervals equaling 10% of the maximum effective running time ( as previously explained ) for each player , corresponding to approximately one - minute time intervals . this approach allowed us to compare data at specific intervals regardless of the final treadmill speed achieved . to test the hypothesis of this study , the players were divided into quartiles according to their maximum hr values . for this initial analysis , we considered the extreme quartiles , q1 and q4 , to represent the lowest and highest maximum hr values . the results of cpet for q1 and q4 were compared in two distinct ways : 1 ) analyzing the hr , relative vo2 and o2 pulse values at intervals representing 10% of each individual 's running time using a two - way anova ( with group and % of the cpet duration as factors ) with bonferroni post - hoc procedures as needed ; and b ) using the coefficient of determination , slope and intercept for the linear regressions of the relative o2 pulse curves ; these were compared using student 's t - test . to determine the normal standards for relative o2 pulse curves in young , healthy individuals with high aerobic fitness , we analyzed data from all 180 players , regardless of the maximum recorded hr . the coefficient of determination , slope and intercept of each relative o2 pulse curve were also calculated following the same criteria adopted for the quartiles . in addition , we verified the plateau frequency in the vo2 and relative o2 pulse curves during cpet in these elite soccer players . the vo2 curve was considered to have reached a plateau when the difference between the averages of the measurements for the last two minutes of the cpet was less than 1.4 mlo2kgmin . this criterion for defining a plateau in the vo2 curve is similar to what has been used in previous studies,3,18 . the o2 pulse curve was considered to have reached a plateau when an absence or decrease at this variable was observed in the last two minutes of cpet . the plateau frequencies between the groups were compared using a chi - squared test to determine whether this behavior could be influenced by extreme hr values . the same procedure was applied to test the hypothesis that there was no difference between q1 and q4 with respect to the positions played by each player . for this analysis , the players were divided according to the following positions : goalkeepers , defenders , midfielders and forwards . finally , we assessed the relationship between maximum hr and age using a linear regression analysis . all descriptive data are presented as mean and standard deviation , and the data from the inferential analyses are reported as the mean and standard error of the mean . the analyses were performed using prism software version 5.01 ( graphpad ; san diego , united states ) . we retrospectively analyzed the results of sports medicine evaluations of 180 professional soccer players from first - division clubs in brazil ( n = 151 ) and angola ( n = 29 ) that were overseen by our research team between 2005 and 2010 . we started with a total sample of 189 players and excluded those who a ) did not provide valid data for a true maximal cpet , i.e. , due to poor motivation and/or limiting muscle / joint pain ; or b ) were prescribed any medication that could affect the physiological response to exercise . the players included in this study were evaluated immediately after the holidays , which is the typical time for pre - season assessment . in the preceding week , subjects did not participate in any formal training or competition . subjects underwent a specialized medical evaluation aimed at identifying relevant diseases or clinical conditions that could affect their performance or competitive eligibility . any abnormalities in the resting electrocardiogram were identified and , when necessary , confirmed as physiological adaptations based on clinical findings and echocardiography.17 after this medical evaluation , all athletes were cleared for professional soccer training and competition . the mean age , weight and height of the players were 24 4 years , 75 8 kg and 178 6 cm ( mean standard deviation ) , respectively . all players provided informed consent explicitly authorizing the evaluation and use of the data ( excluding identifiable information ) for research and statistical purposes . all players were assessed using the same ramp protocol on an atl master treadmill ( inbrasport ; porto alegre , brazil ) programmed to achieve a maximum duration of 10 to 15 minutes . after one minute at 5.5 km / h , the velocity was rapidly increased to 8 km / h and then increased by 0.1 km / h every 7.5 s ( 0.8 km / h every minute ) . considering that the sport of soccer is played on a level field , we intentionally did not incline the treadmill . the criteria we adopted to ensure a maximal test were a ) achievement of maximum voluntary exhaustion , despite verbal encouragement , accompanied by a maximum effort sensation ( a grade of 10 on the borg scale ) ; and b ) a respiratory exchange ratio greater than 1.10 . ventilatory and hr data were collected starting at the third minute of the cpet , at a velocity of 8.8 km / h . we eliminated data collected during the first two minutes , which included the initial walking and running phases . we disregarded these initial phases , which comprise the transition between rest and exercise , because the responses during that time tended to be non - linear . hr was measured every 10 s from a continuous recording on a single derivation ( using cc5 or cm5 chest leads ) measured by a digital micromed electrocardiograph with the elite ergopc software versions 3.2.1.5 or 3.3.6.2 ( micromed ; braslia , brazil ) . later , in an effort to eliminate artifacts , the hr values were visually compared , and when there was a difference between two consecutive measurements that exceeded five beats , the values were confirmed on the electrocardiographic tracing and , if appropriate , corrected from the reading of five r - r intervals ( cardiac cycles ) . in about 3% of the readings , excess electrocardiographic tracing artifacts hindered this measurement and , as a result , the hr values were interpolated . the greatest observed hr value over a 10-s interval during the cpet was considered to be the maximum achieved hr . ventilatory expired gas was collected using a prevent pneumotachograph ( medgraphics ; saint paul , united states ) with the aid of a nose clip and was expressed every 10 s by a vo2000 metabolic analyzer ( medgraphics ; saint paul , united states ) , which was calibrated with known gas concentrations before and after the cpet . the o2 pulse values were collected every 10 s during the maximum cpet and divided by the athlete 's body weight to provide the relative o2 pulse . to facilitate reading of the data , the relative o2 pulse values were multiplied by 100 . to minimize the intrinsic variability of ventilatory measurements , the maximum relative vo2 and the maximum relative o2 pulse were defined as the highest mean values obtained from a 10-s interval during the maximum cpet . the cpet data were analyzed at intervals equaling 10% of the maximum effective running time ( as previously explained ) for each player , corresponding to approximately one - minute time intervals . this approach allowed us to compare data at specific intervals regardless of the final treadmill speed achieved . to test the hypothesis of this study , the players were divided into quartiles according to their maximum hr values . for this initial analysis , we considered the extreme quartiles , q1 and q4 , to represent the lowest and highest maximum hr values . the results of cpet for q1 and q4 were compared in two distinct ways : 1 ) analyzing the hr , relative vo2 and o2 pulse values at intervals representing 10% of each individual 's running time using a two - way anova ( with group and % of the cpet duration as factors ) with bonferroni post - hoc procedures as needed ; and b ) using the coefficient of determination , slope and intercept for the linear regressions of the relative o2 pulse curves ; these were compared using student 's t - test . to determine the normal standards for relative o2 pulse curves in young , healthy individuals with high aerobic fitness , we analyzed data from all 180 players , regardless of the maximum recorded hr . the coefficient of determination , slope and intercept of each relative o2 pulse curve were also calculated following the same criteria adopted for the quartiles . in addition , we verified the plateau frequency in the vo2 and relative o2 pulse curves during cpet in these elite soccer players . the vo2 curve was considered to have reached a plateau when the difference between the averages of the measurements for the last two minutes of the cpet was less than 1.4 mlo2kgmin . this criterion for defining a plateau in the vo2 curve is similar to what has been used in previous studies,3,18 . the o2 pulse curve was considered to have reached a plateau when an absence or decrease at this variable was observed in the last two minutes of cpet . the plateau frequencies between the groups were compared using a chi - squared test to determine whether this behavior could be influenced by extreme hr values . the same procedure was applied to test the hypothesis that there was no difference between q1 and q4 with respect to the positions played by each player . for this analysis , the players were divided according to the following positions : goalkeepers , defenders , midfielders and forwards . finally , we assessed the relationship between maximum hr and age using a linear regression analysis . all descriptive data are presented as mean and standard deviation , and the data from the inferential analyses are reported as the mean and standard error of the mean . the analyses were performed using prism software version 5.01 ( graphpad ; san diego , united states ) . among the 180 soccer players included in this study , there was an inverse and relatively weak relationship between age and maximum hr ( r = -.23 ; p<.01 ) . table 1 shows the demographic data and cardiopulmonary responses for the entire sample and for q1 and q4 based on the maximum cpet results . participants in q1 were slightly older ( p<.01 ) , although the mean difference was only two years . the relative vo2 did not significantly differ between the subjects in q1 and q4 at any of the cpet time intervals . starting when 20% of the cpet duration was complete , the hr was lower among the participants in q1 . conversely , the relative o2 pulse was higher in the q1 when 40% of the cpet duration was complete ( p<0.01 ) ( figure 1 ) . the linear regression model for the relative o2 pulse fit equally well for both quartiles , as shown by the high coefficients of determination ( 0.69 0.03 and 0.67 0.02 for the first and fourth quartiles , respectively ) . the values of the slope and the intercepts of the relative o2 pulse curves for participants in q1 ( lower maximum hr values ) were 0.015 0.001 and 23.2 0.5 , respectively , and for participants in q4 ( higher maximum hr values ) , they were 0.014 0.001 and 21.1 0.6 , respectively . whereas the relative o2 pulse curve slopes were virtually identical between the quartiles ( p = .25 ) , the curve intercepts differed ( p<.01 ) . the average exercise time during cpet was 13.2 1.2 min . the maximal relative o2 pulse ( 100 ) value was 33.4 4.0 mlo2beatkg . the coefficient of determination , slope and intercept for the relative o2 pulse curves were 0.68 0.18 , 0.014 0.006 and 23.0 3.2 , respectively . figure 2 shows the behavior of the relative vo2 , hr and relative o2 pulse in response to the increase in treadmill velocity . the vo2 curves for a total of 67 ( 37% ) subjects reached a plateau according to the criterion defined for this study , and the average relative vo2 variation between two consecutive minutes was 2.2 2.1 mlo2kgmin . among the subjects whose vo2 curves reached a plateau , 10 were from q1 , and 17 were from q4 ( p = .17 ) ; the remaining players were in q2 and q3 . similarly , when the entire sample of 180 soccer players was considered , a plateau was observed for the relative o2 pulse values at the end of cpet in 20% of the study participants , including 9 from q1 and 14 from q4 . as previously described , these subjects showed no increase or reduction in this variable during the last two minutes of cpet ( p = 0.33 ) . additionally , the positional roles on the soccer field were similar between q1 and q4 ( p = .87 ) . among the 180 soccer players included in this study , there was an inverse and relatively weak relationship between age and maximum hr ( r = -.23 ; p<.01 ) . table 1 shows the demographic data and cardiopulmonary responses for the entire sample and for q1 and q4 based on the maximum cpet results . participants in q1 were slightly older ( p<.01 ) , although the mean difference was only two years . the relative vo2 did not significantly differ between the subjects in q1 and q4 at any of the cpet time intervals . starting when 20% of the cpet duration was complete , the hr was lower among the participants in q1 . conversely , the relative o2 pulse was higher in the q1 when 40% of the cpet duration was complete ( p<0.01 ) ( figure 1 ) . the linear regression model for the relative o2 pulse fit equally well for both quartiles , as shown by the high coefficients of determination ( 0.69 0.03 and 0.67 0.02 for the first and fourth quartiles , respectively ) . the values of the slope and the intercepts of the relative o2 pulse curves for participants in q1 ( lower maximum hr values ) were 0.015 0.001 and 23.2 0.5 , respectively , and for participants in q4 ( higher maximum hr values ) , they were 0.014 0.001 and 21.1 0.6 , respectively . whereas the relative o2 pulse curve slopes were virtually identical between the quartiles ( p = .25 ) , the curve intercepts differed ( p<.01 ) . the maximal relative o2 pulse ( 100 ) value was 33.4 4.0 mlo2beatkg . the coefficient of determination , slope and intercept for the relative o2 pulse curves were 0.68 0.18 , 0.014 0.006 and 23.0 3.2 , respectively . figure 2 shows the behavior of the relative vo2 , hr and relative o2 pulse in response to the increase in treadmill velocity . the vo2 curves for a total of 67 ( 37% ) subjects reached a plateau according to the criterion defined for this study , and the average relative vo2 variation between two consecutive minutes was 2.2 2.1 mlo2kgmin . among the subjects whose vo2 curves reached a plateau , 10 were from q1 , and 17 were from q4 ( p = .17 ) ; the remaining players were in q2 and q3 . similarly , when the entire sample of 180 soccer players was considered , a plateau was observed for the relative o2 pulse values at the end of cpet in 20% of the study participants , including 9 from q1 and 14 from q4 . as previously described , these subjects showed no increase or reduction in this variable during the last two minutes of cpet ( p = 0.33 ) . additionally , the positional roles on the soccer field were similar between q1 and q4 ( p = .87 ) . this study assessed trends in vo2 , hr and the relationship between these two variables among healthy young male athletes with high aerobic fitness under controlled exercise conditions , in which the intensity was gradually increased to a maximum level . we compared these responses between athletes at high and low extremes of maximum hr values . the results indicate that the relative vo2 levels at all of the intervals analyzed as percentages of the total exercise time were not significantly different between the groups . nevertheless , as expected , hr values were lower in the group with a lower maximum hr starting at the time point corresponding to 20% of the cpet ; consequently , starting at the time point corresponding to 40% of the cpet , we observed an opposite trend in the relative o2 pulse curve . specifically , the participants in q1 , who had lower maximum hr values , had higher average relative o2 pulse values when compared with the participants in q4 . figure 1 shows the behavior of vo2 , hr and relative o2 pulse expressed as a percentage of the duration of the running time in the cpet . considering the adequacy of the linear regression models for analyzing the relative o2 pulse curves as indicated by the high coefficients , we were able to compare the intercepts and slopes of the curves for the two groups , which had similar slopes and distinct intercepts . interestingly , as illustrated in figure 3 , our results show a weak inverse relationship between the maximum hr and the age of the players , supporting the idea that , although maximum hr tends to decrease with age , this may vary considerably between young individuals and is poorly predicted by general formulae . we therefore chose to analyze the subjects of this study using quartiles that were assigned based on the maximal hr achieved during cpet regardless of age . we found that the trends for the relative vo2 were similar for both groups and that they remained directly related to the exercise intensity during the cpet despite the variability in the hr and relative o2 pulse values . thus , although there was a difference between the relative o2 pulse curves observed in the extreme quartiles , hr compensated for this discrepancy at most of the intervals defined as the percentage of the maximal cpet time ; i.e. , we observed lower hr values in the group with higher relative o2 pulse values . as shown in figure 1 , even at the earliest intervals of the cpet , when relative o2 pulse and hr did not differ statistically between the extreme quartiles , participants in q1 already had a lower hr and higher relative o2 pulse . other studies have shown19 - 21 a strong association between vo2 and cardiac output in incremental exercise tests . this suggests that both vo2 and cardiac output should have been similar between the groups during the cpet . thus , even with the differences in hr and relative o2 pulse between the groups , there were no differences in the demand of the active muscles for oxygen at any time point , and were there no marked differences in mechanical efficiency . in addition , the lack of differences between the slopes of the relative o2 pulse curves suggests that these trends were not affected by differing maximum hr values and that the proportion of the increase in the relative o2 pulse did not vary between the groups during the cpet . on the other hand , the intercept of the relative o2 pulse curves was significantly lower in the group with higher maximal hr values , suggesting that changes in cardiac output in response to incremental increases in exercise intensity occurred with a proportionally smaller sv . for some of the soccer players in both groups , a pronounced plateau or decreasing pattern was observed in the relative o2 pulse curve . the reason for this is that , whereas hr tends to present a linear pattern throughout the cpet , vo2 often damps during the last minutes . when exercise intensity exceeds the anaerobic threshold , a higher proportion of the energy produced will come from anaerobic metabolism , which allows the subject to tolerate the increase in exercise intensity without any further increase in vo2 . in this context , hr continues to increase throughout the entire cpet duration , whereas vo2 follows a less steep pattern or even keeps constant , resulting in a plateau of the relative o2 pulse . because the o2 pulse can be considered a surrogate for sv , this finding corroborates the observations described in other studies15,16,21 - 23 that suggest a trend of continuously increasing sv in high - performance athletes during a maximal cpet . for example , gledhill et al.15 reported a continuous increase in sv in elite cyclists during a maximum cpet . notably , the athletes in the gledhill study had high hr values ( 180 - 190 bpm ) , which were comparable to those found in the athletes who participated in the present study . similarly , zhou et al.16 observed that in elite long - distance runners , the sv increased ( by approximately 52 ml ) between light and maximum exercise intensities during cpets . although it may be possible to observe a continuous sv increase during a cpet with increasing intensity , these studies analyzed individuals with a higher maximal aerobic power than that found in our sample of professional soccer players . based on these results , we sought to compare the sv behavior in individuals with similar aerobic conditioning to our sample . vanfraechem21 assessed 17 well - trained soccer players at 25% , 50% and 75% of the maximum vo2 and reported an sv increase of 37% between 50% and 75% of the maximal aerobic capacity . this suggests that it is possible to produce increases in sv during more intense exercise in healthy individuals with high aerobic fitness . thus , in different studies and populations with similar or even greater aerobic fitness , the linear pattern of the sv response to increasingly intense exercise appears to be feasible , at least for the large majority of the subjects , despite the significant reduction in the ventricular filling time that occurs during a very intense effort . it is important to mention that these studies ' results are in contrast the conventional view that sv tends to plateau starting at 40 - 50% of maximum vo2 in progressive exercise tests.12,13,24 in some of these studies,12,13 the aerobic conditioning of the subjects was relatively low . boutcher et al.,24 however , also failed to observe increases in sv during the final stage of the cpet even among trained subjects , although it was concluded that there were differences in the sv when comparing non - trained , active and aerobically - trained men . however , a detailed analysis of these responses revealed that the hr was only analyzed to 150 bpm due to excessive movements that hindered the continuity of data collection , and the authors were unable to observe whether the sv increased beyond that point . as our sample was composed only of individuals with a high level of aerobic conditioning ( vo2 = 66.2 7.4 mlo2kgmin ) , it is likely that their inotropic and lusitropic cardiac characteristics prevented the sv from being substantially limited at the end of the cpet , even in individuals with high hr values . studies on cardiac structure25,26 show significant differences in the posterior wall and interventricular septum thickness in the heart and differences in the dimensions of the left ventricular in aerobically trained individuals cavity when compared with apparently healthy sedentary individuals . these findings could contribute to the preservation of a high sv in conjunction with a highly elevated hr at peak exercise . additionally , it has been argued16,27 that a lower resistance offered by the pericardium in aerobically trained individuals may explain an elevated end - diastolic volume and a potential for further increases in sv by the frank - starling mechanism , even in the final minutes of the cpet . data obtained in dogs appear to show that this mechanism is physiologically possible,28 as these animals were able to generate an increase in the end - diastolic volume and sv after pericardiotomy . the characteristics of the o2 pulse curve were previously analyzed by other authors29 who scored o2 pulse curve behavior according to reference values . however , the criteria they adopted appear to be subjective and oversimplify the phenomenon ; moreover , this method has not been adopted in other studies since its original publication . another objective of this study was to define a standard for the relative o2 pulse curve in young , healthy males with high aerobic fitness . although the o2 pulse has been consistently reported in the literature in absolute terms ( mlo2beat),7,8,10,30,31 it is known that obese individuals have higher submaximal vo2 values when compared with non - obese individuals at the same exercise intensity due to gravity effects and the greater effort that is required for obese individuals to run at a fixed velocity.5 thus , although two individuals may have the same maximum o2 pulse , their functional capacity ( measured by maximum running speed ) can vary significantly . therefore , to avoid the influence of body dimension variability on o2 pulse values , we analyzed this variable relative to body weight ( mlo2beatkg ) , as was recently done in other studies of our group.7,23,32 therefore , our reported relative o2 pulse values allow the extrapolation of the results to a wider range of individuals and situations . this was somewhat less evident in the last two minutes , and it was independent of the final maximum treadmill speed or the magnitude of the maximum hr . one final salient observation was that the presence of a plateau in vo2 is rather uncommon , as it occurs in only about one third of the players . this is consistent with other studies that have reported similar percentages for vo2 plateaus when trained individuals were evaluated.1,3 concerning relative o2 pulse curves , a flattening or decrease in the last two minutes of cpet was seen in 36 ( 20% ) of the 180 players . additionally , our data indicate that the occurrence of plateaus in the vo2 and relative o2 pulse curves was not significantly influenced by the magnitude of the maximal hr achieved in these professional male soccer players . importantly , we did not directly measure cardiac output , arteriovenous o2 differences or sv . therefore , the behavior of the sv can only be inferred from the relative o2 pulse data . in addition , we did not acquire invasive readings of the mean arterial pressure during cpet . these readings could contribute to a better understanding of the mechanisms that allow an increase in sv in young individuals with good to excellent aerobic conditioning . additional studies employing other methodologies for data collection are necessary to further understand these issues . the major finding of this study is that a shorter diastolic filling time , as seen in young , healthy and fit athletes with high maximum hr values , did not influence the shape of the relative o2 pulse curve , suggesting that the sv profile was likely to be unaffected . relative o2 pulse also tended to increase in a linear fashion throughout a maximal cpet . regardless of the maximal hr achieved , the relative o2 pulse curve did not increase in the last two minutes of cpet , which suggests that there is some physiological limitation of stroke volume in these individuals . mr . raphael perim and gabriel signorelli were supported by the conselho nacional de desenvolvimento cientfico e tecnolgico ( brazil ) . claudio gil is a recipient of research grants / fellowships from conselho nacional de desenvolvimento cientfico e tecnolgico and fundao carlos chagas filho de amparo pesquisa do estado do rio de janeiro ( brazil ) .

the definitive implantation of a permanent impulse generator ( interstim 3023 , medtronic ) months after was performed after a temporary trial period of stimulation . she had regained weight , defecations continued with a good daily rhythm withouth straining or abdominal pain . the patient declared to be fully satisfied by the improvement to her quality of life . we performed an abdominal x - ray showing marked distension of the whole bowel , particularly of the colon . after a brief observation period , air fluid levels increased and the colon reached an enormous dilation . ct scan demonstrated no mechanical obstruction , there seemed to be a deteriorating intestinal pseudoocclusion ( ogilvie syndrome ) . the patient 's serious general condition and the presence of free air in the peritoneum forced us to operate on her . the length and volume of the colon were enormously increased and the wall was thin and perforated in the cecum . histological findings revealed a diffused and dense inflammatory infiltrate restricted to all myenteric plexuses ( fig . 1 , fig . 3 ) , associated with degenerative features and concurrent loss of ganglion cells . in some plexuses , ganglion cells were absent . inflammatory infiltrate was mixed in nature , being composed of lymphocytes , plasma cells and eosinophilis . 4 ) revealed that most lymphocytes were cd3 + and cd4 + and only scattered lymphocytes were cd8 + or cd20 + ; a normal number and distribution of interstitial cells of cajal stained with c - kit ( cd117 ) was demonstrated . no pathological changes were observed in the mucosa and submucosa , with the exception of a mild and specific inflammatory infiltrate in the colonic mucosa . the histological findings , and particularly the presence of the just described subclasses of lymphocytes , allowed the diagnosis of acute pseudoobstruction due to idiopathic immune - mediated myenteric ganglionitis. unfortunately surgery did not resolve the illness . after a period of relative healthiness , three months after the operation the abdomen was again dilated and the peristalsis stopped . the patient was given total parenteral nutrition and at the same time we attempted a course of pulse - dosed steroid treatment ( 100 mg i.v . we attempted a jejunostomy to obtain decompression of the small bowel , but the patient 's condition quickly worsened until she eventually died . although the cause of idiopathic severe chronic constipation has never been completely clarified , almost all pathological clues have been described to be of degenerative character and/or alterations of the neuropeptide levels . an underlying inflammatory neuropathy may be responsible for chronic constipation / megacolon , usually secondary as in chagas and paraneoplastic diseases . recently , idiopathic forms of chronic constipation / megacolon have been described with autoimmune enteric ganglionitis underlying chronic idiopathic constipation and subsequent megacolon . chronic intestinal pseudoobstruction , characterized by a loss or failure of intestinal peristalsis without organic causes occluding the lumen , appears to be characterized by frequent pseudoocclusive episodes , which can simulate mechanical occlusion . in chronic intestinal pseudoobstruction the clues affect either the myogenic and/or the neurogenic part of the intestinal wall in mitochondrial myopathies and in progressive systemic sclerosis ; more frequently an enteric ganglionitis can be secondary to different conditions , including inflammatory and paraneoplastic disease , or more rarely can be idiophatic . because of recent reports in literature [ 2 , 3 ] we speculate that in our case , ganglionitis of the myenteric plexus was present since the first observation with clinical signs of chronic constipation / megacolon with a subsequent dramatic change into an acute pseudoobstruction , because of the worsening of the autoimmune inflammation . pharmacological treatment of severe idiopathic chronic constipation ( and of chronic pseudoobstruction ) is reported in the scientific literature with various types of prokinetics : cisapride , metoclopramide , domperidone , octreotide , prostigmina , erythromycin , etc . however , the disordered motility is resistant to treatment and prokinetic drug therapy is generally disappointing . recently some encouraging results have been reported regarding the first experiences in treatment by sacral neuromodulation . if the cause of severe chronic constipation is an autoimmune ganglionitis , we can also attempt immunosuppressive therapy [ 4 , 5 ] . in hindsight , we should suspect intestinal ganglionitis as a cause of severe constipation . nowadays , a secure diagnosis concerning the existence of enteric ganglionitis can be confirmed only by histological sampling from full - thickness samples of the intestinal wall , and this is possible , except for the rectum , only in patients undergoing intestinal surgery . antineuronal circulating antibodies , such as the antineuronal antinuclear anna-1 , the anti - hu ( antineuronal nuclear antibody ) , the anti - yo ( anti purkinje cell protein cytoplasmatic antibody ) or other circulating antineuronal antibodies expressed in ganglionitis , are not reliable for diagnosis . however , in presence of any suspicion when intestinal occlusion is not secondary to mechanical causes , we should not hesitate to obtain a full - thickness biopsy of the intestinal wall , even if surgery is required . this way a short operation can avoid a long series of useless surgical procedures . once we have a firm diagnosis , patients can benefit immediately from immunosuppressive treatment with high - dosed steroids and/or immunosuppressive drugs , also in association with permanent sacral nerve stimulation , which can help patients with complete failure of intestinal motility . the reported case is exceptionally rare , in fact we found only one analogous case of mortality in a young adult due to acute intestinal pseudoocclusion with known primary visceral myopathy in the literature . however we can draw some conclusions : ( 1 ) when severe chronic constipation is present , we must consider that this can be due to an inflammatory process of the myenteric plexus and thus might evolve into severe pseudoocclusion . ( 2 ) only a full - thickness biopsy can offer a firm diagnosis : if we suspect ganglionitis we should try absolutely to obtain it . ( 3 ) with a firm diagnosis of ganglionitis we can treat the disease in time , not only during the exacerbation but also in the chronic stadium , by high doses of corticosteroids [ 4 , 5 ] , npt , etc . ( 4 ) in a part of rare perforative complications , surgery is not indicated in intestinal pseuodoocclusion , except for full - tickness biopsy or decompressive procedures .

malnutrition is a frequent problem in cancer patients , whose prevalence and degree mainly depend on tumor stage and site 1 . its negative consequences are prolonged hospitalization , a higher degree of treatment - related toxicity , reduced response to cancer treatment , lower activity level , impaired quality of life and a worse overall prognosis 2 . even minimal weight loss during chemo / radiotherapy ( crt ) is associated with significantly reduced survival 3 . nutritional support is a step by step intervention , which should be actively managed and targeted for each patient according to nutritional conditions , clinical status , planned oncologic treatment and expected outcome . its goal is preventing or treating malnutrition , in order to allow the successful completion of oncologic treatments , improve prognosis and preserve functional status and quality of life 4 , 5 . although recommendations on the optimal management of nutritional support for patients with malignancies have been provided 4 , 6 , 7 , the attitude towards this issue varies considerably among oncologists , sometimes even within one center , and an important proportion of malnourished patients is reported not to receive adequate nutritional support 1 . this could be related to the continuing insufficient awareness of nutritional problems among health care professionals 8 , the lack of structured collaboration between oncologists and clinical nutrition specialists and the still limited number of clinical trials aimed at improving our understanding of the nutritional support required in different care settings for cancer patients . another worrying issue , which may hamper the appropriate nutritional care of cancer patients , is the expanding market of alternative anti - cancer diets , which are not supported by scientific evidence and may lead to insufficient protein - calorie intake . the purpose of this paper is to highlight the nutritional issues in cancer patients , thus allowing the italian association of medical oncology ( aiom ) and the italian society of artificial nutrition and metabolism ( sinpe ) to make suitable and concise practical recommendations for appropriate nutritional support in this patients ' population . we reviewed the available literature - prioritizing meta - analyses , systematic reviews and randomized controlled trials where available - and international guidelines on the nutritional management of patients with cancer . in addition , experts from the two societies , who are listed among the authors , provided additional clinical information which helped in clarifying some issues . early recognition of nutritional problems is the first key point for appropriate nutritional management of cancer patients . different tools for nutritional screening have been validated in the oncologic setting and effectively allow the identification of patients at nutritional risk , who are likely to benefit from nutritional support . they are : the nutritional risk screening 2002 ( nrs 2002 ) , the malnutrition universal screening tool ( must ) , the malnutrition screening tool ( mst ) and the mini nutritional assessment ( mna ) 9 . nutritional screening should be performed using a validated tool upon diagnosis and systematically repeated at regular time points during the course of disease in patients with cancer type , stage or treatment potentially affecting nutritional status . patients at nutritional risk should be promptly referred for comprehensive nutritional assessment and support to clinical nutrition services or medical personnel with documented skills in clinical nutrition , specifically for cancer patients . bioelectrical impedance vectorial analysis ( biva ) can be performed in different clinical settings and allow the suitable assessment of patients in whom calculation of body composition fails due to altered hydration 11 . in particular , the primary output measure of this technique , phase angle , was found to be associated with functional status 12 and energy intake 13 , and to be predictive of quality of life and prognosis in cancer patients 12 , 14 . as nutritional therapy is primarily intended to preserve or restore lean body mass , the assessment of body composition by biva should be integrated in the nutritional assessment of cancer patients . indications for nutritional support in cancer patients vary throughout the continuum of care , depending on whether patients are undergoing active oncologic treatment , are in remission or in a palliative stage . this means that regular nutritional monitoring is mandatory in all patients with cancer type , stage or treatment potentially affecting nutritional status . nutritional interventions should compensate for inadequate energy intake with the objective of improving clinical outcomes 4 , 6 , 7 , 15 . the correct identification of candidates for nutritional support relies on the evaluation of current and expected nutritional status and energy intake . accordingly , nutritional support should be provided to malnourished patients and those at nutritional risk , in particular when oral energy intake is already insufficient or expected to be inadequate ( < 60% of estimated caloric requirements ) for more than 7 days 4 , 6 , 7 , 15 . undernourished cancer patients with planned elective surgery should receive at least 7-day pre - operative nutritional support to improve post - operative outcomes , even if this may delay surgery 16 . dietary counseling , including the use of oral nutritional supplements ( ons ) , should be the first - step towards achieving satisfactory energy intake . in presence of normal gut function and inadequate food intake , total or integrative enteral tube feeding must be considered . if enteral nutrition ( en ) is not feasible due to gut dysfunction , symptoms which could be worsened by enteral support ( i.e. nausea , vomiting , diarrhea ) or patients ' refusal , parenteral nutrition ( pn ) is required for delivering nutritional support 4 , 6 , 7 , 15 . nutritional counseling is the first - line of treatment in malnourished cancer patients or in those at nutritional risk , due to its proven efficacy in increasing protein - calorie intake , body weight and improving body composition 17 , 18 . in head and neck cancer patients undergoing crt , nutritional counseling was found to be associated with lower crt toxicity and symptom - induced morbidity 19 , and to have beneficial effects on quality of life 20 . therefore , all malnourished or at nutritional risk cancer patients who are able to eat should be referred to a dietitian with documented skills in cancer patient care for appropriate dietary intervention and its monitoring . while taking into account individual preferences , ethnicity and culture , the optimization of oral diet should consider predominantly the issue of appropriate protein - calorie content and texture , in order to cope with nutritional deficiencies and swallowing difficulties . in addition , any practical suggestions for managing the common symptoms related to cancer treatments , leading to impaired food intake or malabsorption should be included , as well . when dietary measures fail to meet patients ' protein - calorie requirements as detected by nutritional monitoring , the prescription of energy - dense ons should be considered , due to their proven efficacy in increasing protein - calorie intake 6 , 21 . complementary therapies in the form of natural dietary supplements are frequently used and asked for by cancer patients . their purported antitumor effects are not yet demonstrated by appropriate efficacy evaluations , so their use can not be recommended . however , healthcare professionals involved in the nutritional treatment of cancer patients should be knowledgeable on this issue , in order to discuss with the patients the potential risks , benefits and expectations deriving from specific dietary supplement consumption 22 . a healthy dietary pattern is known to be associated with reduced cancer risk 23 , so it is reasonable to argue that it would reduce cancer recurrence , as well . however , the available clinical supporting evidence is limited to reduction of fat intake in women with early - stage breast cancer 24 . since cancer and related treatments may be responsible for metabolic changes affecting nutritional requirements , dietary advice should be tailored to the individual patient and hypocaloric alternative anti - cancer diets ( e.g. macrobiotic or vegan ) are not recommended , as they could worsen protein - calorie intake with no proven benefits on recurrence rates 25 . finally , although recent animal model studies showed that pretreatment short - term starvation could improve chemotherapy ( ct ) efficacy and reduce toxicity by diminishing malignant cells ' resistance to drugs while protecting normal tissues 26 , this hypothesis still needs to be confirmed in humans . therefore , this practice is not recommended , particularly in malnourished patients and those at nutritional risk , since weight and lean body mass loss is associated with dose - limiting toxicity and mortality in patients undergoing ct 3 , 10 . en by means of tube feeding offers the possibility of increasing or ensuring nutrient intake whenever the gastrointestinal tract is functional and oral nutrition is not feasible or remains inadequate despite nutritional counseling and ons consumption 6 , 7 , 27 . en should not be used routinely during anticancer treatment in all patients , but only in those who are malnourished or judged to be unable to eat adequately ( the intention being to introduce an amount of calories 60% of estimated requirements ) for more than 7 days 6 , 7 , 27 . tube feeding can either be delivered via trans - nasal ( nasogastric / nasojejunal tube ) or a percutaneous route ( percutaneous endoscopic / radiologically inserted / surgical gastrostomy or jejunostomy ) . to date , there is insufficient evidence to recommend the best route in terms of efficacy and safety 27 , 28 ; however , gastrostomy should be preferred for long term treatment ( i.e. home artificial nutrition , han ) , as it may be more comfortable for patients and easier to manage for care - givers , whereas trans - nasal tubes need to be replaced approximately every 6 weeks 6 , 7 . whenever trans - nasal tubes or gastrostomy placement is not feasible , as may be the case in severe obstructing esophageal or gastric cancer , needle catheter jejunostomy represents the most appropriate en delivery route 6 , 7 , 27 . with regards to timing , prophylactic feeding does not seem to offer advantages in terms of nutritional outcomes , treatment interruptions and survival compared to reactive feeding , which is initiated once nutritional counseling and ons have failed to satisfy energy requirements 27 , 28 . en represents the first - line peri - operative nutritional treatment also for surgical cancer patients requiring artificial nutrition 6 , 7 . both european and american guidelines recommend preoperative en with immune - enhancing formulas , containing arginine , -3 fatty acids and nucleotides , in cancer patients undergoing major head - neck or abdominal surgery 6 , 7 , 16 , although the grade of this recommendation is still being debated . post - operative en is recommended in surgical patients malnourished at the time of intervention , in those who can not reinitiate oral nutrition early or when this is expected to be inadequate for more than 10 days 16 . the use of pn in cancer patients has been debated because of the risk of infection . both european and american guidelines clearly stated that pn is indicated in patients receiving active cancer treatment who are malnourished or are facing a period longer than 7 days of inadequate energy intake when nutritional counseling , ons or en are not feasible or ineffective 4 , 7 . a short period of pn ( 10 - 15 days ) is indicated in patients with acute and severe mucositis , ileus or intractable vomiting , whereas long - term pn ( more than 30 days ) should be implemented in patients with intestinal failure due to extensive bowel resection , severe malabsorption , mechanical bowel obstruction , in sub - acute or chronic radiation enteritis and in patients with graft versus host disease of the digestive tract 4 , 7 . pn may also aid insufficient oral intake in hypophagic patients with a working gut ( supplemental pn ) 27 . pn is contraindicated in hemodynamically unstable patients , with ascites , severe organ failure , or in the presence of severe glycemic instability and it is rarely appropriate in incurable cancer patients with life expectancy shorter than 3 months , karnowfsky score 50 or ecog performance status 3 4 , 7 , 15 . for long - term pn , a tunneled - catheter or implanted chamber is needed . rigorous monitoring , particularly of glycemia and electrolytes , should be implemented from the time of starting pn , in order to prevent clinical and metabolic complications and to evaluate the impact of pn on clinical outcomes . home artificial nutrition ( han ) is a well established extra - hospital therapy , which helps to decrease the costs of health care , mostly by reducing the number and length of hospitalizations 29 . han can improve the prognosis of patients in several acute and chronic diseases , including cancer , and allows patients to integrate into their families and into society , thus improving their quality of life 30 . due to its organizational complexity , potentially serious complications and the necessity of periodic outcomes assessment , han should be prescribed and regularly monitored using defined protocols shared between oncologists and clinical nutrition specialists . nutritional support , including han , may be also integrated into palliative care programs , when it is expected to be beneficial to quality of life and if it is estimated that patients may die from malnutrition prior to dying from cancer progression 4 , 27 . while patients who are not in the terminal phase of cancer may benefit from nutritional support and other medical therapies for cancer cachexia 31 , low - quality evidence ( i.e. in the absence of randomized trials ) suggests that the administration of en or pn in the last weeks of life does not change the course of the disease , so it may not be indicated 32 . according to international guidelines , artificial nutrition may not be appropriate in incurable cancer patients with life expectancy shorter than 3 months or karnowfsky score 50 or ecog performance status 3 4 , 6 , 7 , 15 . in conclusion , nutritional support , including han , may be integrated into palliative care programs , according to individual - based evaluations , quality of life implications , life expectancy and patients ' awareness . it should be emphasized that malnutrition is an important issue in cancer patients , which should be appropriately managed by structured collaboration between oncologists and clinical nutrition specialists . the aiom and sinpe recommend validated nutritional screening upon diagnosis and at regular time points in all patients with cancer type , stage or treatment potentially affecting nutritional status , together with prompt referral to clinical nutrition services or medical personnel with documented skills in clinical nutrition for comprehensive nutritional assessment and support prescription . well - designed clinical trials are needed to improve the evidence in favour of nutritional support in different care settings for cancer patients . in addition , nutritional parameters should be considered as relevant outcomes or potential confounders in outcome assessment in clinical oncology research .

diabetic retinopathy , a vision - threatening disease , is classically regarded as microvasculopathy . however , recent evidence suggests that diabetic retinopathy is a progressive neurodegenerative disease in which visual dysfunction is initiated early after the onset of diabetes and progresses independently of the vascular lesions [ 14 ] . however , the molecular mechanisms underlying the diabetes - induced retinal neurodegeneration and dysfunction are still not well understood . recent studies revealed that diabetic retinal neurodegeneration is associated with oxidative stress resulting from excess generation of reactive oxygen species as well as inflammation [ 3 , 5 ] . brain - derived neurotrophic factor ( bdnf ) , a protein belonging to the neurotrophin family , is expressed in retinal ganglion cells and mller cells and is important for the survival of retinal ganglion cells . bdnf is important in neural development and cell survival and is essential to molecular mechanisms of synaptic activity . recent studies suggested that the early retinal neuropathy of diabetes involves the reduced expression of bdnf and can be ameliorated by an exogenous supply of this neurotrophin [ 1 , 3 ] . it was also demonstrated that the reduction of bdnf in the diabetic retina was attenuated by the antioxidant lutein , indicating that this change was partly caused by excessive oxidative stress . high - mobility group box-1 ( hmgb1 ) is a nonhistone dna - binding nuclear protein that is highly conserved during evolution . necrotic cell death can result in passive leakage of hmgb1 from the cell as the protein is then no longer bound to dna . in addition , hmgb1 can be actively secreted by different cell types , including activated monocytes and macrophages , mature dendritic cells , natural killer cells , and endothelial cells . extracellular hmgb1 functions as a proinflammatory cytokine [ 912 ] . released hmgb1 signals through the receptor for advanced glycation end products ( rage ) , a member of the immunoglobulin superfamily of receptors , leading to activation of the transcription factor nuclear factor kappa b ( nf-b ) , which may alter gene transcription and lead to the upregulation of proinflammatory cytokines , chemokines , and adhesion molecules and intensifies cellular oxidative stress [ 913 ] , processes that may play a role in the pathogenesis of diabetic retinal neurodegeneration and dysfunction . therefore , recently , rage has been implicated in the pathogenesis of various diabetic complications , via oxidative stress [ 13 , 14 ] . strong evidence indicates that chronic , low - grade inflammation is implicated in the pathogenesis of diabetic retinopathy [ 16 , 17 ] . it was also demonstrated that hmgb1 provides the link between chronic neuroinflammation and progressive neurodegeneration in neurodegenerative diseases , such as parkinson 's disease . in addition , it was reported that extracellularly released hmgb1 protein mediates postischemic damage of the brain and retina and that inhibiting or knockdown of hmgb1 attenuated postischemic neurodegeneration [ 1821 ] . in previous studies , we demonstrated that hmgb1 was upregulated in the vitreous fluid and epiretinal membranes from patients with proliferative diabetic retinopathy ( pdr ) as well as in the retinas of diabetic mice . in addition , we demonstrated significant positive correlations between levels of hmgb1 and levels of inflammatory biomarkers such as monocyte chemoattractant protein-1 ( mcp-1 ) and soluble intercellular adhesion molecule-1 ( sicam-1 ) in vitreous fluid from patients with pdr [ 2224 ] . glycyrrhizin ( ga ) , an ingredient of the licorice roots , has long been known to exhibit glucocorticoid - like anti - inflammatory actions by inhibiting 11-hydroxysteroid dehydrogenase . more recently , ga has also been shown to bind to and inhibit cytokine - like activities of hmgb1 . in this study , we explored the hypothesis that bdnf - mediated neuroprotection is reduced by hmgb1 in the diabetic retina . to test this hypothesis , we measured the levels of bdnf , hmgb1 , soluble rage ( srage ) , biomarkers of inflammation and endothelial dysfunction including mcp-1 , and sicam-1 and the oxidative stress and lipid peroxidation marker thiobarbituric acid reactive substances ( tbars ) in the vitreous fluid and serum from a series of patients with pdr . in addition , we investigated the expression of bdnf , hmgb1 , the synaptic vesicles protein synaptophysin , tbars , and the apoptosis executer enzyme cleaved caspase-3 in the retinas of diabetic rats . we also examined the effect of intravitreal administration of hmgb1 on the retinas of normal rats and whether constant ga intake suppresses diabetes - induced changes in bdnf expression . undiluted vitreous fluid samples ( 0.30.6 ml ) and paired serum samples were obtained from 46 patients with pdr during pars plana vitrectomy . the diabetic patients were 35 males and 11 females , whose ages ranged from 22 to 80 years with a mean of 53.9 12.8 years . the duration of diabetes ranged from 7 to 32 years with a mean of 16.4 5.6 years . twenty - four patients had insulin - dependent diabetes mellitus , and 22 patients had noninsulin - dependent diabetes mellitus . at presentation , the control group consisted of 34 patients who had undergone vitrectomy for the treatment of rhegmatogenous retinal detachment ( rd ) with no proliferative vitreoretinopathy . controls were free from systemic disease and were 23 males and 11 females whose ages ranged from 12 to 82 years with a mean of 47.8 16.8 years . vitreous samples were collected undiluted by manual suction into a syringe through the aspiration line of vitrectomy , before opening the infusion line . the samples were centrifuged ( 5000 rpm for 10 min , 4c ) and the supernatants were aliquoted and frozen at 80c until assay . blood was collected after an overnight fast , and serum was obtained by centrifugation and stored at 70c . the study was conducted according to the tenets of the declaration of helsinki , and informed consent was obtained from all patients . the study was approved by the research centre , college of medicine , king saud university . all procedures with animals were performed in accordance with the arvo statement for use of animals in ophthalmic and vision research and were approved by the institutional animal care and use committee of the college of pharmacy , king saud university . adult male sprague dawley rats , 8 - 9 weeks of age weighting in the range of 210230 g , were overnight fasted and streptozotocin ( stz ; 65 mg / kg in 10 mm sodium citrate buffer , ph 4.5 ; sigma , st . measuring of blood glucose concentrations and body weight was started after 3 days of stz injection . rats with glucose levels > 250 mg / dl were considered to have diabetes . after 4 weeks of diabetes , animals were anesthetized by intraperitoneal injection of an overdose of chloral hydrate and sacrificed by decapitation . sprague dawley rats ( 220230 g ) were kept under deep anesthesia , and sterilized solution of recombinant hmgb1 ( 5 ng/5 l ; r&d systems , minneapolis , mn , usa ) was injected into the vitreous of the right eye . for the control , the left eye received 5 l of sterile phosphate buffer saline ( pbs ) . the animals were sacrificed 4 days after intravitreal administration , and the retinas were carefully dissected , snap frozen in liquid nitrogen , and stored at 80c until analyzed . diabetic rats were divided into 2 groups : the rats in group i received normal drinking water without any supplementation , and group ii received drinking water supplemented with glycyrrhizic acid ( 150 mg / kg / day , santa cruz biotechnology , inc . , santa cruz , ca , usa ) immediately after establishment of diabetes . after 4 weeks of diabetes , the rats were sacrificed , the eyes were removed , and retinas were isolated and frozen immediately in liquid nitrogen and stored at 80c until analyzed . elisa kits for human bdnf ( quantikine brain - derived neurotrophic factors factor , cat . dcp00 ) , human srage ( quantikine human receptor for advance glycation end products , cat . the detection limits for bdnf , mcp-1 , srage , sicam , and hmgb1 were 20 , 5 , 4.12 , 96 , and 200 picograms / ml ( pg / ml ) , respectively . the elisa plate readings were done using fluostar omega - microplate reader from bmg labtech , offenburg , germany . the assay kit for the oxidative stress and lipid peroxidation marker tbars ( cat . 10009055 ) was purchased from cayman chemical company , ann arbor , mi , usa . the quantifications of the level of bdnf , mcp-1 , srage , sicam , and hmgb1 in the vitreous and serum and in the rat retinas were determined using specific elisa kits according to the manufacturer 's instruction . for each elisa kit , the undiluted standard served as the highest concentration and calibrator diluents served as the blank . depending upon the detection range of the elisa kit and the expression level of the particular molecule , vitreous and serum samples were either directly used or diluted with calibrator diluent supplied with elisa kit . for measurement of bdnf in the vitreous , 50 l of undiluted samples was added to elisa plates for analysis . for serum , samples were diluted 25-fold , 5-fold , 2-fold , and 20-fold for bdnf , mcp-1 , srage , and sicam-1 measurements , respectively . 100 l , 200 l , 50 l , and 100 l of diluted sample for bdnf , mcp-1 , srage and sicam were added into each of the elisa plates for the analysis . for measurement of bdnf in rat retinas , 200 g of rat retinal homogenate was used and added into each of the elisa plates for the analysis . for the quantification of hmgb1 within the high sensitivity range , 50 l of diluents buffer ( dilbuf , ibl international ) was added to each well of the plate followed by the addition of 50 l of 2-fold diluted sample . following sample incubation into the wells of elisa plates , secondary antibodies against bdnf , mcp-1 , srage , sicam and hmgb1 the reaction was stopped by the addition of 2 n sulfuric acid and optical density ( od ) was read at 450 nm in microplate reader . each assay was performed in duplicate . using the 4-parameter fit logistic ( 4-pl ) curve equation , the actual concentration for each sample was calculated . for the samples that have been diluted , the correction read from the standard curve obtained using 4-pl the steps for the measurement of tbars in serum and in rat retinal homogenate were followed as per the manufacturer instructions that use the principle of formation of adduct between malondialdehyde ( mda ) and thiobarbituric acid ( tba ) under high temperature ( 95c ) and acidic condition and color developed is measured colorimetrically . color reagent to be used was prepared by mixing tba with tba acetic acid and tba sodium hydroxide ( supplied with the kit ) . in a 10 ml tube , 100 l undiluted serum and retinal homogenate were mixed with 100 l of sodium dodecyl sulphate ( sds ) . the color reagent ( 4 ml ) was added in each respective tube and was boiled for 1 hour . tubes were cooled immediately on ice for 10 minutes and were centrifuged for 10 minutes at 1600 g at 4c . the upper clear solution was loaded on 96-well clear plates and the color was measured at 530 nm using fluostar omega - microplate reader ( bmg labtech ) . retinas were homogenized in a western lysis buffer ( 30 mm tris - hcl ; ph 7.5 , 5 mm edta , 1% triton x-100 , 250 mm sucrose , 1 mm sodium vanadate , and protease inhibitor cocktail ) . the lysate was centrifuged at 14,000 g for 10 min at 4c , and the supernatant was collected . protein content was assayed by dc protein assay ( bio - rad laboratories , hercules , ca , usa ) . the tissue lysates containing 50 g protein were separated on 1015% sds - polyacrylamide gels and were transferred onto polyvinylidene difluoride ( pvdf ) membranes . the blots were blocked with tbst ( 20 mm tris - hcl ; ph 7.6 , 136 mm nacl , and 0.1% tween-20 ) containing 5% nonfat milk . for detection of bdnf , synaptophysin , cleaved caspase-3 , and hmgb1 , the membrane was incubated overnight at 4c with bdnf mouse monoclonal anti - bdnf ( 1 : 500 , cat no . sc-65513 , santa cruz ) , goat polyclonal antisynaptophysin ( 1 g / ml , cat . af-5555 , r&d systems ) , rabbit monoclonal anticleaved caspase-3 ( 1 : 300 , cat . mab835 , r&d systems ) and rabbit polyclonal anti - hmgb1 ( 1 : 1000 , cat . no . the membranes were washed four times with tbs - t ( 5 min each ) . for bdnf , the membrane was incubated at room temperature for 1.5 h with anti - mouse secondary horseradish peroxidase - conjugated antibody ( 1 : 2000 , sc-2005 , santa cruz ) , for synaptophysin with anti - goat secondary horseradish peroxidase - conjugated antibody ( 1 : 2000 , sc-2768 , santa cruz ) , and for cleaved caspase-3 and hmgb1 with anti - rabbit secondary horseradish peroxidase - conjugated antibody ( 1 : 2000 , sc-2004 , santa cruz ) . after incubations with secondary antibodies , membranes were washed four times with tbs - t ( 5 min each ) and the immunoreactivity of bands was visualized on a high - performance chemiluminescence machine ( g : box chemi - xx8 from syngene , synoptic ltd . cambridge , uk ) by using enhanced chemiluminescence plus luminol ( sc-2048 , santa cruz ) and quantified by densitometric analysis using image processing and analysis in genetools ( syngene by synoptic ltd . membranes were stripped and incubated with a mouse monoclonal anti--actin antibody ( 1 : 2000 , sc-2048 , santa cruz ) and all the remaining steps were followed as detailed above . all data from the three independent experiments were expressed as a ratio to mean intensity . the nonparametric mann - whitney u test was used to compare means from two independent study groups . bdnf was not detected in vitreous samples from patients with pdr and nondiabetic control patients . bdnf , hmgb1 , srage , sicam-1 , mcp-1 , and tbars were detected in all serum samples from patients with pdr and nondiabetic controls . mean levels of bdnf in serum samples from patients with pdr were significantly lower than those in nondiabetic control patients ( p = 0.015 ) . on the other hand , mean levels of hmgb1 , srage , sicam-1 , and tbars were significantly higher in serum samples from patients with pdr than those in nondiabetic controls ( p < 0.001 ; p = 0.008 ; p = 0.019 ; p = 0.011 , resp . ) . mean levels of mcp-1 did not differ significantly between patients with pdr and nondiabetic control patients ( p = 0.836 ) ( table 1 ) . there was a significant inverse correlation between serum levels of bdnf and hmgb1 ( r = 0.324 ; p = 0.049 ) ( figure 1 ) . there were significant positive correlations between serum levels of tbars and srage ( r = 0.335 ; p = 0.018 ) , sicam-1 ( r = 0.303 ; p = 0.032 ) , and mcp-1 ( r = 0.344 ; p = 0.012 ) . there was a significant positive correlation between serum levels of srage and sicam-1 ( r = 0.431 ; p < 0.001 ) ( table 2 ) . we examined the correlations between serum bdnf , hmgb1 , srage , sicam-1 , mcp-1 , and tbars levels and clinical parameters at presentation including fasting blood sugar , triglycerides , total cholesterol , hdl - cholesterol , ldl - cholesterol , and creatinine . there were significant positive correlations between serum levels of mcp-1 , and triglycerides ( r = 0.402 ; p = 0.0231 ) and total cholesterol ( r = 0.519 ; p = 0.003 ) . there were no significant relationships between serum levels of bdnf , hmgb1 , srage , sicam-1 , mcp-1 and tbars and type of diabetes treatment ( table 3 ) . the mean serum levels of srage in patients receiving antihypertensive agents were significantly higher than those in other patients ( table 4 ) . the body weights of the rats with diabetes were lower and their blood glucose levels were more than fourfold higher compared with age - matched normal control rats ( 180 22 versus 250 28 g and 453 32 versus 111 12 mg / dl , resp . ) . treatment of the diabetic rats with ga for one month did not change these metabolic variables in diabetic rats ( 167 25 versus 178 22 g and 449 36 versus 475 32 mg / dl , resp . ) . quantification of bdnf levels in nondiabetic controls and the retinas of rats with diabetes was done with the use of elisa . bdnf protein levels in the retinas of animals with diabetes ( 0.03 0.01 pg/g protein ) were significantly lower than those in nondiabetic controls ( 0.06 0.02 pg/g protein ) ( p = 0.014 ; mann - whitney u test ) ( figure 2 ) . the generation of tbars in diabetic retinas ( 11.88 8.2 mole/g protein ) significantly increased compared with nondiabetic controls ( 5.27 1.8 mole/g protein ) ( p = 0.005 ) ( figure 3 ) . we quantified the expression of hmgb1 , bdnf , synaptophysin , and cleaved caspase-3 by western blot analysis . densitometric analysis of the bands revealed a significant increase in hmgb1 ( p = 0.01 ) and cleaved caspase-3 ( p = 0.004 ) and a significant decrease in bdnf ( p = 0.046 ) and synaptophysin ( p = 0.003 ) in diabetic retinas compared with nondiabetic controls ( figure 4 ) . elisa demonstrated that intravitreal administration of hmgb1 in normal rats induced significant downregulation of the expression of bdnf in the retinas ( 0.038 0.01 pg/g protein ) compared with controls ( 0.05 0.01 pg/g protein ) ( p = 0.01 ) ( figure 2 ) . hmgb1 injection induced significant upregulation of the generation of tbars in the retinas ( 8.24 1.5 mole/g protein ) compared with controls ( 5.51 1.9 mole/g protein ) ( p = 0.045 ) ( figure 3 ) . western blot analysis for hmgb1 , bdnf , synaptophysin , and cleaved caspase-3 showed that intravitreal administration of hmgb1 in normal rats significantly increased the expression of hmgb1 ( p = 0.001 ) and cleaved caspase-3 ( p = 0.004 ) and significantly decreased the expression of bdnf ( p = 0.001 ) and synaptophysin ( p = 0.001 ) compared with controls ( figure 5 ) . western blot analysis was used to assess the effect of ga on diabetes - induced alterations of bdnf in the retinas of rats . constant ga intake from the onset of diabetes significantly attenuated diabetes - induced downregulation of bdnf ( p = 0.048 ) ( figure 6 ) . in a previous study , we demonstrated that constant ga intake from the onset of diabetes significantly attenuated diabetes - induced upregulation of hmgb1 in the retinas of rats . in the present study , we investigated the correlations between the levels of bdnf and the levels of hmgb1 in the vitreous fluid and serum from patients with pdr and in the retinas of rats with diabetes . we also investigated the effect of intravitreal administration of hmgb1 on the retinas of rats . we demonstrated that bdnf levels were below the detection limit of our test system in the vitreous fluid . bdnf was significantly downregulated in the serum from patients with pdr and in the retinas of rats with diabetes , whereas hmgb1 was significantly upregulated . we also found a significant inverse correlation between the levels of bdnf and hmgb1 in the serum . our results are consistent with previous reports that demonstrated reduced levels of the protein and mrna of bdnf in streptozotocin - induced diabetic rat retinas [ 1 , 3 ] . sasaki et al . reported that excessive oxidative stress is responsible for the reduced bdnf levels in the retinas from rats with diabetes . the antioxidant lutein prevented reactive oxygen species generation , visual impairment , bdnf depletion , and neuronal cell apoptosis in the diabetic retina . furthermore , intraocular administration of bdnf rescued retinal dopaminergic amacrine cells from neurodegeneration in rats with diabetes . the findings suggest that the early retinal neuropathy of diabetes involves the reduced expression of bdnf , whose deficiency is associated with a number of neurodegenerative disorders . we showed that the protein level of bdnf was also reduced in the retinas by intravitreal injection of hmgb1 , whose levels are elevated in the vitreous fluid and epiretinal membranes from patients with pdr as well as in the retinas of diabetic animals [ 2224 ] . in addition , the hmgb1 inhibitor ga attenuated diabetes - induced upregulation of hmgb1 and downregulation of bdnf in the retinas of rats . our findings suggest that diabetes - induced decrease of bdnf in the retina seems to be mediated by hmgb1 . bdnf , in addition to its role in neuronal health , plays a systemic role in glucose metabolism . in animals , bdnf is involved in insulin resistance , reduces food intake , and lowers blood glucose levels in obese diabetic mice . it was also demonstrated that , compared with thiazolidinediones , bdnf potently ameliorates pancreatic dysfunction , fatty liver , and energy expenditure , thereby exerting favourable antidiabetic effects in type 2 diabetic mice . it was also reported that high levels of glucose , but not insulin , inhibit the output of bdnf from the human brain . these findings suggest that bdnf may have a potential as a unique hypoglycemic agent for the treatment of diabetes . in line with earlier reports [ 30 , 31 ] , serum levels of bdnf in patients with pdr were significantly reduced compared to control nondiabetic patients . in contrast to bdnf , we demonstrated elevated levels of hmgb1 in the serum from patients with pdr . our results are consistent with previous reports that demonstrated elevated levels of hmgb1 in the serum from patients with type 1 and type 2 diabetes [ 32 , 33 ] and that higher serum hmgb1 levels were associated with greater prevalence and severity of albuminuria . in addition , hmgb1 levels were positively associated with markers of low - grade inflammation and endothelial dysfunction . it possibly serves multiple functions in synaptic vesicle formation and exocytosis , playing an important role in neurotransmitter delivery . it is widely used as one of the synaptic function markers and is also thought to be closely related to synaptogenesis and synaptic plasticity during neural tissue development . synaptophysin knockout mice exhibited a significant decrease in synaptic vesicles in retinal rod photoreceptors , which disturbs neurotransmitter release and synaptic network activity . previous studies demonstrated that 1 month of diabetes decreases the retinal expression of synaptophysin [ 3 , 35 , 36 ] and that the antioxidant lutein prevented synaptophysin reduction and avoided increase in cleaved caspase-3 in the diabetic retina suggesting that local oxidative stress has a neurodegenerative influence in diabetic retina . in the present study , we demonstrated that , similar to diabetes , hmgb1 caused a significant decrease in the synaptic vesicle protein synaptophysin and a significant increase in the activated cleaved caspase-3 in the retina of normal rats . activation of hmgb1/rage signaling axis is important in promoting proinflammatory pathways considered to play an important role in diabetes - induced retinal neuroinflammation [ 912 , 16 , 17 ] . interaction of hmgb1 with rage results in activation of nf-b , release of cytokines and chemokines , expression of adhesion molecules , and induction of oxidative stress [ 914 ] . in our laboratory , we recently demonstrated that diabetes induced significant upregulation of the expression of hmgb1 , rage , activated nf-b , and intercellular adhesion molecule-1 ( icam-1 ) in the retinas of rats and that intravitreal administration of hmgb1 in normal rats mimics the effect of diabetes . in addition , coimmunoprecipitation studies showed that diabetes increases the interaction between hmgb1 and rage . these findings suggest a pathogenic role of hmgb1 in the development of diabetic retinopathy through rage and activation of nf-b . recently , it was reported that chronic neuroinflammation may be a driving force of progressive neurodegeneration and that hmgb1 provides the link between chronic neuroinflammation and progressive neurodegeneration in neurodegenerative diseases , such as parkinson 's disease . in the present study , we report that srage and sicam-1 levels were significantly upregulated in the serum from patients with pdr and that mcp-1 levels did not differ between patients with pdr and nondiabetic control subjects . our results are consistent with previous reports that demonstrated increased circulating levels of srage [ 32 , 37 , 38 ] and sicam-1 [ 32 , 3941 ] in both patients with type 1 and type 2 diabetes mellitus compared to nondiabetic controls . pham et al . demonstrated increased serum concentration of sicam-1 in patients with type 2 diabetes mellitus compared with healthy subjects , whereas serum concentrations of the chemokine mcp-1 were not increased . it has been suggested that human srage production might be derived from alternative rna splicing as well as by release from the full - length rage receptor by proteinases . it was also demonstrated that the engagement of hmgb1 with rage promotes the shedding of the receptor and that high levels of soluble forms of rage correlate with high levels of chronic ongoing inflammation . furthermore , circulating srage levels may reflect enhanced tissue rage expression in diabetic vasculature . in the present study , we identified a significant positive correlation between the serum levels of srage and the biomarker of endothelial activation and dysfunction sicam-1 consistent with a previous study . these findings suggest that rage signaling pathway is involved in the pathogenesis of endothelial activation and dysfunction in diabetes and that these factors may be coregulated in diabetic retinopathy . srage , a truncated form of the receptor , binds ligands with affinity equal to that of cellular rage . it , therefore , has the ability to prevent rage signaling acting as a decoy by binding ligands and preventing them from reaching the cell surface rage . in vitro , srage added to cultured cells blocked the effects of rage ligands on expression of inflammatory markers , cellular migration and proliferation , and cytotoxicity . srage has successfully been used in a variety of animal disease models to antagonize rage - mediated pathologic processes [ 44 , 45 ] . several studies showed that srage might beneficially impact early vascular and neuronal dysfunction in the diabetic retina . systemic administration of srage inhibits blood - retinal barrier breakdown , leukostasis , and expression of icam-1 in the retina of diabetic animals . in addition , attenuation of the rage axis with srage ameliorated retinal neuronal dysfunction and reduced the development of capillary lesions in a murine model of nonproliferative diabetic retinopathy . our results suggest that elevated levels of srage in the serum from patients with pdr potentially negatively regulate inflammation and that srage is secreted extracellularly as a negative feedback mechanism to limit diabetes - induced retinal vascular and neuronal dysfunction . , there was a significant increase in lipid peroxidation in the serum from patients with pdr and in the retinas of diabetic rats as measured by tbars formation . in addition , intravitreal administration of hmgb1 to normal rats induced significant upregulation of tbars in the retina . these results are in agreement with several studies that reported elevated levels of circulating tbars in diabetic subjects [ 4850 ] and that the expression of tbars is increased in the retinas of diabetic rats . our analysis showed significant positive correlations between the serum levels of tbars and srage , sicam-1 , and mcp-1 . our results are consistent with a previous report that demonstrated a significant positive correlation between plasma levels of tbars and sicam-1 in patients with type 1 diabetes mellitus and that hyperglycemia - induced oxidative stress induces an increase of circulating icam-1 levels and the expression of rage in human endothelial cells . on the basis of our findings , we propose a causal relationship linking hyperglycemia , activation of hmgb1/rage signaling axis , neuroinflammation , oxidative stress , and diabetic retinal neurodegeneration . in conclusion , these data suggest that diabetes - induced increased oxidative stress , downregulation of bdnf and synaptophysin , and upregulation of cleaved caspase-3 were also induced by hmgb1 . the hmgb1 inhibitor ga attenuated diabetes - induced downregulation of bdnf in the retinas of rats collectively , our present data suggest that blocking hmgb1 signaling pathways might be a novel therapeutic strategy for neuronal dysfunction in vision - threatening diabetic retinopathy .

invasive gas infections have increased worldwide during the past decade despite organism remaining sensitive to penicillin and other commonly used beta - lactam antibiotics . gas have been described frequently as an emerging cause of severe invasive infections in population - based surveillance studies , whereas the descriptions of group b , c , and g streptococci ( gbs , gcs , and ggs ) have been less frequent . it is not possible to differentiate between invasive infections caused by gas , gbs , gcs , or ggs solely on the clinical presentation . bhs from groups other than group a have been associated with outbreaks of pharyngitis , but their importance in causing endemic pharyngitis remains uncertain . since non - group a , bhs are inhabitants of the oropharynx during both health and disease , proof of their pathogenicity requires the isolation of strains more frequently among symptomatic patients with pharyngitis than among healthy person . while reports of the recovery of ggs / gcs from normally sterile sites are increasing , studies describing ggs / gcs throat colonization rates relative to gas in the same population are very few . the present study has been undertaken to estimate the prevalence of different serogroups of streptococci , among patients with the signs of pharyngotonsillitis . furthermore , to determine antibiotic susceptibility pattern to delineate any differences between infections caused by gcs , ggs , and gas in the same area during the same period . all the consecutive patients suffering from acute pharyngitis , attending outpatient department of otorhinolaryngology and medicine of karnataka institute of medical sciences hospital , hubli were included in the prospective study conducted during a 1-year period , from december 2011 to november 2012 . throat swabs were collected from a total of 218 patients and also from 82 appropriate age and sex matched healthy controls . two swabs were collected from each patient using sterile cotton tipped swab and transported in nutrient broth . the isolation and identification of bhs the gram - stained smears were observed under the microscope for the presence of pus cells and gram - positive cocci in pairs and chains . the second swab was inoculated on to 5% sheep blood agar plate and streptococcus selective agar using streak - stab technique . streptococcus selective media is used to inhibit the growth of throat commensals by the addition of inhibitory agents such as colistin , nalidixic acid , crystal violet , and support the growth of streptococci by enriching the media with blood . the plates were incubated for 2448 h using a candle jar with a 510% co2 . subcultures were made from beta - hemolytic colonies using fresh blood agar plates . from the subcultured plates , identification of the beta - hemolytic colonies were done based on conventional biochemical reactions ; catalase test and arginine dihydrolase test ; fermentation of sugars ; glucose , lactose , mannitol , ribose , sorbitol , and trehalose . presumptive identification of bhs was based on susceptibility and the resistant pattern of isolates using differential discs such as bacitracin and trimethoprim - sulfamethoxazole . clipper boulevard west , crossways , dartford , kent , da2 6pt , united kingdom . streptex is a rapid latex agglutination test system for use in the qualitative detection and identification of the lancefield group of streptococci . reagents are provided for groups a , b , c , d , f , and g covering the majority of clinical isolates . antimicrobial susceptibility testing was done on mha with 5% sheep blood agar by kirby - bauer disc diffusion method as per clinical and laboratory standards institute guidelines . microbial type culture collection ( mtcc ) number 1926 streptococcus pyogenes obtained from mtcc and gene bank , institute of microbial technology , chandigarh was used as a control . isolates were tested for ampicillin 10 g , amoxicillin / clavulanic acid 20/10 g , erythromycin 15 g , clindamycin 2 g , levofloxacin 5 g , tetracycline 30 g , cephotaxime 30 g , and vancomycin 30 g . all the consecutive patients suffering from acute pharyngitis , attending outpatient department of otorhinolaryngology and medicine of karnataka institute of medical sciences hospital , hubli were included in the prospective study conducted during a 1-year period , from december 2011 to november 2012 . throat swabs were collected from a total of 218 patients and also from 82 appropriate age and sex matched healthy controls . two swabs were collected from each patient using sterile cotton tipped swab and transported in nutrient broth . the isolation and identification of bhs the gram - stained smears were observed under the microscope for the presence of pus cells and gram - positive cocci in pairs and chains . the second swab was inoculated on to 5% sheep blood agar plate and streptococcus selective agar using streak - stab technique . streptococcus selective media is used to inhibit the growth of throat commensals by the addition of inhibitory agents such as colistin , nalidixic acid , crystal violet , and support the growth of streptococci by enriching the media with blood . the plates were incubated for 2448 h using a candle jar with a 510% co2 . subcultures were made from beta - hemolytic colonies using fresh blood agar plates . from the subcultured plates , identification of the beta - hemolytic colonies were done based on conventional biochemical reactions ; catalase test and arginine dihydrolase test ; fermentation of sugars ; glucose , lactose , mannitol , ribose , sorbitol , and trehalose . presumptive identification of bhs was based on susceptibility and the resistant pattern of isolates using differential discs such as bacitracin and trimethoprim - sulfamethoxazole . clipper boulevard west , crossways , dartford , kent , da2 6pt , united kingdom . streptex is a rapid latex agglutination test system for use in the qualitative detection and identification of the lancefield group of streptococci . reagents are provided for groups a , b , c , d , f , and g covering the majority of clinical isolates . antimicrobial susceptibility testing was done on mha with 5% sheep blood agar by kirby - bauer disc diffusion method as per clinical and laboratory standards institute guidelines . microbial type culture collection ( mtcc ) number 1926 streptococcus pyogenes obtained from mtcc and gene bank , institute of microbial technology , chandigarh was used as a control . isolates were tested for ampicillin 10 g , amoxicillin / clavulanic acid 20/10 g , erythromycin 15 g , clindamycin 2 g , levofloxacin 5 g , tetracycline 30 g , cephotaxime 30 g , and vancomycin 30 g . a maximum number of cases 58 ( 26.6% ) were in the age group of 110 years . majority 110 ( 50.45% ) of patients presented with fever and cough , 47 ( 21.55% ) with sore throat , 83 ( 38.07% ) with throat pain , 36 ( 16.51% ) with difficulty in swallowing , and 2 ( 0.91% ) with change of voice . severity of infections among serogroups bhs were isolated from 34 ( 15.59% ) patients with pharyngitis and 11 ( 13.41% ) healthy carriers . among pharyngitis cases , the incidence of gas was 20 ( 9.17% ) , gcs 7 ( 3.21% ) , and ggs 7 ( 3.21% ) . carriage rate of gas was 6 ( 7.31% ) and gcs 5 ( 6.09% ) . antibiotic resistant pattern of group a , group c , and group g streptococci isolated from throat swab presented in table 2 . antibiotic resistant pattern of group a , group c and group g streptococci isolated from patients throat swab equisimilis ( ggs / gcs ) are evolutionarily related , share the same tissue niche in humans , exchange genetic material , share up to half of their virulence associated genes , and cause a similar spectrum of diseases . yet , ggs / gcs are often considered as a commensal bacterium and its role in streptococcal disease burden is under recognized . the etiological agents of acute pharyngitis include viruses , isolated in approximately 4060% of cases and primary bacterial pathogens , which account for approximately 530% of cases . in around 30% of cases , the most important source of concern is infection with group a beta - hemolytic s. pyogenes , that is , associated with acute glomerulonephritis and acute rheumatic fever . in the present study , among adults overall the prevalence of pharyngitis we have isolated 83 ( 38.07% ) bacterial pathogens from pharyngitis cases . among these bhs were predominant organisms 34 ( 40.96% ) . slightly , the lower prevalence rate of gas was observed by sindhulina et al . from cmc vellore . maximum number 157 ( 72.01% ) of pharyngitis cases occurred in the month of october ( winter season ) . some studies have noted two peak incidences in may and september with sporadic cases of pharyngitis throughout the year . by latex agglutination test , 20 ( 58.82% ) isolates were identified as gas , 7 ( 20.58% ) as gcs and , 7 ( 20.58% ) as ggs . the roles of gcs and ggs in causing endemic pharyngitis are still controversial , although gcs are implicated in the outbreak of pharyngitis and associated disorders . gcs and ggs have also been described as frequent invasive pathogens in elderly patients , often in association with alcohol abuse , diabetes mellitus , malignant diseases , cardiac , and peripheral vascular diseases . the prevalence of gcs was 7 ( 3.21% ) and ggs 7 ( 3.21% ) . the frequency of gcs and ggs isolates from acute pharyngitis from different regions ranges from 0.7% to 6.24% and 3.74% to 5.1% , respectively . the percentage of asymptomatic gas carriers was 6 ( 7.31% ) and gcs was 5 ( 6.09% ) . carriage rate of gas ranging from 1.3% to14.9% , gcs ranging from 0.97% to 11% , and ggs ranging from 2.4% to 11% from asymptomatic individuals have been reported by various studies . all 20 ( 100% ) gas , 5 ( 71.42% ) of gcs , and 4 ( 57.14% ) of ggs isolates from acute pharyngitis were sensitive to bacitracin . all 6 ( 100% ) gas and 2 ( 40% ) gcs of isolates from healthy control throat swabs were also sensitive to bacitracin . all the gas isolates from acute pharyngitis and healthy controls were resistant to sulfamethoxazole - trimethoprim . reported that 9.8% strains of gas were resistant to bacitracin , whereas 85.7% strains of ggs and 90% strains of gcs were sensitive to bacitracin . antibiotic susceptibility test revealed the majority of gas and gcs isolates from acute pharyngitis patients and healthy control group were sensitive for amoxyclavulanic acid , levofloxacin , and cephotaxime . most of the ggs isolates were sensitive to amoxyclavulanic acid , tetracycline , and cephotaxime . the majority of gas 4 ( 66.66% ) and 2 ( 40% ) of gcs isolates from healthy controls were resistant to tetracycline . most of the studies have quoted all the gas isolates being sensitive to penicillin , cefotaxime , and vancomycin . al - charrakh et al . quoted high bacterial resistance to beta - lactam antibiotics - ampicillin , third generation cephalosporins - cefotaxime , ceftriaxone , and fourth generation cephalosporin - cefepime . macrolides have been effective in the treatment of individuals with s. pyogenes infection and a good alternative in patients who are allergic to penicillin . unfortunately , because of the general use of these agents , macrolide - resistant s. pyogenes have been isolated by many different studies from india and other countries . we have observed 26 ( 55.31% ) of bhs isolates resistant to both erythromycin and clindamycin . resistance to erythromycin ranging from 3.4% to 91.8% has been quoted by various studies . comparatively , the low rate of resistance was observed with clindamycin ranging from 0.6% to 80% and tetracycline ranging from 24% to 50% . none of the isolate in our study revealed inducible clindamycin resistance . in comparison with gas , gcs exhibited a higher range of drug resistance to all the antibiotics tested in our study , except for levofloxacin and tetracycline . ggs also exhibited a higher range of drug resistance to all drugs tested except for levofloxacin . healthy carriers of bhs are sources for bacterial dissemination and even lead to severe epidemics . invasive infections caused by gas , gbs , gcs , and ggs are still a major challenge for clinicians . . continued epidemiological and microbiological surveillance is necessary to assess the development of infections by different serogroups of bhs for better management of patients and to improve preventative strategies .

mitochondria are organelles present in every cell of the body ( except red blood cells ) and generate almost all of the energy needed by the cells to grow and sustain life . in addition to adenosine triphosphate ( atp ) generation , mitochondria are involved in a large number of specialized functions in major cellular pathways including apoptosis , urea cycle , pyrimidine biosynthesis , heme synthesis , etc . the proteins that take part in these pathways are encoded both by mitochondrial dna ( mtdna ) and nuclear dna ( ndna ) . mtdna encodes only a limited number of genes ( 37 ) which code for 13 proteins , two rrnas and 22 trnas . these proteins code for four respiratory complexes of the oxidative phosphorylation ( oxphos ) system . the only non - coding segment of mtdna is the displacement loop ( d - loop , 1121 bp ) that contains the origin of replication of the h - strand ( oh ) and the promoters for l and h - strand transcription . the diseases related to mitochondrial dysfunction are due to mutations in both the mtdna and ndna encoded components . genetically , mitochondrial diseases are characterized as ( i ) those with sporadic or maternally inherited mtdna mutations , ( ii ) those with abnormalities with mendelian transmission of the trait , i.e. , disorders believed to be due to mutations in nuclear genes that control mitochondrial biogenesis , and ( iii ) those that are caused by nuclear genes but are misinterpreted as mitochondrial based on the biochemical findings 4 , 5 . mtdna point mutations which can either be maternally inherited or generated somatically have been associated with many diseases like a3243 g for melas , a8344 g for merrf , t8993 g for narp , etc . also , there appears to be a class of slightly deleterious mutations that modify the risks of developing certain complex diseases or trait . human mtdna and ndna mutations causing mitochondrial dysfunction are implicated in a broad spectrum of diseases affecting various tissues like brain , heart , liver , skeletal muscles , etc . the clinical symptoms of the disease depend on the cell type affected and range from loss of motor control , muscle weakness , cardiac disease to visual or hearing loss , etc . . given that mitochondria are involved in a large number of cellular pathways , it is always challenging to correlate the exact causative role of genome variation with the observed phenotype . additionally , the varying spectrum of disease symptoms is a major deterrent in early disease diagnosis . this is clear from the fact that with over 5000 mtdna variation reported across databases , pathogenicity assignments for most of the variation is only limited to the association with the phenotype without any conclusive evidence on its causative role . while there are a large number of resources available on various aspects of human mtdna , a major bottleneck is the lack of documentation of genomic variation data across populations with clinical details to evaluate these variations for disease association . the need of the hour is to curate these resources using standards for data exchange over the web as also using standard ontologies for data analysis across platforms . most of the mitochondrial diseases have rare occurrence in the population and hence are termed as rare diseases. as of now there are nearly 7000 rare diseases reported worldwide arising from mutations in either ndna or mtdna . it is estimated that nearly 300 million people in the world are affected by rare diseases . in india alone there are nearly 70 million people diagnosed with rare diseases ( http://www.rarediseasesindia.org/ ) , despite the fact that there are no standard diagnostic tests available for most rare diseases . in addition , the definition of rare diseases vary , for e.g. , the european union considers diseases to be rare when they affect no more than 5 in 10,000 people , while the unites states of america ( usa ) consider a disease to be rare when affecting fewer than 200,000 people . in asia , however , the prevalence of most mitochondrial diseases is not known 9 , 10 . this review elaborates on the available resources , the bottlenecks in rare disease research and proposes innovative ways to address these challenges . there are many reviews discussing the challenges involved in establishing genotype - phenotype correlations with mitochondrial dysfunction given its involvement in multiple pathways in a spatio - temporal manner 3 , 5 , 8 , 11 . here we review our current understanding of the rare disease initiatives and efforts that are ongoing globally along with specific focus on mitochondrial disease resources . the first human dna to be completely sequenced was the human mitochondrial dna ( 16569 base pairs ) in 1981 12 , 13 . given the relatively small size and absence of repeats , sequencing and assembling the mitochondrial human genome was not as challenging and difficult as sequencing the human nuclear genome using the sanger sequencing technology . however , mitochondrial genome sequencing has its own unique problems given the high mutation rate and high levels of heteroplasmy . precise determination of the levels of heteroplasmy is crucial since the level of heteroplasmy determines both the penetrance and severity of expression of some mitochondrial diseases . the next generation sequencing ( ngs ) technologies like virtual terminator sequencing ( illumina ) , pyrosequencing ( roche ) and solid have allowed to overcome these limitations by providing massive parallelization , high coverage , high accuracy as compared to sanger sequencing . specific protocols , including long range pcr with mitochondria specific primers , and algorithms for reference based and de novo assembly have been developed to sequence mitochondrial dna using ngs technologies . ngs - based clinical targeted gene assay for the mitochondrial genome and 108 selected nuclear genes associated with mitochondrial disorders have also been designed to facilitate the analysis and understanding of nuclear and mitochondrial variations in mitochondrial diseases . these emerging technologies offer an excellent opportunity to further dissect the molecular basis of disease manifestation . with an increasing number of individuals that may be genetically screened across different populations the genomics data along with clinical and biochemical profiles may also be used to identify disease biomarkers with high sensitivity and specificity , which is a major challenge in diagnosing mitochondrial dysfunction . over the years , a large number of web - based resources have been developed on various aspects of mitochondrial diseases , most of them focusing on the data from mtdna . some of these include , mitomap , a database on human mitochondrial variation , mitolsdb , the largest curated data on mtdna variation with phenotype using lovd , mitocarta , a resource on mitochondrial proteins based on localization , mitominer , a mitochondrial protein identification system based on multiple evidences 18 , 19 , mitobreak , a curated dataset on mtdna rearrangements , hmtdb , an online resource for data on mitochondrial genome sequences annotated with population and variation data , mitochondrial database ( mitodb ) , the mitochondrial database on clinical features seen in mitochondrial diseases , to name a few . analysis pipelines and platforms have also been developed , including the mtsnpscore which assesses the role of variation in context of disease association using a combined evidence approach , mit - o - matic , an analysis pipeline for clinical evaluation of mitochondrial variations from the ngs datasets . more recently , the united mitochondria disease foundation ( umdf ) ( http://www.umdf.org/site/c.8qkoj0mvf7lug/b.7929671/k.bdf0/home.htm ) along with the national institute of child health and human development ( nichd ) ( http://www.nichd.nih.gov/pages/index.aspx ) launched the mitochondrial disease sequence data resource ( mseqdr ) consortium . the goals of this consortium is to facilitate deposition , curation , annotation and integrated analysis of genomic data for mitochondrial diseases for clinical and research communities . there are a large number of resources developed both for mitochondrial community and rare diseases community . the first section lists the resources available on mitochondrial diseases including support and advocacy groups , databases and analysis pipelines , research and patient networks . globally , attempts have also been made to systematically address the problem of rare diseases by establishing focused programs and consortia - based approaches . therapeutics of rare and neglected diseases ( trnd ) ( http://www.ncats.nih.gov/research/rare-diseases/trnd/trnd.html ) , a program led by the national center for advancing translational sciences ( ncats ) ( http://www.ncats.nih.gov/ ) , supports the development of potential treatments for rare and neglected diseases to first - in - human trials . this approach provides a de - risking strategy making the downstream development efforts commercially viable . trnd also supports the pre - clinical studies including medicinal chemistry optimization , drug metabolism and pharmacokinetics , toxicology formulation , and others studies required to file investigational new drug ( ind ) application for regulatory approvals . the other initiatives by ncats for rare diseases include office of rare disease research ( ordr ) ( http://www.orphadata.org/cgi-bin/inc/ordo_orphanet.inc.php ) which coordinates a large number of collaborative research efforts towards rare diseases including support to institutes and centers , managing patient registry , human bio - specimen repository , to name a few major activities . rare diseases clinical research network ( rdcrn ) ( http://rarediseases.info.nih.gov/research/pages/41/rare-diseases-clinical-research-network ) , from ordr , focuses on advancing medical research on rare diseases by facilitating collaboration , study enrollment and data sharing . it also connects scientists from multiple disciplines across various clinical sites globally to work with patient advocacy groups . the north american mitochondrial disease consortium ( namdc ) ( http://www.rarediseasesnetwork.org/namdc/ ) , a part of rdcrn , specially works towards collecting information from mitochondrial disease patients in a clinical patient registry . in addition to periodically updating the patients on mitochondrial diseases , namdc also helps researchers to identify and recruit patients for future studies . data generated as part of the various initiatives involving patient information are managed by the ncats global research patient registry data repository ( grdr ) ( http://www.ncats.nih.gov/research/rare-diseases/grdr/grdr.html ) . this is a web - based resource that aggregates de - identified patient information across many registries and provides a globally unique identifier ( guid ) to each patient data . guid allows for patient follow - up across different registries , diseases , studies and countries and also ensures that clinical information is also mapped to bio - specimen datasets . bridging interventional development gaps ( brids ) ( http://www.ncats.nih.gov/research/rare-diseases/bridgs/bridgs.html ) is a division of ncats for pre - clinical innovation towards the development of new therapeutic agents both for common and rare diseases . a recent perspective on the ncats trnd and bridgs programs highlights the role of team effort where academia , biotech and pharma industries , patient communities , advocacy groups , regulators , and government support , all are needed to navigate through the translational valley of death . national organization for rare disorders ( nord ) ( http://www.rarediseases.org/ ) is a non - profit organization with the aim to improve the lives of all people affected by rare diseases . the services offered by nord include identification , treatment and cure of rare disorders through advocacy , research and programs of education . similarly , global genes is a non - profit organization for patient advocacy and work towards building awareness and providing connections and resources for rare disease patients ( http://globalgenes.org/ ) . similar efforts in the european subcontinent have led to the establishment of orphanet , a consortium of 40 countries coordinated by the french inserm team and which hosts a reference portal for rare diseases and orphan drugs . orphanet hosts a directory of information of expert clinics , medical labs , clinical trials , patient organizations , etc . the joint effort of the european commission and the nih established irdirc , the international rare diseases research consortium , in 2011 . this international consortium of researchers and organizations aims to deliver 200 therapies and means to diagnose most rare diseases by 2020 , and as per their reports , the targets are being delivered ( http://www.irdirc.org/ ) . the list of various rare diseases resources may be seen in table 1 . as mentioned earlier , there are no clear estimates of the number of mitochondrial rare diseases . in order to get an approximation , the list of 6537 rare diseases was taken from global genes website . to find out which rare disease is also a mitochondrial disease , a list of mitochondrial diseases was taken from the mitochondrial database ( mitodb ) ( 51 ) . in addition , information on mitochondrial diseases is also referred from umdf ( 41 ) and mitomap ( 51 ) . on comparing rare disease list with the mitochondrial diseases lists , it is important to mention here that since an exact match was performed we could have missed diseases for which abbreviation or synonyms are used . this also highlights that standard ontologies and terms are not systematically followed making data intractable for automated data analysis . we further checked the prevalence of these 18 rare diseases from the rare diseases india ( http://www.rarediseasesindia.org/ ) and orphanet portals . as may be seen on table 2 , data are available for few diseases only . the table provides a list of diseases that cause mitochondrial dysfunction and are also reported rare diseases . * * indicates birth prevalence it is evident from a simple search in the clinical trials registry ( https://clinicaltrials.gov/ ) that only 0.2% of clinical trials ever done , ongoing , terminated or planned are for rare diseases . this clearly indicates that the major challenge for rare diseases research is the cost involved in research and development given the poor return on investment . it is also well known that clinical trials are prohibitively expensive even for common diseases where finding expert clinicians and acceptable number of patients is not as challenging as for rare diseases . these issues are addressed by de - risking research , developing platforms for sharing patient data , generating centralized patient registries and offering various incentives for making the development commercially viable . we discuss the various models that have been attempted to bridge the last mile of developing novel therapeutics . one of the major challenges in the treatment of rare diseases is clinical trials patient recruitment . to overcome this challenge , patient advocacy groups and websites like patientslikeme are turning out to be a major game changer . patientslikeme is a patient powered research network that allows people to connect and share their experience with other people having the same disease or condition . patientslikeme started its first online community for amyotrophic lateral sclerosis ( als ) 27 29 in 2006 where participants could ask specific questions about the treatment options and what to expect to fellow users ( http://www.patientslikeme.com/ ) . in addition , the patients also got involved in experimenting with drugs that have not received regulatory approval . thus data generated by these self - reporting platforms can be used to establish the efficacy and safety of a compound for rare diseases . the self - reported data help provide evidence to support or refute treatment outcomes . despite several limitations of self - reported data , like unmeasured covariates , data reliability and controlled settings , these approaches are promising as has been shown with the quality of data gathered with smartphone games which were comparable to data obtained from controlled laboratory environments 30 , 31 . in 2011 , the company expanded its scope and allowed any patient with any condition to join the community . currently there are more than 300,000 members registered on the patientlikeme community with more than 2000 health conditions ( http://en.wikipedia.org/wiki/patientslikeme ) . besides , interactions of the patient advocates from the rare diseases group with major regulatory bodies has led to expedite review and approval process for rare disease treatments ( http://www.forbes.com/sites/medidata/2014/09/25/rare-disease-patient-voices-bring-change-to-the-clinical-trials-process/ ) . using another interesting strategy , pfizer inc . used a web - based interactive platform to evaluate the efficacy of a drug ( tolterodine tartrate extended release capsules ) to treat overacting bladder . this project called remote is a phase iv trial under an investigational new drug ( ind ) application . the participants for the trial were recruited through an interactive web - based platform from one clinical site overseen by physician . despite poor patient participation , the study reports that the trial outcomes are consistent with the results from the conventional trial . the observations from remote is a learning experience for the trial community on the challenges involved at various levels from patient understanding to technical issues . these learnings are used to conduct another trial in europe , remote2.0 , to overcome the bottlenecks faced with the first attempt . these initiatives are of immense significance in establishing the robustness of trial strategies where patient recruitment is a challenge . the mobile technologies in form of wearable sensors also make the trial monitoring and patient diagnosis at different time points more affordable . it is also proposed that the n - of-1 trial method may be used to evaluate new treatments . under this model a single patient is the entire trial and the treatment outcome is measured at different time points for pre - treatment , treatment duration and post - treatment . if the treatment outcome is measurable and the patient is cured , the treatment may be considered effective . thus , the crowdsourcing strategy is an attractive tool for patient recruitment as it is affordable and time effective . likewise , crowdfunding is also proposed as a viable option for financing various aspects of rare diseases research . crowdfunding initiatives not only generate awareness about rare diseases but also lead to funding for supporting research activities . one such example is the als ice bucket challenge leading to 1.2 million videos on facebook and 2.2 million tweets in a matter of 3 months , which raised over $ 100 million of donations ( http://en.wikipedia.org/wiki/ice_bucket_challenge ) . through innovative ideas like als ice bucket challenge it is possible to raise funds and also increase public awareness about the rare diseases . the rare genomics institute ( rgi ) ( http://raregenomics.org/ ) is an international non - profit providing expert network and an online crowdfunding mechanism to assist families pursue personalized research projects for diseases which would not be studied otherwise . rgi offers sequencing facilities for diagnosis , expert guidance on sequencing and systematic interpretation of the data generated . a recently published success story is that of a 3-year old girl showing symptoms of involuntary eye movement , small - sized head , involuntary muscle contraction , development delay and progressive decline . whole exome sequencing of the family revealed a novel mutation that causes mental retardation and severe developmental delays . through the rgi platform , $ 5000 was raised in 50 days to carry out the genome sequencing in the girl and her family , which led to identification of the novel mutation . these examples underline the potential of crowdfunding in addressing scientifically challenging and socially important problems . with the increasing disease burden , the need of the hour is to establish new models for ensuring that clinical trials are sustainable and transparent . it is not possible to apply the conventional methods to rare disease trials given the shear costs involved . to overcome these bottlenecks , efforts are being made lately to reverse the approach of rare disease patient recruitments for clinical trials by taking the trials to the patients and not vice versa . this approach demands establishing patient - centric sites which is again challenging given their extremely sparse distribution . telemetry innovations may offer solutions to this problem , which allows health monitoring of the patients remotely . this model is not just suitable for rare diseases but is also applicable to common diseases trials given that many clinical trial sites never recruit patients ( http://www.clinicalleader.com/doc/how-rare-disease-know-how-can-shape-big-pharma-clinical-trials-0001 ) . open access platforms which allow for data integration and exchange of ideas to facilitate the process of bringing new therapeutic interventions and diagnostic methods to the market are needed to drive sustained research and development for rare diseases . it is also imperative to synchronize the efforts at all levels of research , diagnosis and regulatory guidelines for evidence - based clinical decision - making . as part of the platform , it is also important to involve regulatory agencies from very early on in the discovery pipeline so that the progression through development and approvals is quicker and more affordable , which is the bottleneck with rare diseases . one strategy which seems to work well is to apply approaches of drug repurposing where it is crucial to understand the mechanism of disease manifestation and subsequently applying various modeling approaches to find new indications of existing drugs in the market ( http://www.fda.gov/forindustry/developingproductsforrarediseasesconditions/howtoapplyfororphanproductdesignation/ucm216147.htm ) . this strategy will be even more successful where one can systematically list the phenotypic parameters provided by patient groups in close collaboration with clinicians . there is a need for innovation in technology that allows for interfacing the different stakeholders , namely , researchers , clinicians and patient groups . such resources will go a long way in future to allow patients to perform diagnostic procedures with greater accuracy and help clinicians identify the best possible route for therapeutic interventions . this is only possible if the participating members use standard terms to share the data . various ontologies have been developed over years for capturing function of genes like the gene ontology , page - om and vario for capturing features of variation , umls which includes mesh , rxnorm and snomed ct for capturing clinical details and human phenotype ontology for phenotype data , to name a few . these ontologies are meant to function as a common set of vocabulary that needs to be shared on a community collaborative platform . the semantic network of these terms will allow deciphering and interpreting novel patterns in understanding disease biology . such a semantic - based platform will be amenable for plugging in data and new methods with increasing understanding of disease biology . it will also ensure that the data generated as part of negative results are also shared systematically with scientific community . orphanet rare disease ontology ( ordo ) ( http://www.orphadata.org/cgi-bin/inc/ordo_orphanet.inc.php ) is an effort to achieve these goals . ordo offers definitions , classification of rare diseases , gene - disease relations , epidemiological data and connections with other terminologies like mesh , umls ( http://www.nlm.nih.gov/research/umls/ ) , omim , uniprotkb , hgnc , ensembl , reactome , etc . it is imperative that existing resources utilize these ontologies for data sharing allowing for data interoperability across different platforms . figure 1 illustrates the scope of the proposed integrative platform describing the scientific challenges involved in establishing genotype - phenotype correlations and how the existing resources and community collaborations may be converged towards a systems level understanding of the disease biology . ( a ) this section describes the challenges involved in dissecting the impact of genomic variation in disease association . as shown , mitochondria are involved in a large number of cellular processes including energy metabolism . the protein subunits of the complexes of the electron transport chain are encoded both by mtdna ( red ) and ndna ( green ) . in order to understand the complex genotype - phenotype correlation it is imperative to identify the molecular interactions at systems level ( protein - protein interaction network , metabolic network , signaling network , regulatory network ) . it is important to curate the information needed to generate these networks from literature and existing resources . ( b ) the resources currently available for mitochondrial dysfunction include databases , web servers and analysis pipelines as shown . for generating systems level models ( c ) there are various ongoing efforts involving researchers , clinicians and or patient groups . it is proposed that a community collaborative open access platform is a must to interface these communities . in order to establish such a platform that allows geographically different communities to work together , internet of dna is listed as one of the top 15 breakthrough technologies of 2015 ( http://www.technologyreview.com/featuredstory/535016/internet-of-dna/?utm_campaign=newsletters&utm_source=newsletter-weekly-biomedicine&utm_medium=email&utm_content=20150224 ) . the technology needed to harness the power of genomics lies in comparing the genetic information from a large number of individuals with medical records . this currently is a huge challenge , partly because of the technical reasons of moving petabytes of data across different labs , but especially due to the privacy issues surrounding patient information . both these issues should be addressed to ensure that the ever - increasing amount of genomic and clinical data piling up in laboratories and hospitals are utilized optimally . upcoming initiatives like the matchmaker exchange are aiming to bring the genotype and phenotype data together on a common platform ( http://matchmakerexchange.org/ ) . global alliance for genomics and health also known as ga4gh is an organization which provides protocols , apis and file formats for effective and responsible sharing of genomic and clinical data ( http://genomicsandhealth.org/ ) . the organization goal is to overcome challenges likes ethics and privacy involved with sharing of genomics data and to accelerate the potential of genomic medicine for advancement of human health . as discussed before , the need of the hour is to let the patients decide on who will access their data and how these may be used . this is only possible if the information generated using patient samples is made available in real - time . it is also important that other components of this major collaborative strategy are also part of a community platform that allows for gated access to patient data with patients deciding how and with whom their data should be shared . in the changing paradigms of disease treatment , a very recent approval is made by britain on mitochondrial donation ( http://www.theguardian.com/politics/2015/feb/24/uk-house-of-lords-approves-conception-of-three-person-babies ) . in this approach in vitro fertilization ( ivf ) technique is used with biological material coming from three parents , mother and father ( contributing 98.8% genetic material ) and a female donor ( contributing 0.2% genetic material ) . this three - parent ivf approval in uk has received mixed reactions and only time will address the concern of the long - term implications of the same . the first human dna to be completely sequenced was the human mitochondrial dna ( 16569 base pairs ) in 1981 12 , 13 . given the relatively small size and absence of repeats , sequencing and assembling the mitochondrial human genome was not as challenging and difficult as sequencing the human nuclear genome using the sanger sequencing technology . however , mitochondrial genome sequencing has its own unique problems given the high mutation rate and high levels of heteroplasmy . precise determination of the levels of heteroplasmy is crucial since the level of heteroplasmy determines both the penetrance and severity of expression of some mitochondrial diseases . the next generation sequencing ( ngs ) technologies like virtual terminator sequencing ( illumina ) , pyrosequencing ( roche ) and solid have allowed to overcome these limitations by providing massive parallelization , high coverage , high accuracy as compared to sanger sequencing . specific protocols , including long range pcr with mitochondria specific primers , and algorithms for reference based and de novo assembly have been developed to sequence mitochondrial dna using ngs technologies . ngs - based clinical targeted gene assay for the mitochondrial genome and 108 selected nuclear genes associated with mitochondrial disorders have also been designed to facilitate the analysis and understanding of nuclear and mitochondrial variations in mitochondrial diseases . these emerging technologies offer an excellent opportunity to further dissect the molecular basis of disease manifestation . with an increasing number of individuals that may be genetically screened across different populations the genomics data along with clinical and biochemical profiles may also be used to identify disease biomarkers with high sensitivity and specificity , which is a major challenge in diagnosing mitochondrial dysfunction . over the years , a large number of web - based resources have been developed on various aspects of mitochondrial diseases , most of them focusing on the data from mtdna . some of these include , mitomap , a database on human mitochondrial variation , mitolsdb , the largest curated data on mtdna variation with phenotype using lovd , mitocarta , a resource on mitochondrial proteins based on localization , mitominer , a mitochondrial protein identification system based on multiple evidences 18 , 19 , mitobreak , a curated dataset on mtdna rearrangements , hmtdb , an online resource for data on mitochondrial genome sequences annotated with population and variation data , mitochondrial database ( mitodb ) , the mitochondrial database on clinical features seen in mitochondrial diseases , to name a few . analysis pipelines and platforms have also been developed , including the mtsnpscore which assesses the role of variation in context of disease association using a combined evidence approach , mit - o - matic , an analysis pipeline for clinical evaluation of mitochondrial variations from the ngs datasets . more recently , the united mitochondria disease foundation ( umdf ) ( http://www.umdf.org/site/c.8qkoj0mvf7lug/b.7929671/k.bdf0/home.htm ) along with the national institute of child health and human development ( nichd ) ( http://www.nichd.nih.gov/pages/index.aspx ) launched the mitochondrial disease sequence data resource ( mseqdr ) consortium . the goals of this consortium is to facilitate deposition , curation , annotation and integrated analysis of genomic data for mitochondrial diseases for clinical and research communities . there are a large number of resources developed both for mitochondrial community and rare diseases community . the first section lists the resources available on mitochondrial diseases including support and advocacy groups , databases and analysis pipelines , research and patient networks . globally , attempts have also been made to systematically address the problem of rare diseases by establishing focused programs and consortia - based approaches . therapeutics of rare and neglected diseases ( trnd ) ( http://www.ncats.nih.gov/research/rare-diseases/trnd/trnd.html ) , a program led by the national center for advancing translational sciences ( ncats ) ( http://www.ncats.nih.gov/ ) , supports the development of potential treatments for rare and neglected diseases to first - in - human trials . this approach provides a de - risking strategy making the downstream development efforts commercially viable . trnd also supports the pre - clinical studies including medicinal chemistry optimization , drug metabolism and pharmacokinetics , toxicology formulation , and others studies required to file investigational new drug ( ind ) application for regulatory approvals . the other initiatives by ncats for rare diseases include office of rare disease research ( ordr ) ( http://www.orphadata.org/cgi-bin/inc/ordo_orphanet.inc.php ) which coordinates a large number of collaborative research efforts towards rare diseases including support to institutes and centers , managing patient registry , human bio - specimen repository , to name a few major activities . rare diseases clinical research network ( rdcrn ) ( http://rarediseases.info.nih.gov/research/pages/41/rare-diseases-clinical-research-network ) , from ordr , focuses on advancing medical research on rare diseases by facilitating collaboration , study enrollment and data sharing . it also connects scientists from multiple disciplines across various clinical sites globally to work with patient advocacy groups . the north american mitochondrial disease consortium ( namdc ) ( http://www.rarediseasesnetwork.org/namdc/ ) , a part of rdcrn , specially works towards collecting information from mitochondrial disease patients in a clinical patient registry . in addition to periodically updating the patients on mitochondrial diseases , namdc also helps researchers to identify and recruit patients for future studies . data generated as part of the various initiatives involving patient information are managed by the ncats global research patient registry data repository ( grdr ) ( http://www.ncats.nih.gov/research/rare-diseases/grdr/grdr.html ) . this is a web - based resource that aggregates de - identified patient information across many registries and provides a globally unique identifier ( guid ) to each patient data . guid allows for patient follow - up across different registries , diseases , studies and countries and also ensures that clinical information is also mapped to bio - specimen datasets . bridging interventional development gaps ( brids ) ( http://www.ncats.nih.gov/research/rare-diseases/bridgs/bridgs.html ) is a division of ncats for pre - clinical innovation towards the development of new therapeutic agents both for common and rare diseases . a recent perspective on the ncats trnd and bridgs programs highlights the role of team effort where academia , biotech and pharma industries , patient communities , advocacy groups , regulators , and government support , all are needed to navigate through the translational valley of death . national organization for rare disorders ( nord ) ( http://www.rarediseases.org/ ) is a non - profit organization with the aim to improve the lives of all people affected by rare diseases . the services offered by nord include identification , treatment and cure of rare disorders through advocacy , research and programs of education . similarly , global genes is a non - profit organization for patient advocacy and work towards building awareness and providing connections and resources for rare disease patients ( http://globalgenes.org/ ) . similar efforts in the european subcontinent have led to the establishment of orphanet , a consortium of 40 countries coordinated by the french inserm team and which hosts a reference portal for rare diseases and orphan drugs . orphanet hosts a directory of information of expert clinics , medical labs , clinical trials , patient organizations , etc . the joint effort of the european commission and the nih established irdirc , the international rare diseases research consortium , in 2011 . this international consortium of researchers and organizations aims to deliver 200 therapies and means to diagnose most rare diseases by 2020 , and as per their reports , the targets are being delivered ( http://www.irdirc.org/ ) . the list of various rare diseases resources may be seen in table 1 . as mentioned earlier , there are no clear estimates of the number of mitochondrial rare diseases . in order to get an approximation , to find out which rare disease is also a mitochondrial disease , a list of mitochondrial diseases was taken from the mitochondrial database ( mitodb ) ( 51 ) . in addition , information on mitochondrial diseases is also referred from umdf ( 41 ) and mitomap ( 51 ) . on comparing rare disease list with the mitochondrial diseases lists , it is important to mention here that since an exact match was performed we could have missed diseases for which abbreviation or synonyms are used . this also highlights that standard ontologies and terms are not systematically followed making data intractable for automated data analysis . we further checked the prevalence of these 18 rare diseases from the rare diseases india ( http://www.rarediseasesindia.org/ ) and orphanet portals . as may be seen on table 2 , data are available for few diseases only . the table provides a list of diseases that cause mitochondrial dysfunction and are also reported rare diseases . * it is evident from a simple search in the clinical trials registry ( https://clinicaltrials.gov/ ) that only 0.2% of clinical trials ever done , ongoing , terminated or planned are for rare diseases . this clearly indicates that the major challenge for rare diseases research is the cost involved in research and development given the poor return on investment . it is also well known that clinical trials are prohibitively expensive even for common diseases where finding expert clinicians and acceptable number of patients is not as challenging as for rare diseases . these issues are addressed by de - risking research , developing platforms for sharing patient data , generating centralized patient registries and offering various incentives for making the development commercially viable . we discuss the various models that have been attempted to bridge the last mile of developing novel therapeutics . one of the major challenges in the treatment of rare diseases is clinical trials patient recruitment . to overcome this challenge , patient advocacy groups and websites like patientslikeme are turning out to be a major game changer . patientslikeme is a patient powered research network that allows people to connect and share their experience with other people having the same disease or condition . patientslikeme started its first online community for amyotrophic lateral sclerosis ( als ) 27 29 in 2006 where participants could ask specific questions about the treatment options and what to expect to fellow users ( http://www.patientslikeme.com/ ) . in addition , the patients also got involved in experimenting with drugs that have not received regulatory approval . thus data generated by these self - reporting platforms can be used to establish the efficacy and safety of a compound for rare diseases . the self - reported data help provide evidence to support or refute treatment outcomes . despite several limitations of self - reported data , like unmeasured covariates , data reliability and controlled settings , these approaches are promising as has been shown with the quality of data gathered with smartphone games which were comparable to data obtained from controlled laboratory environments 30 , 31 . in 2011 , the company expanded its scope and allowed any patient with any condition to join the community . currently there are more than 300,000 members registered on the patientlikeme community with more than 2000 health conditions ( http://en.wikipedia.org/wiki/patientslikeme ) . besides , interactions of the patient advocates from the rare diseases group with major regulatory bodies has led to expedite review and approval process for rare disease treatments ( http://www.forbes.com/sites/medidata/2014/09/25/rare-disease-patient-voices-bring-change-to-the-clinical-trials-process/ ) . using another interesting strategy , pfizer inc . used a web - based interactive platform to evaluate the efficacy of a drug ( this project called remote is a phase iv trial under an investigational new drug ( ind ) application . the participants for the trial were recruited through an interactive web - based platform from one clinical site overseen by physician . despite poor patient participation , the study reports that the trial outcomes are consistent with the results from the conventional trial . the observations from remote is a learning experience for the trial community on the challenges involved at various levels from patient understanding to technical issues . these learnings are used to conduct another trial in europe , remote2.0 , to overcome the bottlenecks faced with the first attempt . these initiatives are of immense significance in establishing the robustness of trial strategies where patient recruitment is a challenge . the mobile technologies in form of wearable sensors also make the trial monitoring and patient diagnosis at different time points more affordable . it is also proposed that the n - of-1 trial method may be used to evaluate new treatments . under this model a single patient is the entire trial and the treatment outcome is measured at different time points for pre - treatment , treatment duration and post - treatment . if the treatment outcome is measurable and the patient is cured , the treatment may be considered effective . thus , the crowdsourcing strategy is an attractive tool for patient recruitment as it is affordable and time effective . likewise , crowdfunding is also proposed as a viable option for financing various aspects of rare diseases research . crowdfunding initiatives not only generate awareness about rare diseases but also lead to funding for supporting research activities . one such example is the als ice bucket challenge leading to 1.2 million videos on facebook and 2.2 million tweets in a matter of 3 months , which raised over $ 100 million of donations ( http://en.wikipedia.org/wiki/ice_bucket_challenge ) . through innovative ideas like als ice bucket challenge it is possible to raise funds and also increase public awareness about the rare diseases . the rare genomics institute ( rgi ) ( http://raregenomics.org/ ) is an international non - profit providing expert network and an online crowdfunding mechanism to assist families pursue personalized research projects for diseases which would not be studied otherwise . rgi offers sequencing facilities for diagnosis , expert guidance on sequencing and systematic interpretation of the data generated . a recently published success story is that of a 3-year old girl showing symptoms of involuntary eye movement , small - sized head , involuntary muscle contraction , development delay and progressive decline . whole exome sequencing of the family revealed a novel mutation that causes mental retardation and severe developmental delays . through the rgi platform , $ 5000 was raised in 50 days to carry out the genome sequencing in the girl and her family , which led to identification of the novel mutation . these examples underline the potential of crowdfunding in addressing scientifically challenging and socially important problems . with the increasing disease burden , the need of the hour is to establish new models for ensuring that clinical trials are sustainable and transparent . it is not possible to apply the conventional methods to rare disease trials given the shear costs involved . to overcome these bottlenecks , efforts are being made lately to reverse the approach of rare disease patient recruitments for clinical trials by taking the trials to the patients and not vice versa . this approach demands establishing patient - centric sites which is again challenging given their extremely sparse distribution . telemetry innovations may offer solutions to this problem , which allows health monitoring of the patients remotely . this model is not just suitable for rare diseases but is also applicable to common diseases trials given that many clinical trial sites never recruit patients ( http://www.clinicalleader.com/doc/how-rare-disease-know-how-can-shape-big-pharma-clinical-trials-0001 ) . open access platforms which allow for data integration and exchange of ideas to facilitate the process of bringing new therapeutic interventions and diagnostic methods to the market are needed to drive sustained research and development for rare diseases . it is also imperative to synchronize the efforts at all levels of research , diagnosis and regulatory guidelines for evidence - based clinical decision - making . as part of the platform , it is also important to involve regulatory agencies from very early on in the discovery pipeline so that the progression through development and approvals is quicker and more affordable , which is the bottleneck with rare diseases . one strategy which seems to work well is to apply approaches of drug repurposing where it is crucial to understand the mechanism of disease manifestation and subsequently applying various modeling approaches to find new indications of existing drugs in the market ( http://www.fda.gov/forindustry/developingproductsforrarediseasesconditions/howtoapplyfororphanproductdesignation/ucm216147.htm ) . this strategy will be even more successful where one can systematically list the phenotypic parameters provided by patient groups in close collaboration with clinicians . there is a need for innovation in technology that allows for interfacing the different stakeholders , namely , researchers , clinicians and patient groups . such resources will go a long way in future to allow patients to perform diagnostic procedures with greater accuracy and help clinicians identify the best possible route for therapeutic interventions . this is only possible if the participating members use standard terms to share the data . various ontologies have been developed over years for capturing function of genes like the gene ontology , page - om and vario for capturing features of variation , umls which includes mesh , rxnorm and snomed ct for capturing clinical details and human phenotype ontology for phenotype data , to name a few . these ontologies are meant to function as a common set of vocabulary that needs to be shared on a community collaborative platform . the semantic network of these terms will allow deciphering and interpreting novel patterns in understanding disease biology . such a semantic - based platform will be amenable for plugging in data and new methods with increasing understanding of disease biology . it will also ensure that the data generated as part of negative results are also shared systematically with scientific community . orphanet rare disease ontology ( ordo ) ( http://www.orphadata.org/cgi-bin/inc/ordo_orphanet.inc.php ) is an effort to achieve these goals . ordo offers definitions , classification of rare diseases , gene - disease relations , epidemiological data and connections with other terminologies like mesh , umls ( http://www.nlm.nih.gov/research/umls/ ) , omim , uniprotkb , hgnc , ensembl , reactome , etc . it is imperative that existing resources utilize these ontologies for data sharing allowing for data interoperability across different platforms . figure 1 illustrates the scope of the proposed integrative platform describing the scientific challenges involved in establishing genotype - phenotype correlations and how the existing resources and community collaborations may be converged towards a systems level understanding of the disease biology . ( a ) this section describes the challenges involved in dissecting the impact of genomic variation in disease association . as shown , mitochondria are involved in a large number of cellular processes including energy metabolism . the protein subunits of the complexes of the electron transport chain are encoded both by mtdna ( red ) and ndna ( green ) . in order to understand the complex genotype - phenotype correlation it is imperative to identify the molecular interactions at systems level ( protein - protein interaction network , metabolic network , signaling network , regulatory network ) . it is important to curate the information needed to generate these networks from literature and existing resources . ( b ) the resources currently available for mitochondrial dysfunction include databases , web servers and analysis pipelines as shown . for generating systems level models , these resources may be integrated systematically . ( c ) there are various ongoing efforts involving researchers , clinicians and or patient groups . it is proposed that a community collaborative open access platform is a must to interface these communities . in order to establish such a platform that allows geographically different communities to work together , globally accepted ontologies in a language independent representation internet of dna is listed as one of the top 15 breakthrough technologies of 2015 ( http://www.technologyreview.com/featuredstory/535016/internet-of-dna/?utm_campaign=newsletters&utm_source=newsletter-weekly-biomedicine&utm_medium=email&utm_content=20150224 ) . the technology needed to harness the power of genomics lies in comparing the genetic information from a large number of individuals with medical records . this currently is a huge challenge , partly because of the technical reasons of moving petabytes of data across different labs , but especially due to the privacy issues surrounding patient information . both these issues should be addressed to ensure that the ever - increasing amount of genomic and clinical data piling up in laboratories and hospitals are utilized optimally . upcoming initiatives like the matchmaker exchange are aiming to bring the genotype and phenotype data together on a common platform ( http://matchmakerexchange.org/ ) . global alliance for genomics and health also known as ga4gh is an organization which provides protocols , apis and file formats for effective and responsible sharing of genomic and clinical data ( http://genomicsandhealth.org/ ) . the organization goal is to overcome challenges likes ethics and privacy involved with sharing of genomics data and to accelerate the potential of genomic medicine for advancement of human health . as discussed before , the need of the hour is to let the patients decide on who will access their data and how these may be used . this is only possible if the information generated using patient samples is made available in real - time . it is also important that other components of this major collaborative strategy are also part of a community platform that allows for gated access to patient data with patients deciding how and with whom their data should be shared . in the changing paradigms of disease treatment , a very recent approval is made by britain on mitochondrial donation ( http://www.theguardian.com/politics/2015/feb/24/uk-house-of-lords-approves-conception-of-three-person-babies ) . in this approach in vitro fertilization ( ivf ) technique is used with biological material coming from three parents , mother and father ( contributing 98.8% genetic material ) and a female donor ( contributing 0.2% genetic material ) . this three - parent ivf approval in uk has received mixed reactions and only time will address the concern of the long - term implications of the same . rare diseases affect over 300 million people globally , however the true burden of these diseases on human health remains to be determined . thus , it is time to review the disease definition considering both the molecular mechanisms involved and environmental factors leading to differential phenotypes . on the other end , a paradigm shift in drug discovery and development is also needed to translate the effort in understanding disease mechanisms to identify potential therapeutic routes . newer models and platforms that allow involvement of patient communities in research and development is also expected to offer solutions to patients suffering from rare diseases who may then benefit from appropriate treatment options . community collaborative approaches for research and funding offer an unprecedented opportunity for making new discoveries and translating to therapeutic interventions .

in recent years , astrocytes have attracted great interest for their capacity to release neuroactive molecules , among which are neurotransmitters like glutamate , because these molecules could modulate neural activity and lead to a possible role for astrocytes in neural information processing [ 13 ] . indeed , astrocyte - derived neurotransmitters , also called gliotransmitters for their astrocytic origin , have been shown to act on neurons and to regulate synaptic transmission and plasticity through a variety of mechanisms . the binding of receptors located on either pre- or postsynaptic terminals by astrocyte - released glutamate has historically been the first pathway for gliotransmission to be discovered and , arguably , the most studied one experimentally for its several possible functional implications . activation of extrasynaptic receptors on presynaptic terminals by astrocytic glutamate modulates the probability of neurotransmitter release from those terminals . in particular , depending on receptor type , such modulation may be either toward an increase or toward a decrease of the frequency of spontaneous [ 711 ] and evoked neurotransmitter release in both excitatory [ 2 , 8 , 10 , 12 ] and inhibitory synapses [ 1315 ] . because synaptic release probability characterizes how a synapse filters or , in other words , processes presynaptic action potentials [ 16 , 17 ] , modulations of synaptic release probability by astrocytic glutamate are suggested to alter the computational properties of neural circuits . glutamate released by astrocytes may also bind to extrasynaptically located postsynaptic nmda receptors , evoking slow inward currents ( sics ) in nearby neurons [ 11 , 1926 ] . the depolarizing action of these currents modulates neural excitability with the potential to affect neuronal action potential firing . moreover , because single astrocytes are in close proximity to a large number ( ~100 ) of neurons , it has been suggested that an inward current can be generated in many adjacent neurons , thereby promoting synchrony of neuronal firing [ 1921 ] . although modulations of both synaptic release and sics mediated by glutamatergic gliotransmission have been recorded in the cortex and the hippocampus , as well as in several other brain regions , their physiological relevance remains elusive . in particular , beyond regulation of synaptic filtering and neuronal firing , theoretical arguments support a further possible role for both pathways in the regulation of nmdar - mediated spike - timing - dependent plasticity ( stdp ) . both pathways have the potential to regulate activation of postsynaptic nmda receptors and , in doing so , glutamatergic gliotransmission could ultimately regulate the stdp outcome , that is , either potentiation ( ltp ) or depression ( ltd ) [ 30 , 31 ] . consistent with this hypothesis , experiments have reported a lower threshold for ltp induction at hippocampal synapses when synaptic release is increased by astrocytic glutamate . moreover , long - term potentiation responses of neurons in the primary visual cortex by cholinergic activation of surrounding astrocytes has also been reported to be correlated with an increase of sic frequency in those neurons . while the potential impact on stdp of pre- or postsynaptic activity - dependent modulations of synaptic efficacy has widely been addressed both experimentally and theoretically [ 34 , 35 ] , the possible effect on plasticity of the regulation of these modulations by glutamatergic gliotransmission ( and by gliotransmission in general ) has been investigated by very few theoretical studies . these studies suggest a potential role in ltp induction both for large increases of synaptic release and for large sics mediated by astrocytic glutamate [ 36 , 37 ] . this scenario seems however at odds with the majority of recent experimental observations that report modest signaling magnitudes for these two routes of gliotransmission . it is thus not clear under what biophysical conditions modulations of synaptic release or sics mediated by glutamatergic gliotransmission could affect stdp . astrocyte - mediated sics , for example , are known to occur sporadically , being recorded in single neurons only as often as < 5/min [ 26 , 32 ] , raising the question of whether and how , by occurring at such low rates , they could effectively play a role in stdp . we thus set to investigating what conditions are required for glutamatergic gliotransmission to affect stdp by presynaptic modulations of neurotransmitter release or through postsynaptic sics . we extend the model of an astrocyte - regulated synapse originally introduced by de pitt et al . to include a biophysically realistic description of synaptically evoked gliotransmitter release by the astrocyte as well as a mechanism for the generation of postsynaptic sics and stdp . first , glutamatergic gliotransmission could change the nature of stdp by modifying the parameter ranges for ltp and ltd induction . second , this effect crucially depends on the nature of gliotransmission , that is , whether it is release - increasing or release - decreasing , its strength , and its rate of occurrence and when it occurs with respect to pre / post pairs . thus , while glutamatergic gliotransmission could potentially play a role in stdp and learning , in practice this effect must satisfy several biophysical and activity - dependent constraints , supporting the existence of specialized dynamic interactions between astrocytes and neurons . although there may be several possible routes by which astrocytes release glutamate [ 3840 ] , ca - dependent glutamate release is likely the main one in physiological conditions [ 41 , 42 ] . from a modeling perspective , as illustrated in figure 1 , ca - dependent glutamatergic gliotransmission consists of three distinct signaling pathways . one pathway ( black arrows ) initiates the release - triggering ca signal in the astrocyte and may be either exogenous or heterosynaptic or be triggered by the very synapses that are modulated by glutamatergic gliotransmission in a homosynaptic fashion . the other two pathways are instead represented by the two recognized routes for the action of glutamatergic gliotransmission on synaptic terminals : the presynaptic pathway whereby astrocytic glutamate modulates synaptic release ( magenta arrows ) and the postsynaptic pathway which mediates sics in nearby neurons ( orange arrows ) . although both pathways could coexist at the same synapse in principle , their functional regulation is probably through different ca - dependent pathways , both in terms of spatiotemporal ca - dynamics and in terms of pools of releasable glutamate resources and/or mechanism of release for these latter . thus , in the following , we set to investigating the effect of synaptic transmission of each pathway independently of the other . we begin our study by a description of a biophysically realistic model of synaptically evoked ca - dependent glutamate release from an astrocyte . at excitatory and inhibitory synapses , astrocytes can respond to synaptically released neurotransmitters by intracellular ca elevations and release glutamate in turn . although morphological and functional details of the coupling between synaptic terminals and the surrounding astrocytic processes remain to be fully elucidated , the current hypothesis is that synaptically evoked glutamate - releasing astrocytic ca signaling is mainly by spillover of synaptic neurotransmitters and/or other factors , which bind to high - affinity astrocytic g protein - coupled receptors ( gpcrs ) and thereby trigger inositol 1,4,5-trisphosphate ( ip3 ) production and ca release from the endoplasmic reticulum ( er ) [ 4648 ] . while early work mainly monitored somatic ca increases concluding that astrocytes respond only to intense neuronal firing patterns , recent experiments in astrocytic processes revealed that astrocytes may also respond to low levels of synaptic activity by ca elevations confined in subcellular regions of their processes [ 10 , 48 , 50 ] , suggesting that the profile of astrocytic ca signaling and thus glutamate release could span the whole spectrum of neuronal ( synaptic ) activity . to realistically describe synaptic release in the whole spectrum of neuronal firing , we consider the model of an activity - dependent synapse first introduced by tsodyks and markram . this model captures the dependence of synaptic release on past activity , that is , presynaptic short - term plasticity , which substantially influences synaptic transmission at high enough rates of neuronal firing . in particular , synaptic release results from the product of two quantities : ( i ) the probability of neurotransmiter - containing vesicles to be available for release and ( ii ) the probability of such vesicles to be effectively released by an action potential , which correlates with intrasynaptic ca . at rest , it is assumed that all vesicles are available for release . the arrival of an action potential opens presynaptic voltage - dependent ca channels that trigger a transient increase of intrasynaptic ca which promotes release of a fraction us of available vesicles . following release , the emptied vesicles are refilled in some characteristic time d , while intrasynaptic ca and thus vesicle release probability decay to zero with a different time constant f . for multiple action potentials incoming at time intervals of the order of these two time constants , neither vesicle replenishment nor intrasynaptic ca are restored to their resting values , so that the resulting synaptic release depends on the history of synaptic activity . we illustrate the response of the synapse model to a train of action potentials in figures 2(a)2(c ) . the low rate of stimulation of the first four action potentials ( figure 2(a ) ) allows for the reintegration of most of the released neurotransmitter in between action potentials thereby keeping vesicle depletion limited ( figure 2(b ) , orange trace ) . in parallel , intrasynaptic ca grows , and so does vesicle release probability ( figure 2(b ) , blue trace ) , resulting in progressively larger release of neurotransmitter per action potential or , in other words , in short - term facilitation of synaptic release ( figure 2(c ) , t < 500 ms ) . on the contrary , the presentation of a series of action potentials in rapid succession at t = 500 ms results in a sharp increase of vesicle release probability to a value close to saturation ( i.e. , nt . pr.0 ) . in this scenario , therefore , from one spike to the next one , progressively fewer neurotransmitter resources are available for release and the amount of released resources decreases with incoming action potentials , leading to depression of synaptic transmission . such depression is short - lived , since synaptic release tends to recover after a sufficiently long period in which no action potentials occur , that is , the case , for example , of the last action potential at t = 800 ms . once released into the synaptic cleft , synaptic neurotransmitter is rapidly cleared by diffusion as well as by other mechanisms , including uptake by transporters and/or enzymatic degradation [ 56 , 57 ] . in the simplest approximation , the contribution of these mechanisms can be modeled by a first - order reaction which accounts for the exponentially decaying profile of neurotransmitter concentration in figure 2(c ) after synaptic release at each action potential . a fraction of released neurotransmitter molecules also spills out of the synaptic cleft to the perisynaptic space ( figure 2(d ) ) where it binds to gpcrs on the astrocyte ( figure 2(e ) ) , therein triggering ca signaling ( figure 2(f ) ) . to quantitatively describe this process , we modify the model of gpcr - mediated ca signaling originally introduced by de pitt et al . to account for dynamic regulation of astrocytic receptors by synaptic activity ( see appendix a , section a.1 ) . accordingly , as illustrated in figure 2(f ) , gpcr - mediated ca signaling is a result of the nonlinear interplay of three processes : ( i ) ip3 production by gpcrs bound by synaptic neurotransmitter ( magenta trace ) , ( ii ) ca release from the er into the cytosol , which is triggered by ip3-bound ca channels ( ip3rs ) and also modulates cytosolic ip3 ( black trace ) , and ( iii ) the effective fraction of available or , more exactly , deinactivated ip3rs that can take part in ca release from the er ( yellow trace ) . depending on the choice of parameter values , the astrocyte model may display both large , long - lasting somatic ca elevations and smaller and shorter ca increases , akin to those reported in astrocytic processes ( see appendix b ) . glutamate release from the astrocyte is then assumed to occur every time that ca increases beyond a threshold concentration ( figure 2(g ) , cyan dotted line ) , in agreement with experimental observations [ 60 , 61 ] . although different mechanisms for glutamate release by the astrocyte could be possible , a large amount of evidence points to vesicular exocytosis as the main one to likely occur on a physiological basis . because astrocytic glutamate exocytosis bears several similarities with its synaptic homologue ( reviewed in de pitt et al . ) , we model it in the same fashion . thus , in line with experimental observations [ 63 , 64 ] , we postulate the existence of an astrocytic vesicular compartment that is competent for regulated glutamate exocytosis . then , upon a suprathreshold ca elevation , a fixed fraction of astrocytic glutamate - containing vesicles releases glutamate into the extracellular space . glutamate is then reintegrated into the astrocyte with some characteristic time constant ( figure 2(h ) ) . in this fashion , glutamate concentration in the extracellular space abruptly increases by exocytosis from the astrocyte and then exponentially decays akin to neurotransmitter concentration in the synaptic cleft , yet , in general , at a different rate ( figure 2(h ) ) ( appendix b ) . the description of gliotransmitter release hitherto introduced ignores the possible stochastic nature of astrocytic glutamate release and reproduces the total amount of glutamate released , on average , by a single ca elevation beyond the release threshold . this description provides a simplified general framework to realistically capture synaptically evoked glutamate release by the astrocyte independently of the underlying mechanism of astrocytic exocytosis , which may either be in the form of a burst of synchronous vesicle fusion events that peaks within the first 50500 ms from the ca rise underneath the plasma membrane [ 61 , 65 , 66 ] or occur at slower fusion rates in an asynchronous fashion [ 67 , 68 ] . once released , astrocyte - derived glutamate can diffuse in the extracellular space and bind extrasynaptic receptors located on presynaptic terminals . in particular , ultrastructural evidence suggests colocalization of glutamate - containing vesicles in perisynaptic astrocytic processes with those receptors , hinting a focal action of astrocytic glutamate on these latter . such action is likely spatially confined and temporally precise , akin to that of a neurotransmitter on postsynaptic receptors , and is not affected by synaptic neurotransmitters . both ionotropic and metabotropic presynaptic receptors may be activated by astrocytic glutamate , yet their differential recruitment likely depends on developmental , regional , physiological , and cellular ( synaptic ) factors ( reviewed in ) . the details of the biochemical mechanisms of action of these receptors on synaptic physiology are not fully understood , but the simplest explanation is that they all modulate intrasynaptic ca levels , eventually increasing or decreasing synaptic release probability , although in a receptor - specific fashion [ 52 , 69 , 70 ] . from a modeling perspective , as originally proposed by de pitt et al . , the common effect on synaptic release shared by different receptors allows expressing , in the simplest approximation , the synapse 's resting release probability proportionally to the fraction of presynaptic receptors activated by astrocytic glutamate ( appendix a , section a.1 ) . in this fashion , as illustrated in figure 3 , the time evolution of the fraction of activated presynaptic receptors ensuing from a series of glutamate release events by the astrocyte ( figures 3(a ) and 3(b ) ) is reflected by the dynamics of synaptic release probability at rest averaged across different trials ( figures 3(c ) and 3(e ) ) . the value of the coefficient of proportionality for the dependence of synaptic release probability on receptor activation sets the type of modulation of synaptic release by astrocytic glutamate which can be either release - decreasing ( figure 3(c ) ) , such as in the case of astrocytic glutamate - binding presynaptic kainate receptors or group ii / iii metabotropic receptors ( mglurs ) [ 13 , 14 , 71 ] , or release - increasing ( figure 3(e ) ) , when astrocytic glutamate binds nmdars or group the functional implications of these modulations of synaptic release by glutamatergic gliotransmission on synaptic transmission have been widely addressed in a series of previous studies [ 18 , 29 , 73 ] , and the remainder of this section reviews and extends the main results from those studies about short - term synaptic plastic and synaptic filtering . figure 3(d ) ( left panel ) shows how postsynaptic currents ( pscs ) change in the presence of release - decreasing glutamatergic gliotransmission when elicited by two consecutive action potentials arriving to the resting synapse 20 ms after the onset of gliotransmission at t = 5 s ( figure 3(c ) ) . two differences with respect to the case without gliotransmission ( black trace ) may be observed . first the psc amplitude overall decreases ( red trace ) , consistent with a decrease of synaptic efficacy caused by the reduction of synaptic release by astrocytic glutamate . then , the second psc is larger than the first one , which is the opposite of what would be measured in the absence of gliotransmission . in other words , in agreement with experimental observations , the release - decreasing effect of astrocytic glutamate results in an increased pair pulse ratio ( ppr ) with respect to the case without gliotransmission ( ppr0 ) . notably , as shown in figure 3(d ) ( right panel ) , this change in the ppr ratio is only transient and vanishes together with the effect of gliotransmission on synaptic release . similar considerations also hold in the case of a release - increasing effect of astrocytic glutamate on synaptic transmission : while psc amplitude increases ( figure 3(f ) , left panel , green trace ) , this occurs to the detriment of ppr , which decreases instead ( figure 3(f ) , right panel ) . thus , synapses whose release probability is increased by glutamatergic gliotransmission are likely to run out of neurotransmitter faster , exhibiting rapid onset of short - term depression , consistent with lower ppr values . on the contrary , synapses whose release probability is reduced by astrocyte - released glutamate deplete their neurotransmitter resources slower and may exhibit progressive facilitation ( i.e. , potentiation ) of their efficacy to transmit action potentials and so larger ppr values . that is , the plasticity mode of a synapse , namely , whether it is depressing or facilitating , may not be fixed but rather be modulated by glutamatergic gliotransmission by surrounding astrocytes in an activity - dependent fashion [ 29 , 73 ] . an important consequence of short - term synaptic dynamics is that synapses can act as filters [ 16 , 17 , 75 ] . hence , modulations of synaptic dynamics by glutamatergic gliotransmission are also expected to affect the synapse 's filtering characteristics . this scenario is illustrated in figure 4 where the effect of release - decreasing versus release - increasing glutamatergic gliotransmission , respectively , on depressing and facilitating synapses , is shown in terms of changes of the filtering characteristics of these synapses , that is , their steady - state release as a function of the frequency of presynaptic stimulation . in the absence of gliotransmission , depressing synapses , which are characterized by intermediate - to - high initial probability of release ( figure 4(a ) , black circles ) , predominantly act as low - pass filters ( figure 4(b ) , black circles ) that are most effective at transmitting low frequency presynaptic spike trains ( figure 4(c ) , black traces ) . on the contrary , facilitating synapses , with a low - to - intermediate initial probability of neurotransmitter release ( figure 4(a ) , black circles ) , function as high - pass or band - pass filters ( figure 4(b ) , black circles ) ; that is , they are mostly effective at transmitting action potentials in an intermediate range of presynaptic activity ( figure 4(c ) , black trace ) . in the presence of glutamate release by the astrocyte , these two scenarios could be reversed . consider indeed the simple heterosynaptic case where glutamatergic gliotransmission is stimulated by other means compared to by the very synapses it impinges on . it may be noted that release - decreasing gliotransmission flattens the synaptic steady - state release towards zero for all frequencies of stimulation ( figure 4(b ) , red circles ) , ensuing in synaptic transmission that resembles the one of a facilitating , band - pass synapse ( compare the red psc trace in figure 4(c ) with the black psc trace in figure 4(f ) ) . vice versa , release - increasing gliotransmission could turn band - pass features of transmission by a facilitating synapse ( figure 4(e ) , green circles ) into low - pass , reminiscent of a more depressing synapse ( compare the green psc trace in figure 4(f ) with the black psc trace in figure 4(c ) ) . on the other hand , when gliotransmission is stimulated by the same synapses that it modulates , that is , in the homosynaptic scenario of gliotransmission , inspection of the ensuing synaptic filtering characteristics ( figures 4(b ) and 4(e ) , cyan circles ) reveals that these latter coincide with those obtained in the absence of gliotransmission for low frequencies of presynaptic activity , while they tend to equal those observed with heterosynaptic gliotransmission as the frequency of stimulation increases . this coexistence of mixed features from apparently opposite scenarios , that is , no gliotransmission versus heterosynaptic gliotransmission , can be explained by the fact that the release of glutamate from the astrocyte requires intracellular ca to cross a threshold concentration . hence , in the homosynaptic scenario , synapses that impinge on the astrocyte must be stimulated at rate sufficiently high to allow astrocytic ca to increase beyond such a threshold . the modulation of synaptic filtering by glutamatergic gliotransmission offers the possibility that the same stimulus could be differently filtered ( i.e. , processed ) and transmitted by a synapse in the presence ( or not ) of glutamate release by surrounding astrocytic processes , ultimately endowing that synapse with processing versatility with respect to incoming action potentials . moreover , to the extent that synaptic dynamics critically shape the computations performed by the neural circuitry , such versatility could also be reflected at the network level , leading to the possibility that the same neuron - glia network could be involved in different computational tasks defined , time by time , by activity - dependent gliotransmitter release by astrocytes in the network . induction of slow inward ( i.e. , depolarizing ) currents ( sics ) by activation of extrasynaptically located postsynaptic nmda receptors is the other mechanism considered in this study whereby glutamatergic gliotransmission could affect synaptic information transfer . while astrocyte - mediated sics have been reported in several brain regions , the pathway underlying glutamate release by astrocytes has not been fully elucidated [ 76 , 77 ] . it is likely that , similar to the presynaptic route for glutamatergic gliotransmission discussed above , multiple pathways for glutamate release could be used by the same astrocyte , but their deployment depends on developmental , regional , and physiological factors . astrocytic ca activity seems to be a crucial factor in the regulation of astrocyte - mediated sics [ 1923 , 25 , 78 ] . in particular , sic frequency and amplitude have been shown to increase upon ca elevations mediated by gpcrs on astrocytes such as mglurs [ 1923 , 72 , 79 ] , the metabotropic purinergic p2y1 receptor , the endocannabinoid cb1 receptor , or the protease - activated receptor 1 ( par1 ) . remarkably , stimulation of par1s on hippocampal astrocytes was shown to trigger , under physiological conditions , ca - dependent glutamate release from these cells through bestrophin-1 anion channel [ 81 , 82 ] , and this pathway of glutamate release has been suggested as a candidate mechanism for sics . channel - mediated glutamate release is expected to account for prolonged ( > 10 s ) release of transmitter but in small amounts per unit time thus ensuing in modest , very slow rising and decaying inward currents . while similar sics have indeed been recorded [ 71 , 84 ] , most experiments reported sics within a wide range of amplitudes to last only few seconds at most and rise in correlation with astrocytic ca increases , with rise time much shorter than their decay [ 2022 , 24 , 26 , 32 , 85 , 86 ] akin to currents that would ensue from a quantal mechanism of gliotransmitter release . based on these arguments , we assume glutamate exocytosis as the candidate mechanism for glutamate release by astrocytes that mediates sics . accordingly , we adopt the description of astrocytic glutamate exocytosis previously introduced ( figures 2(g)2(i ) ) to also model astrocyte - mediated sics . in this fashion , glutamate exocytosis by the astrocyte into the extracellular space ( figure 5(a ) ) results in activation of extrasynaptically located nmdars on nearby neuronal dendrites which trigger sics ( figure 5(b ) ) that cause slow depolarizing postsynaptic potentials ( psp , figure 5(c ) ) . an important functional consequence of sic - mediated depolarizations is that they can modulate neuronal excitability [ 2123 , 85 ] . as illustrated in figures 5(d ) and 5(e ) , astrocyte - mediated sics ( cyan trace ) may add to regular synaptic currents ( black trace ) resulting in depolarizations of postsynaptic neurons closer to their firing threshold . in turn , these larger depolarizations could dramatically change generation and timing of action potentials by those neurons in response to incoming synaptic stimuli ( figure 5(f ) ) . this could ultimately affect several neurons within the reach of glutamate released by an astrocyte , leading to synchronous transient increases of their firing activity . remarkably , this concerted increase of neuronal excitability has often been observed in correspondence with large amplitude ( i.e. , > 100 pa ) sics [ 21 , 25 , 85 , 87 ] , but experiments report the majority of sics to be generally smaller , with amplitudes < 80 pa [ 11 , 21 , 22 , 26 , 32 , 87 ] . it is therefore unclear whether sic - mediated increase of neuronal excitability could occur or not [ 87 , 89 , 90 ] in physiological conditions . in figure 5(g ) , we consider postsynaptic firing in a standard leaky integrate - and - fire neuron model [ 91 , 92 ] as a function of presynaptic activity for sics of different amplitudes ( 3045 pa , see appendix b ) randomly occurring at an average rate of 1 hz based on a binomial process for glutamate release from astrocytes as suggested by experiments ( see appendix a ) . in line with experimental evidence , the input - output transfer function in the absence of gliotransmission has a typical sigmoidal shape ( black dots ) which reflects the following : ( i ) gradual emergence of firing for low ( > 10 hz ) fluctuating synaptic inputs ; ( ii ) the progressive , quasi - linear increase of the firing rate for presynaptic activity beyond ~30 hz ; and finally , ( iii ) saturation of the firing rate for sufficiently strong synaptic inputs such that timing of action potential generation approaches the neuron 's refractory period ( which was fixed at 2 ms in the simulations , appendix b ) . the addition of astrocyte - mediated sics alters the firing characteristics of the neuron due to the ensuing larger depolarization . in particular the neuron could generate action potentials for lower rates of presynaptic activity ( cyan / blue dots ) . clearly , the larger the sic is , the more the postsynaptic firing increases with respect to the case without sics , for a given level of presynaptic activity . as previously mentioned , these results assume an average 1 hz rate for astrocyte - mediated sics . while such a rate can not be excluded , it seems unlikely for the following reasons . the weak correlation of sic amplitude with somatic ca elevations observed in experiments favors indeed the idea that glutamate - mediated sics are highly localized events , occurring within subcellular domains at astrocytic processes . in turn , ca elevations in astrocytic processes could be as short - lived as ~0.5 s [ 10 , 50 ] , thus in principle allowing for glutamate release rates of the order of 1 hz . however , in practice , reported sic frequency is much lower , that is , < 5/min ( i.e. , ~0.08 hz ) [ 11 , 22 ] . hence , it may be expected that the effect of sics on neuronal firing could be considerably reduced with respect to the case in figure 5(g ) . we consider this possibility more closely in figure 5(h ) , where we analyze postsynaptic firing in function of the average frequency of astrocyte - mediated sics , both in the absence of synaptic activity ( black and dark blue dots ) and in the case of presynaptic activity at an average rate ~ 1 hz , which corresponds to typical levels of spontaneous activity in vivo ( grey and light blue dots ) . it may be noted that the effect of sics of typical amplitudes on postsynaptic firing rate is generally small , that is , < 0.5 hz , except for unrealistic ( > 0.1 hz ) sic rates , while it gets stronger in association with synaptic activity . in this latter case however the possible increase in postsynaptic firing by astrocyte - mediated sics is limited by the rate of reintegration of released glutamate resources in the astrocyte ( fixed at ~1 hz , appendix b ) . analogously to short - term synaptic depression in fact , our description of gliotransmitter release predicts that , for release rates that exceed the rate of reintegration of released glutamate by the astrocyte , exhaustion of astrocytic glutamate resources available for further release will result in sics of smaller amplitude . in this fashion , due to depletion of astrocytic glutamate , the effect of large rates of glutamate release and thus of sics on neuronal firing tends to be equivalent to that of considerably lower ones . taken together , the above results do not exclude a possible role of sics in modulation of neuronal excitability and firing but suggest that such modulation could effectively occur only in coincidence with proper levels of synaptic activity . in this fashion , astrocyte - mediated sics could be regarded to operate a sort of coincidence detection between synaptic activity and astrocytic glutamate release , whose readout would then be a temporally precise , cell - specific increase of neuronal firing ( figure 5(f ) ) . the strength of a synaptic connection between two neurons can be modified by activity , in a way that depends on the timing of neuronal firing on both sides of the synapse , through a series of processes collectively known as spike - timing - dependent plasticity ( stdp ) . as both pre- and postsynaptic pathways of glutamatergic gliotransmission potentially change epsc magnitude , thereby affecting postsynaptic firing although the molecular mechanisms of stdp remain debated , and different mechanisms could be possible depending on type of synapse , age , and induction protocol , at several central excitatory synapses postsynaptic calcium concentration has been pointed out as a necessary factor in induction of synaptic changes by stdp [ 31 , 9699 ] . remarkably , amplitude and , likely , time course of postsynaptic ca could control the direction of plasticity : smaller , slower increases of postsynaptic ca give rise to spike - timing - dependent long - term depression ( ltd ) , whereas larger , more rapid increases cause spike - timing - dependent long - term potentiation ( ltp ) [ 31 , 96 , 97 ] . in calcium - based stdp models , this is also known as the ca - control hypothesis [ 35 , 100 , 101 ] . according to this hypothesis , no modification of synaptic strength occurs when ca is below a threshold d that is larger than the resting ca concentration . if calcium resides in an intermediate concentration range , between d and a second threshold p > d , the synaptic strength is decreased . finally , if calcium increases above the second threshold , p , the synaptic strength is potentiated . figures 6(a1 ) and 6(b1 ) exemplify the operational mechanism of the ca - control hypothesis within the framework of a nonlinear ca - based model for stdp at glutamatergic synapses originally introduced by graupner and brunel . at most glutamatergic synapses , postsynaptic ca is mainly regulated by two processes : ( i ) postsynaptic ca entry mediated by nmdars and ( ii ) ca influx by voltage - dependent ca channels ( vdccs ) [ 31 , 33 , 99 , 104 ] . in this fashion , each presynaptic action potential generates a long - lasting ca transient by opening nmdar channels , while postsynaptic firing results in a short - lasting ca transient due to opening of vdccs by dendritic depolarization through back - propagating action potentials ( baps ) . presynaptic action potentials alone do not trigger changes in synaptic strength , but they do so in correlation with postsynaptic baps . notably , in a typical stdp induction pairing protocol , ltd is induced if the postsynaptic neuron fires before the presynaptic one , that is , post pre pairing at negative spike - timing intervals t ( figure 6(a1 ) ) . contrarily , ltp is induced when the presynaptic cell fires before the postsynaptic cell , that is , for pre post pairing at positive t intervals ( figure 6(a1 ) ) . this is possible because , when a presynaptic action potential is followed shortly after by a postsynaptic bap , the strong depolarization by this latter drastically increases the voltage - dependent nmdar - mediated ca current due to removal of the nmdar magnesium block [ 107 , 108 ] , thereby resulting in supralinear superposition of the nmdar- and vdcc - mediated ca influxes . in the framework of the ca - control hypothesis , pre- and postsynaptic ca transients do not interact , and the contributions from potentiation and depression by pre / post pairs ( or vice versa ) cancel each other , leading to no synaptic changes on average ( figure 6(c ) , black curves ) . for short , negative t , the presynaptically evoked ca transient rises instead above the depression threshold ( d ) but not beyond the potentiation threshold ( p ) . consequently , depression increases whereas potentiation remains constant , which leads to ltd induction . for short , positive t however the postsynaptically evoked calcium transient rises on top of the presynaptic transient by the nmdar nonlinearity and increases activation of both depression and potentiation . because the rate of potentiation is larger than the rate of depression ( appendix c ) , this results in ltp induction . for the same number of pre / post pairs ( or vice versa ) , mapping of the average synaptic modification as a function of the spike - timing interval t ultimately provides an stdp curve that qualitatively resembles the classic curve originally described by bi and poo ( figure 6(c ) , right panel , black curve ) . in the following , we will focus on parameters that lead to such a stdp curve and investigate how this curve is affected in the presence of glutamatergic gliotransmission , through the pre- and postsynaptic pathways of regulation discussed above . the very nature of synaptic transmission crucially depends on the synapse 's initial probability of neurotransmitter release , insofar as this latter sets both the tone of synaptic transmission , that is , how much neurotransmitter is released per action potential by the synapse on average , and whether the synapse displays short - term depression or facilitation . synapses with low - to - intermediate values of initial neurotransmitter release probability , for example , schaffer collateral synapses , or some cortical synapses , are indeed prone to display facilitation , whereas synapses that are characterized by large release probability are generally depressing . because synaptic release probability also dictates the degree of activation of nmdars and consequently the magnitude of postsynaptic ca influx , it is expected that both the tone of synaptic transmission and its short - term dynamics could affect stdp . the relative weight of these two factors in shaping synaptic changes however likely depends on the protocol for stdp induction . short - term plasticity could indeed sensibly regulate stdp induction only for rates of presynaptic action potentials high enough to allow facilitation or depression of synaptic release from one ap to the following one [ 109 , 110 ] . in this study , we consider low pre / post frequencies of 1 hz . at such frequencies we expect short - term plasticity to have a negligible effect , and thus we only focus on how changes in the tone of synaptic transmission by glutamatergic gliotransmission affect stdp . figures 6(a2 ) and 6(b2 ) , respectively , show the outcome of ltd and ltp induction for two consecutive pre post and pre post pairings preceded by the onset of release - decreasing gliotransmission at 0.1 s ( top panels , black marks ) . a comparison of the ensuing postsynaptic ca dynamics with respect to the case without gliotransmission ( figures 6(a1 ) and 6(b1 ) ) reveals that the strong decrease of synaptic release probability ( srp , top panels , red curves ) caused by gliotransmission remarkably reduces the nmdar - mediated contribution to postsynaptic ca influx ( middle panels ) , resulting in smaller variations of synaptic strength ( bottom panels ) . in this fashion , at the end of the pairing protocol , release - decreasing gliotransmission accounts for less time spent by ca above both ltd and ltp thresholds ( figure 6(c ) , left panel , red traces ) . the resulting stdp curve thus displays strongly attenuated ltd and ltp ( figure 6(c ) , right panel , red curve ) , with ltp windows spanning a considerably smaller range of ts values than in the curve obtained without gliotransmission ( black curve ) . similar considerations apply to the case of release - increasing gliotransmission ( figures 6(a3 ) and 6(b3 ) ) . in this case , the nmdar component of postsynaptic ca could increase by gliotransmission even beyond the d threshold ( dashed blue line ) , thus favoring depression while reducing potentiation ( bottom panels ) . in particular , for short positive t , the maximal ltp does not change but the t range for ltp induction shrinks . for t > 40 ms in fact , the time that ca spends above the ltd threshold increases with respect to the time spent by ca above the ltp threshold , thereby resulting in ltd induction ( figure 6(c ) , left panel , green traces ) . in this fashion , the stdp curve in the presence of release - increasing gliotransmission displays a narrow 040 ms ltp window outside which ltd occurs instead ( figure 6(c ) , right panel , green curve ) . figure 6(d ) summarizes how the stdp curve changes for the whole spectrum of glutamatergic gliotransmission . in this figure , a y - axis value of gliotransmission type equal to 0 corresponds to maximum release - decreasing gliotransmission ( red curve in figure 3(c ) ) ; a value equal to 1 stands instead for maximum release - increasing gliotransmission ( as in the case of the green curve in figure 3(c ) ) ; finally , a value of 0.5 corresponds to no effect of gliotransmission on synaptic release ( black curve in figure 3(c ) ) . it may be noted that gliotransmission may affect the stdp curve in several ways , changing both strength of plastic changes ( color code ) and shape and areas of ltp and ltd windows . in particular , as revealed by figure 6(e ) , maxima of ltp ( cyan circles ) and ltd ( yellow circles ) decrease with decreasing values of gliotransmission type , consistently with smaller postsynaptic ca influx for larger decreases of synaptic release by gliotransmission . this suggests that release - decreasing gliotransmission ( red - shaded area ) could attenuate stdp yet in a peculiar fashion , counteracting ltd more than ltp induction , as reflected by increasing values of ltp / ltd area ratio ( magenta curve ) . on the contrary , the effect of release - increasing gliotransmission ( figure 6(e ) , green - shaded area ) could be dramatically different . for sufficiently strong increases of synaptic release by gliotransmission in fact , the ltp / ltd area ratio drops to zero ( hatched area ) in correspondence with the appearance of two open ltd windows , one for t < 0 and the other for sufficiently large positive spike - timing intervals . in parallel , consistently with the fact that release - increasing gliotransmission tends to increase the fraction of time spent by postsynaptic ca above the threshold for ltd thereby promoting this latter ( figure 6(c ) ) , the range for ltp induction also tends to shrink to lower t values as release - increasing gliotransmission grows stronger ( figure 6(d ) , red color - coded areas for gliotransmission type > 0.5 ) . in summary , our analysis reveals that modulation of synaptic release by glutamatergic gliotransmission could change stdp both quantitatively and qualitatively , from hindering its induction for release - decreasing modulations to altering both shape and existence of ltd windows for release - increasing modulations . however , whether and how this could effectively be observed in experiments remain to be investigated . supported both by experimental evidence and theoretical arguments is the notion that regulations of the tone of synaptic transmission by glutamatergic gliotransmission likely require specific morphological and functional constraints to be fulfilled by the nature of astrocyte - synapse coupling [ 5 , 18 ] . similar arguments may ultimately hold true also for modulation of stdp ; insofar as for this modulation to be measured in our simulations , we required both a sufficiently strong increase / decrease of synaptic release by gliotransmission and a decay time of such increase / decrease long enough for this latter to be present during the induction protocol . should these two aspects not have been fulfilled in our simulations , then modulation of stdp by gliotransmitter - mediated changes of synaptic release would likely have been negligible or even undetectable . we now turn our analysis to the possible impact of astrocyte - mediated sics on stdp . because sics are through extrasynaptic nmda receptors and these receptors are mainly permeable to ca ions , then sics could contribute to postsynaptic ca thereby affecting stdp nevertheless , we should note that it is unclear whether and how extrasynaptic nmdars contribute to plasticity , independently of the occurrence of sics . for example , theta - burst ltp induction in ca1 neurons of rat hippocampal slices is turned into ltd when extracellular nmdars are selectively stimulated , but it is unknown whether these receptors have a role in stdp . in general , for a given stdp induction protocol , two factors that could crucially regulate how ca transients mediated by extrasynaptic nmdars are involved in stdp are the location of these receptors on the spine and the morphology of this latter in terms of spine head and neck [ 114 , 115 ] . unfortunately both these factors remain unknown in the current knowledge of sic - mediating extrasynaptic nmdars and , for the remainder of this study , we assume that , in spite of their possible location away from the postsynaptic density along the spine neck or the dendritic shaft , sic - mediating extrasynaptic nmdars could still regulate spine ca dynamics . based on the above rationale , we thus model sics as slow postsynaptic ca transients that will add to presynaptically and postsynaptically triggered ones and study their effect on the induction of sdtp by classic pairing protocols . for the sake of generality , we express the peak of sic - mediated ca transients in units of nmdar - mediated epscs . however , since in our stdp description individual epscs do not trigger any synaptic modification , then we may expect that only sics sufficiently larger than epscs could effectively affect stdp . on the other hand , smaller sics could also combine with ca transients by pre / post pairings , resulting in ca elevations beyond either ltd or ltp thresholds that would ultimately cause synaptic changes ( figures 7(a ) and 7(b ) ) . hence , based on these considerations , we deem amplitude and timing of sics , in terms of both frequency of occurrence and onset with respect to stdp - inducing stimuli , to be crucial factors in shaping how sics affect stdp , and thus we set to analyzing these three factors separately . figure 7(c ) summarizes the results of our simulations for sics as large as 0.5 , 1 , or 1.5 times typical epscs , occurring at a fixed rate of 0.1 hz and starting 100 ms before the delivery of 60 stdp - inducing pre / post pairings at 1 hz . as illustrated in figures 7(a ) and 7(b ) , for the same sic kinetics , these simulations guarantee superposition between ca influxes mediated by sics and pre / post pairings such that the extension of the ensuing ca transient beyond ltd and ltp thresholds ( dashed lines ) merely depends on sic amplitude . in this fashion , it may be noted that sics of amplitude smaller than or equal to typical epscs ( figure 7(c ) , turquoise circles and black circles , resp . ) , which alone would not produce any synaptic modification , do not sensibly change the stdp curve with respect to the previously considered case of an alike synapse in the absence of gliotransmission ( figure 6(c ) , black circles ) . conversely , large sics could dramatically affect stdp , shifting the stdp curve towards negative synaptic changes ( blue circles ) , and this negative shift increases the larger sics grow beyond the d threshold ( results not shown ) . in this case , stdp generally results in ltd with the exception of a ltp window that is comprised between ~0 ms and positive t values that are smaller than those in the absence of gliotransmission ( figure 6(c ) , green circles ) . this resembles what was previously observed for stdp curves in the presence of release - increasing gliotransmission , with the only difference that , for large |t| values , ltd strength in the presence of astrocyte - mediated sics is found to be the same , regardless of t ( compare the blue curve in figure 7(c ) with the green curve in figure 6(c ) ) . in figure 7(d ) we consider the alternative scenario where only sics as large as typical epscs impinge on the postsynaptic neuron at different rates , yet always 100 ms before stdp - inducing pairings . akin to what happens for sic amplitudes , the larger the sic frequency is , the more the stdp curve changes . indeed , as sic frequency increases above sic decay rate ( i.e. , 1/a , appendix a , section a.1.4 ) , sic - mediated ca transients start adding up , so that the fraction of time spent by ca beyond the ltd threshold increases favoring ltd induction . in this fashion , the ensuing stdp curve , once again , consists of a narrow ltp window for t 0 , outside which only ltd is observed ( red curve ) . in practice however , because sics occur at rates that are much slower than their typical decay ( appendix b ) , they likely affect stdp in a more subtle fashion . this may be readily understood considering the pink stdp curve obtained for sics at 0.1 hz , that is , the maximum rate experimentally recorded for these currents . inspection of this curve indeed suggests that sics could effectively modulate ltd and ltp maxima as well as the outer sides of the ltd / ltp windows , which dictate how fast depression / potentiation decay for large |t| , but overall the qualitative features of the stdp curve are preserved with respect to the case without gliotransmission ( black curve ) . clearly , the extent of the impact of sic amplitude and frequency on stdp discussed in figures 7(c ) and 7(d ) ultimately depends on when sics occur with respect to ongoing stdp - inducing pairings . had we set sics to occur ~200 ms after pre / post ca transients in our simulations , then , as illustrated in figures 8(a ) and 8(b ) , we would have not detected any sensible alteration of stdp , unless sics were larger than typical epscs and/or occurred at sufficiently high rate to generate ca transients beyond the plasticity thresholds ( results not shown ) . to seek understanding of how timing of sics versus pre / post pairings could alter ltd and/or ltp , we simulated stdp induction by pairing as the time interval ( ) between sic and pre / post pairs was systematically varied ( with sic rate fixed at 0.2 hz ) ( figures 8(c)8(e ) ) . in doing so , we adopted the convention that negative values stand for sics preceding pre / post ( or post / pre ) pairings while positive values refer to the opposite scenario of sics that follow pairings ( figure 8(c ) , top schematics ) . then , it may be observed that , for approximately in between 300 ms and 0 ms , ltd could be induced for any negative t as well as for large positive t ( figure 8(c ) , blue tones ) , in this latter case to the detriment of the ltp window , whose upper bound moves to lower t values ( figure 8(c ) , red tones ) . this results in stdp curves ( e.g. , figure 8(f ) , yellow curve for = 75 ms ) that bear strong analogy with the blue and red curves in figures 7(c ) and 7(d ) , respectively , obtained for sics of large amplitude and frequency and suggest that depression grows as sics tend to concur with pre / post pairings . an inspection of postsynaptic ca transients ( figures 8(d ) and 8(e ) ) indeed reveals that coincidence of sics and pre / post pairings , which occurs at negative of the order of sic rise time ( see appendix b ) , corresponds to the longest time spent by ca above the ltd threshold , thereby resulting in maximum ltd ( figure 8(g ) ) and thus minimum ltp ( figure 8(h ) ) . clearly , the range for which coincidence of sics with pre / post pairings enhances ltd induction ultimately depends on kinetics of sics , as reflected by their rise ( s ) and/or decay time constants ( s ) , and spans values approximately comprised within sic duration ( i.e. , s + s ) . as sic duration increases in fact , because of either larger s or larger s or both , so does the range for ltd enhancement , as reflected by the orange and blue curves in figures 8(f)8(h ) . in conclusion the simulations in figures 8(c)8(h ) point to both timing and duration of sics with respect to pre / post pairing - mediated ca transients as a further , potentially crucial factor in setting strength and polarity of stdp at glutamatergic synapses . it is noteworthy to emphasize that , however , to appreciate some effect on stdp , we had to assume in those simulations sics occurring at 0.2 hz , that is , twofold the maximum sic rate ( i.e. , ~0.1 hz ) experimentally observed . indeed , analogous simulations run with realistic sic rates 0.1 hz did produce only marginal changes to stdp curves , akin to those previously observed for the pink stdp curve in figure 7(d ) . the potential functional implications ( or lack thereof ) of this perhaps puzzling result are addressed in discussion . a large body of evidence has accumulated over the last years suggesting an active role of astrocytes in many brain functions . collectively , these data fuelled the concept that synapses could be tripartite rather than bipartite , since in addition to the pre- and postsynaptic terminals , the astrocyte could be an active element in synaptic transmission [ 1 , 49 , 117 ] . using a computational modeling approach , we showed here that glutamatergic gliotransmission could indeed play several roles in synaptic information transfer , either modulating synaptic filtering or controlling postsynaptic neuronal firing , as well as regulating both short- and long - term forms of synaptic plasticity . supported by experimental observations [ 8 , 9 , 13 , 23 , 118 ] , these results complement and extend previous theoretical work on astrocyte - mediated regulations of synaptic transmission and plasticity [ 18 , 29 ] and pinpoint biophysical conditions for a possible role of glutamatergic gliotransmission in spike - timing - dependent plasticity . an important prediction of our model indeed is that both pathways of regulation of synaptic transmission by astrocytic glutamate considered in this study , presynaptic modulation of transmitter release and postsynaptic sics , could affect stdp , potentially altering induction of ltp and ltd . this alteration could encompass changes in the timing between pre- and postsynaptic firing that is required for plasticity induction , as well as different variations of synaptic strength in response to the same stimulus . with this regard , the increase of ltp observed in our simulations , when moving from release - decreasing to release - increasing gliotransmission ( figure 6(e ) ) , agrees with the experimental observation that ltp induction at hippocampal synapses requires weaker stimuli in the presence of endogenous glutamatergic gliotransmission rather than when gliotransmission is inhibited thereby decreasing synaptic release probability . notably , spike - timing - dependent plasticity in the hippocampus is not fully understood insofar as stdp induction by pairing protocols has produced a variety of seemingly contradicting observations for this brain region . recordings in hippocampal slices , for example , showed that pairing of single pre- and postsynaptic action potentials at positive spike - timing intervals could trigger ltp [ 120122 ] , as effectively expected by the classic stdp curve , but also induce either ltd or no plasticity at all . although different experimental and physiological factors could account for these diverse observations [ 119 , 125 ] , we may speculate that glutamatergic gliotransmission by astrocytes , which in those experiments was not explicitly taken into account , could also provide an alternative explanation . for example , the prediction of our model that release - increasing glutamatergic gliotransmission could account for multiple ltd windows , at either positive or negative spike - timing intervals ( figure 6 ) , indeed supports the possibility that ltd in the hippocampus could also be induced by proper presentations of pre post pairings sequences . on the same line of reasoning , the possibility that astrocyte - mediated sics could transiently increase postsynaptic firing ( figure 5(f ) ) could explain why , in some experiments , precise spike timing in the induction of synaptic plasticity in the hippocampus could exist only when single epsps are paired with postsynaptic bursts [ 124 , 126 ] . moreover , it was also shown that postsynaptic firing is relatively less important than epsp amplitude for the induction of stdp in the immature hippocampus compared to the mature network , possibly due to a reduced backpropagation of somatic aps in juvenile animals . remarkably , these diverse modes of plasticity induction could also ensue from different dynamics of glutamatergic gliotransmission , as likely mirrored by the developmental profile of somatic ca signals in hippocampal astrocytes , which have been reported to be much more frequent in young mice . insofar as somatic ca signals may result in robust astrocytic glutamate release that could trigger , in turn , similar increases of synaptic release and/or sics [ 5 , 62 ] , the frequent occurrence of these latter could then ultimately guarantee a level of dendritic depolarization sufficient to produce ltp in mice pups . high amplitude / rate sics , or large increases of synaptic release mediated by glutamatergic gliotransmission , result , in our simulations , in ltd induction for any spike - timing interval except for a narrow ltp window at small - to - intermediate t > 0 . this is in stark contrast with stdp experiments , where the observed plasticity always depends , to some extent , on the coincidence of pre- and postsynaptic activity , as epsps or postsynaptic action potentials fail to induce plasticity by their own [ 95 , 105 ] . apart from the consideration that large sic amplitudes / rates and large increases of synaptic release by astrocytic glutamate may not reflect physiological conditions [ 76 , 90 ] , this contrast may be further resolved on the basis of the following arguments . a first consideration is that we simulated plasticity induction assuming either persistent occurrence of sics or continuous modulations of synaptic release during the whole induction protocol . while this rationale proved useful to identify the possible mechanisms of regulation of stdp by glutamatergic gliotransmission , it may likely not reflect what occurs in reality . indeed , modulations of synaptic release by glutamatergic gliotransmission could last only few tens of seconds [ 7 , 8 ] and thus be short - lived with respect to typical induction protocols which are of the order of minutes [ 33 , 105 , 129 ] . moreover , the morphology of astrocytic perisynaptic processes is not fixed but likely undergoes dynamical reshaping in an activity - dependent fashion during plasticity induction [ 118 , 130 ] , thereby potentially setting time and spatial range of action of gliotransmission on nearby synaptic terminals . in this fashion , ltd for large spike - timing intervals could be induced only transiently and at selected synapses , focally targeted by glutamatergic gliotransmission , while leaving unchanged the qualitative features of the classic stdp curve obtained by somatic recordings in the postsynaptic neuron . a further aspect that we did not take into account in our simulations is also the possible voltage dependence of astrocyte - triggered sics . the exact nature of this dependence remains to be elucidated and likely changes with subunit composition of nmda receptors that mediate sics in different brain regions and at different developmental stages . regardless , it may be generally assumed that slow inward currents through nmda receptors become substantial only for intermediate postsynaptic depolarizations when the voltage - dependent mg block of these receptors is released . in this fashion , the possible effect of sics on stdp would be confined in a time window around t 0 for which coincidence with pre- and postsynaptic spikes allows for robust depolarization of postsynaptic spines . outside this window instead , sics would be negligible , and plasticity induction would essentially depend on mere pre- and postsynaptic spiking rescuing the experimental observation of no synaptic modification for large spike - timing intervals [ 95 , 105 ] . on the other hand , even without considering voltage dependence of sic - mediating nmdars , the precise timing of sics with respect to pre / post pairs is predicted by our analysis , to be potentially critical to determine strength and sign of plasticity . similar considerations could also hold for the onset time and duration of modulations of synaptic release triggered by gliotransmission with respect to the temporal features of plasticity - inducing stimuli . this ultimately points to timing of glutamate release by the astrocyte ( and its downstream effects on synaptic transmission ) as a potential additional factor for associative ( hebbian ) learning , besides sole correlation between pre- and postsynaptic activities [ 131 , 132 ] . remarkably , this could also provide a framework to conciliate the possibility that modest , sporadic sics that we predict would not substantially affect stdp ( figure 7 ) could do so instead . indeed our predictions are based on the average number of sics within a given time window , as documented in literature , rather than on the precise timing of those sics in that time window . in this fashion , for example , there is no distinction in terms of effect on stdp in our simulations , between a hypothetical scenario of three sics randomly occurring on average every ~10 s in a 30 s time frame and the alternative scenario of three sics taking place within the same time frame but in rapid succession ( figure 5b in ) , as could happen following an exocytic burst of glutamate release by the astrocyte [ 61 , 62 , 65 ] . yet the latter case could result in a dramatically different plasticity outcome with respect to the former . while individual sics likely fail to induce synaptic modification alone in fact , their occurrence in rapid succession would instead allow postsynaptic ca levels to quickly increase beyond one of the thresholds for plasticity induction . furthermore , this increase could further be boosted by coincidence of sics with pre- and postsynaptic activity , ultimately accounting for robust ltp , as indeed predicted by other theoretical investigations . however , to complicate this intriguing scenario is the observation that glutamatergic gliotransmission and even more so astrocyte - mediated sics [ 19 , 25 ] are likely not deterministic but rather stochastic processes . therefore , it would ultimately be interesting to understand how this stochasticity could affect neuronal activity and shape learning . to conclude , our analysis provides theoretical arguments in support of the hypothesis that , beyond neuronal firing , astrocytic gliotransmission could represent an additional factor in the regulation of activity - dependent plasticity and learning [ 18 , 134 , 135 ] . this could occur in a variegated fashion by both presynaptic and postsynaptic elements targeted by glutamatergic gliotransmission , with possibly diverse functional consequences . nonetheless , the practical observation in future experiments of a possible mechanism of action of glutamatergic gliotransmission on activity - dependent plasticity will depend on the implementation of novel specific plasticity - inducing protocols that match possible stringent temporal and spatial dynamical constraints defining the complex nature of neuron - astrocyte interactions .

balance impairment greatly affects activities of daily living and other essential activities in stroke patients . the assessment of balance is therefore extremely important in clinical practice for managing stroke patients . currently available assessment tools for balance in stroke patients include the functional reach test ( frt)1 , the functional balance scale ( fbs)2 , 3 and the timed up and go test4 . these tests , all of which are performance tests , are widely used in clinical practice and research , and have the advantage that measurement can be easily performed without specific devices or instruments . however , it has been noted that patients with reduced physical fitness or paralysis due to stroke may not be able to perform these tasks , and the test results thus have limitations in terms of statistical analysis5,6,7 . the hand - held dynamometer ( hhd ) is a clinically useful device , based on its easy - to - use and easy - to - carry features , and allows quantitative measurement8 , 9 . to overcome the limitations of the conventional balance tests mentioned above , we previously developed a standing balance assessment index using this hhd ( the hhd assessment index)10 , 11 . in 2004 , we reported that the hhd assessment index correlated with the brunnstrom recovery stages and walking ability , and had a satisfactory intrarater reliability measured with the intraclass correlation coefficient ( icc ) . in 2012 , we confirmed the criterion - related validity of the hhd assessment index using the fbs as a criterion variable . however , the participants in these studies were not limited to stroke patients and the number of participants who had suffered a stroke was small . therefore , this study aimed to examine the reliability and validity of the hhd assessment index of standing balance in stroke patients . the participants were 60 cerebral stroke hemiplegic patients ( 30 with right hemiplegia and 30 with left hemiplegia ) who were able to walk indoors independently or with supervision ( table 1table 1.general characteristics of all the subjects included in this studydemographic valuesgender ( male / female)29/31age ( mean sd)74.6 10.1etiology ( hemorrhage / infarct)14/46affected hemisphere ( right / left)30/30 ) . patients unable to maintain a standing position , those who could walk outdoors independently or run , those with difficulty following the testing instructions due to sensory aphasia or advanced dementia etc . , and those with severe pain that prevented participation , were excluded . the objective and methods of the study were fully explained verbally and in writing to potential participants , and those who consented to participate were included in the study . the ethics committee of ibaraki prefectural university of health sciences approved all study protocols , and each participant provided written informed consent prior to enrollment . intrarater reliability of the hhd assessment index was evaluated by icc for 30 randomly selected participants . interrater reliability of the hhd assessment index was evaluated by icc between the scores determined by two physical therapists ( at the start and at the end of the session ) for the 10 randomly selected participants . validity was evaluated by spearman s rank correlation coefficient between the hhd assessment index and the fbs ( the criterion variable of standing balance ) for all 60 participants . with regard to the evaluation of intrarater reliability of the hhd assessment index , one physical therapist conducted the measurements for each participant twice , and the correlation coefficient for the pairs of scores was calculated as the reliability coefficient for the 30 participants . with regard to the evaluation of interrater reliability , two physical therapists conducted the measurements for each participant under the same conditions , and the correlation coefficient between the two raters scores the measurement device used was the microfet 2 hhd ( hoggan health industries inc . we conducted the assessment in the physical therapy room according to the method described by iwamoto et al11 . the measurements were performed under the following conditions : the patient stood 2 m from both the anterior and the lateral walls , with eyes open ; the upper limbs were down at the sides of the body , and the inner edges of the feet were parallel and at a distance of 10 cm from each other . we chose the right and left iliac crests ( ic ) , anterior superior iliac spines ( asis ) , and posterior superior iliac spines ( psis ) ( a total of six sites ) as measurement sites because they were easily palpated and good reliability of landmarks could be expected . the participant was instructed to maintain the standing posture , while remaining as still as possible , against a gradually increased force exerted by the examiner with the hhd : the force on the ic was applied from the lateral direction ; that on the asis was applied from the anterior side , and that on the psis was applied from the posterior side , horizontally to the floor . the break test was adopted as a uniform method of measurement : a participant taking a fixed posture was given pressure through hhd until he lost the posture . as the measurement value was highest when the measurement site was pressured by hhd until the participant could no longer hold his body posture , the hdd value at that moment ( n ) was adopted as a measurement value . it was often the case that a part of the sole of the foot left the floor at the moment when the participant lost control of his posture , which provided a cue to stop the pressure . therefore , the best efforts were made to set the eye level of examiners at the height of the participant s pelvis in order to make it easier for them to see the imbalance of the posture or the sole of the foot . the make test to measure the pressure to hdd which was generated by the participants pressuring of fixed hdd was not adopted . as was already pointed out , the participants who could not understand the direction of movement involved in the break test or the make test and those who could not comply with the reminder were excluded . to determine the validity of the hhd assessment index , all 60 participants underwent the hhd assessment and the fbs . as for the hhd assessment , the examiner performed the measurement three times for each site , and the mean values served as the scores of the hhd assessment index . , with a 5-stage scale for each item ( a total of 56 points ) . the icc values were classified into five categories according to the criteria of landis et al.12 : 0.000.20=slight , 0.210.40=fair , 0.410.60=moderate , 0.610.80=substantial , 0.811.00=almost perfect . to confirm the validity of the hhd assessment index as a balance test , the statistical analysis was performed using a statistical software package ( ibm spss statistics 19 ) . in addition , based on the fact that there was no relation between the evaluation - figure value measured in this study and the body weight , the ratio of the body weight was not adopted . the scores of the hhd assessment index and the icc values calculated to determine the intrarater and interrater reliabilities are shown in table 2table 2.reliability of hhd assessment index ( n=30)sidemeasurement sitesrater 1rater 2re - testtesticc(2,1)icc(1,1)mean standard deviationasis29.6 14.723.7 13.726.3 13.127.4 13.20.950.97affected ic43.8 19.243.1 18.040.9 16.840.6 19.30.910.98psis38.1 16.537.0 13.638.1 16.538.0 14.70.920.96asis24.6 11.424.0 11.324.6 11.424.3 9.90.910.97unaffectedic41.5 15.339.2 18.039.6 15.840.0 17.50.970.97psis29.7 12.128.4 8.8 27.8 8.628.4 10.40.910.94asis : anterior superior iliac spine , ic : iliac crest , psis : posterior superior iliac spine . icc(1,1 ) was used for intra - rater reliability .. the icc values for both intrarater and interrater reliabilities were 0.88 or higher , indicating almost perfect correlations according to the criteria of landis et al . asis : anterior superior iliac spine , ic : iliac crest , psis : posterior superior iliac spine . icc(1,1 ) was used for intra - rater reliability . to confirm the validity of the hhd assessment index as a balance test , spearman s rank correlation coefficient with the fbs was calculated . a significant positive correlation was found between the hhd assessment index and the fbs ( r=0.83 , p<0.01 ) . however , there have been no studies focusing on the usefulness of hhd in testing balance in stroke patients . we previously reported the reliability and validity of the standing balance assessment index using a hhd in healthy individuals and patients with impaired standing balance , but did not explore this issue in stroke patients . in the present study , we evaluated the reliability of the hhd assessment index in stroke patients and confirmed high reliability : 0.940.98 for intrarater reliability and 0.910.97 for interrater reliability . we also evaluated the validity of the hhd assessment index using the fbs as a reference variable , and demonstrated a significant positive correlation , showing a strong association between the hhd assessment index and the fbs . these findings confirmed the reliability and validity of the hhd assessment index as a balance test for stroke patients . bohannon et al.13 , developed a trunk muscle test using a hhd in the sitting position in hemiplegic patients after cerebrovascular accidents . they demonstrated both the accuracy and the reliability of their measurement method . according to their report , the icc values for the measurement of lateral trunk flexion muscle strength using a hhd were 0.987 on the paretic side and 0.996 on the non - paretic side . although the icc values calculated in the present study were slightly lower than those reported by bohannon et al . , they were within the range of almost perfect correlations ( 0.811.00 ) according to the criteria of landis et al . therefore , the results of this study showed the hhd assessment index to have satisfactory reliability . possible explanations for our relatively low , as compared to bohannon et al.s study , icc values might involve differences in the testing position ( sitting position vs. standing position ) , directions of the applied forces ( two lateral directions in bohannon et al.s study and six anterior - posterior and lateral directions in our study ) , and measurement sites ( shoulder vs. pelvis ) . future studies comparing measurements at the pelvis and other sites , as well as measuring the shift in center of gravity using a stabilometer during the test , may shed light on this issue . regarding validity , the present study showed a significant correlation between the hhd assessment index and the fbs . we therefore concluded that the criterion - related validity of the hhd assessment index as a balance test was satisfactory . the fbs , used as a reference variable in the present study , is a performance test of balance that provides scores by asking patients to perform test movements . this test has the following limitations : the test comprises multiple test movements , taking approximately 25 minutes , and may cause fatigue in frail patients with low endurance ; the test may not be suitable for patients with mild motor impairment because of the ceiling effect ; the ordinal scale , which is employed for the test , limits carrying out statistical analysis ; and the test does not allow identification of the cause or the mechanism of balance problems14 . in contrast , the balance assessment using a hhd evaluates the degree to which the patient can maintain the standing posture against the force exerted by the examiner and has the advantage that the measurements can be performed in less time than performance tests , although the examinees are required to be able to maintain a standing position without aid . at present , we are considering approaches and modifications that would allow the time required for the hhd assessment index to be reduced , by minimizing the measurement times to once per site because the present results showed good reliability . in addition , the score of the hhd assessment index is an interval scale that provides a broader range of options for statistical analysis15 . furthermore , the hhd assessment index is a disturbance load test , which may contribute to analyzing the causes of and mechanisms underlying balance impairment in the future . this study determined the reliability and validity of the hhd assessment index of standing balance in stroke patients . the criterion - related validity was evaluated based on correlations with the fbs , which is an established balance test with evidence of both reliability and validity . the results confirmed the reliability and validity of the hhd assessment index suggesting it to be a useful balance test not only for healthy , including elderly , people but also for stroke patients . it can be used for a wide variety of subjects11 , including healthy individuals who may have ceiling effects when conventional balance tests are employed2 , 3 , 16 and stroke patients with poor standing balance due to paralysis . we consider the hhd assessment index to be a potentially useful part of the test battery for balance employed in clinical practice . finally , the significance of measuring hhd evaluation indicators is considered to be that , different from fbs2 , 3 and the tinetti balance test ( tbt)16 , it is available even for healthy people who can not undergo the measurement because of the ceiling effect , it requires less time and is convenient , and it is an interval scale . its different from tug with the same scale as the interval scale is that it is available even for cases where the subject can stand erect but can not walk . its difference from other evaluation indicators , such as frt , is that it can sense the pressure on the sensor of the pressure receiver of hhd as a resistance that is the body s reaction to the subject . however , given that the hhd evaluation indicator is specialized only for standing balance and that the balancing ability during walking can not be measured , the author et al . consider that it is important to compare the processes as an interval scale by using other evaluation indicators .

pancreatic cancer represents the fourth leading cause of cancer - related death in the western world and still has a poor prognosis , in spite of the progresses achieved in cancer treatment over the last decades . surgical resection is considered the only curative treatment for localized pancreatic adenocarcinoma , although it is known that only 15 to 20% of patients are candidates for resection surgery . local recurrences are linked with poor prognosis and possible onset of severe symptoms , such as excruciating pain , jaundice and hemorrhage . the management of these lesions is challenging due to both the objective difficulties of redoing surgical resection with a curative intent and the common coexistence of distant metastases . a 58-year - old year lady underwent open distal pancreatectomy for a moderately differentiated pancreatic adenocarcinoma measuring 20x10 mm with no peripancreatic lymph node metastases , followed by adjuvant chemotherapy . she had been well without recurrence of cancer for six years following primary treatment , when she started to complain of moderate to severe abdominal pain , dyspepsia and weight loss . the patient was then sent for a contrast - enhanced computed tomography ( ct ) scan that showed an irregular mass located in the head of the pancreas ( figure 1 ) . a ct - guided fine - needle aspiration cytology confirmed the lesion to be a cancer recurrence . a fluorodeoxyglucose positron emission tomography / ct study showed intense radiotracer accumulation in the pancreatic head . since no signs of distant metastases were present at metastatic work - up , the patient underwent an exploratory celiotomy for salvage surgery . however , the cancer recurrence was deemed unresectable at the time of surgery because of tight adhesions and the lack of any cleavage planes with the hepatic artery and the portal vein . therefore , first - line chemotherapy with cisplatin - gemcitabine combination was administered . since the lesion in the pancreatic head had increased to 25x30 mm at the follow - up nonetheless , a ct scan revealed a progression of disease ( tumor mass maximum diameter 30x30 mm ) . fourteen months after the diagnosis of cancer recurrence the patient underwent ct - guided percutaneous cryoablation ( pca ) under local anesthesia and conscious sedation , using a tabletop argon gas - based cryoablation system ( galil medical , yokeneam , israel ) . two cryoprobes ( icesed ) were inserted under ct - guidance ( figure 2 ) into the tumor for double intraprocedurally freezing thawing cycles . under ct guidance , the needle s position and the ice ball dimensions ( figure 3 ) were assessed . we only observed a low ( and asymptomatic ) increase of serum amylase and lipase the day after the procedure , which returned to normal in five days . one month after the treatment the patient was asymptomatic and contrast - enhanced ct scan showed no enhancement of the treated lesion ( figure 4 ) . local recurrence following pancreatic resection for cancer occurs in 35 - 86% of patients operated on with curative intent . while local relapses are often detected in conjunction with disseminated disease , there is a smaller group of patients ( approximately 30% ) , where isolated local recurrence is observed without evidence of distant metastasis . to date , no standard treatment or supportive care guidelines exist for the management of this severe condition . systemic chemotherapy has historically resulted in a median survival rate of 5 - 11 months , and also radiation therapy has been shown to result in significant local control and to have some survival effects in selected patients . some authors suggest that redo surgery could be effective and prolong survival in selected patients ; however morbidity rates for repeated pancreatic resection can be associated to high morbidity rates due to adhesions , anatomical complexities and post - radiation fibrosis . moreover , patients with local recurrences are frequently not good surgical candidates because of the poor general conditions . in the case described herein , a re - resection was attempted , but unfortunately it was not possible to remove the recurrence located in the pancreatic head because of the adhesions and possible infiltration of the hepatic artery and portal vein . during the last ten years different minimally invasive ablative methods have been emerging for the palliative treatment of pancreatic cancer . cryoablation is an ablative method which has been widely used for the treatment of several benign and malignant solid tumors . in essence , its effectiveness is based on the specific cytotoxic effects of cold on tissue that produces both instant and delayed destruction of cellular ultrastructure at temperatures lower than 40c . ultrasound or ct - guided pca is a relatively new method used for the treatment of various solid malignancies . acceptance of pca for pancreatic malignancies has been delayed by concerns regarding both technical difficulties and risk of complications , in that the pancreas has a soft texture , has a profound location in the abdomen , and is close to structures such as the duodenum , portal vein , superior mesenteric artery , hepatic artery and common bile duct . however , recent advances in image - guided percutaneous techniques and instruments have led to a surge in the application of pca in patients with pancreatic cancer . in fact , some studies and literature reviews have shown the safety and feasibility of pca for unresectable or not amenable to surgery pancreatic cancer , although its use remains restricted to very selected cases in expert hands . the risk of vascular injuries from cold temperatures is hampered by the fact that the rapid blood flow in the large vessels close to the pancreas has a hot pool effect . optimal tumor freezing and prevention of injuries to adjacent organs can be obtained with intraprocedural monitoring using ultrasonography , ct , or magnetic resonance imaging . ultrasound can be used to guide pca , but posterior acoustic shadowing limits visualization . at ct - scan the frozen lesion appears as an ice ball that is hypodense with respect to unfrozen tissue . for these reasons , we used ct guidance to assess the size and the location of the ice ball during the procedure . possible minor and major adverse effects have been described after cryoablation of pancreatic tumor , the main ones being abdominal pain , fever , increased serum amylase , bleeding , and leakage of pancreatic juice . however , rates of severe complications due to cryoablation appear to be lower than observed in other ablative techniques . in our patient , the postoperative period was uneventful and the patient was discharged 2 days after the treatment . the majority of authors report the patients undergoing pca for pancreatic cancer under general anesthesia . it should be noted that we performed pca on this patient under local anesthesia and conscious sedation without any intraoperative complications . unfortunately , the patient was lost to follow - up after 3 months , thus it was not possible to evaluate further the clinical course and response to treatment . we were just able to notice that she was asymptomatic at the time of the one - month ct scan , hence we can assume that the procedure had a role in ameliorating her initial symptoms ( moderate to severe pain and dyspepsia ) . nonetheless , we believe that this case is worthy of attention , since pca , as well as other ablative procedures , has been gaining popularity as a minimally invasive palliative procedure in patients with unresectable locally advanced pancreatic cancer , and several authors reported its effectiveness and safety in selected patients . to the best of our knowledge , however , ours is the first description of the use of pca in the setting of an isolated local recurrence of pancreatic cancer . besides , the patient underwent pca with no complications and was discharged on postoperative day 2 . the case described herein , together with the evidence of the feasibility of cryoablation in locally advanced pancreatic tumors , suggests that pca under ct - guidance is feasible and safe , and deserves further attention in the multimodality treatment of patient with local recurrence after primary surgery for pancreatic carcinoma .

spindle epithelial tumor with thymus - like differentiation ( settle ) is a malignant tumor of the thyroid gland , which shows thymic or related branchial pouch differentiation . this tumor is believed to be derived from the third or fourth branchial pouch and thymic remnants . settle is regarded as a low - grade malignant neoplasm because of its slow - growing nature and protracted clinical behavior . to the best of our knowledge , we present the cytologic , histologic , and immunohistochemical findings and the review of literature including its differential diagnosis . a 22-year - old woman complained of a bulging neck mass at the primary clinic . she was diagnosed with papillary carcinoma on fine - needle aspiration at a local pathology laboratory . the ultrasonography demonstrated a well - defined hypoechoic mass , measuring 3.9 3.4 cm in the left lobe of the thyroid . the frozen section slide showed mostly papillary epithelial configuration intermixed with focal spindle cell component . the touch preparation slides showed tight clusters of spindle or ovoid tumor cells with papillary configuration . on the histologic examination , the tumor was a highly cellular biphasic tumor characterized by fasciculated spindle cells with streaming pattern and tubulopapillary epithelial structures . the epithelial cells of the tubulopapillary structures showed abundant cytoplasm and round to ovoid nuclei . the tumor cells were positive for cytokeratin , vimentin , c - kit , epithelial membrane antigen ( ema ) , and thyroid transcription factor-1 ( ttf-1 ) . however , the tumor cells were negative for thyroglobulin , calcitonin , cd99 , s-100 protein , cd34 , smooth muscle actin , hbme-1 , and galectin-3 . the spindle cells showed elongated and cigar - shaped nuclei with fine chromatin and inconspicuous nucleoli [ figure 1 ] . no intranuclear cytoplasmic inclusions or nuclear grooves were seen . after the operation , the patient is doing well without any evidence of recurrence or metastasis for 12 months . ( a and b ) the smears were highly cellular and showed a biphasic pattern composed of dense groups of spindle cells and intermixed epithelial clusters . the spindle cells revealed scanty cytoplasm and uniform , elongated , or cigar - shaped nuclei . ( c ) the epithelial cells showed abundant cytoplasm and variable sized oval nuclei with indistinct nucleoli . ( d ) the tumor showed a biphasic histologic pattern composed of a spindle cell component and a tubulopapillary epithelial cell component . ( a : 100 , b : 200 , c : 400 , d : h and e , 100 ) we experienced a touch preparation cytology of settle during the frozen section diagnosis and described the characteristic cytologic features . we searched reports including the cytologic findings of settle in pubmed and found only eight cases in the english literature . most reports have described the cytologic findings of settle as highly cellular smears composed of spindle cells and/or epithelial cells . the previously reported cases , along with their characteristic cytologic findings , are summarized in table 1 . the cytologic findings of settle described in the literature recently , recondo et al . reported a case of settle with a comprehensive review of the literature . grossly , settle usually presents as an encapsulated or partially circumscribed mass with grayish to tan cut surface . microscopically , settle shows a highly cellular biphasic pattern and is composed of cellular sheets , short fascicles , interlacing bundles or attenuated storiform arrangement of spindle cells , and glandular epithelial component . the nuclei of spindle cells are oval to elongated and characterized by distinct nuclear membrane , inconspicuous nucleoli , and evenly distributed chromatin . the glandular tumor cells show narrow tubular , tubulopapillary , trabecular , or pseudopapillary structures . immunohistochemically , the tumor cells show immunoreactivity for pan - cytokeratin , smooth muscle actin , c - kit and vimentin , and no immunoreactivity for thyroglobulin , calcitonin , s-100 protein , chromogranin , synaptophysin , cd34 , cd99 , and ttf-1 . because settle is a rare tumor and not often considered , only eight reports have described the cytologic features of settle in fine - needle aspiration cytology samples of settle . the cytology shows a highly cellular smear and a biphasic pattern composed of spindle cells and intermixed epithelial cells . as summarized in table 1 , the main differential diagnoses in cytologic smears of settle are a spindle cell variant of medullary carcinoma ( mc ) and synovial sarcoma ( ss ) . the smear of mc shows a mixed population of spindle , plasmacytoid , and epithelioid neoplastic cells . the neoplastic cells of mc have an abundant or moderate amount of fine granular cytoplasm . the nuclei show the neuroendocrine features with eccentric location in both epithelioid and spindle tumor cells . however , the cytologic features of ss reveal severe cytological atypia , many mitotic figures , apoptotic bodies , and necrosis . we described a case of settle of the thyroid gland with touch preparation cytologic findings . although the incidence is very rare , setlle should be included in the differential diagnosis when a spindle cell neoplasm is encountered in touch preparation cytology in young patients with a thyroid mass . the touch preparation cytology during the frozen section diagnosis may be helpful to confirm the diagnosis of thyroid cancer .

the human bone marrow is often evaluated in patients with various hematological disorders , including anemia , thrombocytopenia , pancytopenia , leukemia and other hematological malignancies . multiple bone marrow procedures ( bmp ) are often required in patients with hematological malignancies to guide their treatment . the instrument customarily employed , the jamshidi needle , is designed to yield both an aspirate and a marrow biopsy . the bmp has changed very little in the last 40 years and involves a manual rotary insertion of the jamshidi needle into the marrow cavity of the posterior aspect of the iliac bone . although local anesthesia for the skin , subcutaneous tissues and periosteum is universally administered , the bmp is regarded by patients and physicians alike as a painful and uncomfortable procedure . in addition , suboptimal specimens including dilute aspirates and small core biopsies are often obtained , limiting the diagnostic potential of the procedure . in addition , especially in heavy patients , the biopsy length is often suboptimal due to limitation of depth that can be reached in the bone by manual pressure . a new fda - approved device for performing bone marrows , the oncontrol bone marrow biopsy system ( obm ) was recently introduced by the vidacare corporation ( shavano park , tx , usa ) . the obm utilizes a battery - powered drill to insert the marrow needle into the iliac bone of adult hematology patients . initial clinical studies utilizing the obm system indicated that it was faster and easier to use for bone marrow aspirations than the traditional method . a few prospective studies comparing the obm with the standard bone marrow procedure ( sbm ) have been carried out to date . while the duration of the procedure has been consistently shorter , and the core samples larger for patients undergoing obm in these reports , no studies have been carried out in teaching hospitals to determine whether or not the obm system will be more readily mastered by hematologists - in - training . instruction of hematology fellows in the bmp technique has never been standardized , varies greatly between different fellowship programs and seldom receives high priority . as a result , fellows completing training are sometimes not well versed in the performance of the bmp and tend to avoid it in their post - fellowship careers . we conducted a prospective , randomized study to compare the obm procedure with the sbm procedure in adults . the length of marrow biopsy specimens ( a surrogate for marrow quality ) , aspirate quality and spicule content and procedure time were assessed objectively by the attending hematologist and pathologist . the patient , fellow and attending also completed questionnaires grading the pain , procedure difficulty , specimen quality and patient acceptability . the study protocol was approved by the biomedical research alliance of new york institutional review board . the obm system consists of a battery - powered driver and a biopsy needle set . the driver resembles a small hand - held drill , and powers a single lumen needle into the medullary cavity of the adult iliac bone . the needle set consists of two parts : an outer cannula , 11 gauge by 4 or 6 inches ( 102 or 152 mm ) long ; and a bevel - tip inner stylet- used to penetrate the cortex . the sbm device used in the study was typically a jamshidi bone marrow biopsy needle ( 11 gauge by 4 or 6 inches ) , which has a two - piece t - handle design , a trocar - tapered stylet point and a triple - crown cannula tip . all fellows satisfactorily completed at least one sbm and obm procedure under the supervision of an attending hematologist , before they were deemed certified to start enrolling to the study . after the initial randomization the fellow then alternated between obm and sbm procedures in a sequential fashion . randomization was performed for the fellows rather than the patients to minimize differences between individual fellows as to previous experience and variable aptitude . after giving informed consent , adult patients requiring bone marrow sampling procedures underwent either a sbm or an obm . bone marrow aspiration and core biopsy were obtained utilizing a one needle / one puncture approach . the fellows were observed and supervised throughout the procedure by an attending hematologist or research technologist . the primary endpoint of the study was the mean length in millimeters of the bone marrow specimens yielded by the two techniques . the measured length of the marrow biopsy specimen is a generally accepted surrogate for the quality of the marrow biopsy . it was chosen as the primary endpoint because that measurement was made in the pathology department by a person who had no knowledge of whether the specimen was obm or sbm and who was not involved in the study in any way . secondary endpoints included the skin to skin procedure time in seconds as well as other endpoints derived from the questionnaires . the questionnaires were completed by the patients , fellows and the attending hematologist / research technologist immediately after the procedure . the patient questionnaire included questions regarding the level of pain experienced , the patient - perceived ease / difficulty of the procedure and the degree of patient willingness to have a repeat bmp if medically recommended . the questionnaires completed by the fellows included questions on the patient 's level of pain , the ease / difficulty of the procedure and the perceived quality of the bone marrow aspirate and biopsy obtained . the questionnaires completed by the attending hematologist or by the research hematology technologist ( who observed the procedure ) included questions regarding the patient 's apparent level of pain , the ease / difficulty of the procedure and the perceived quality of the bone marrow aspirate and biopsy obtained . complications or adverse events were recorded during the procedure and at patient follow - up evaluation . statistical testing was conducted using sas version 9 for windows ( sas institute , cary , nc ) . continuous parameters were summarized and compared between groups using a 2 sample t - test . categorical parameters were summarized as proportions and compared using fisher 's exact test . because most fellows used obm and sbm multiple times , linear mixed effects models were fit to the data to evaluate differences between the oncontrol and standard methods and adjust for the potential correlation in repeated measurements from the same fellow . an interim analysis was planned after the first 51 patients were accrued to determine if significant endpoints had been reached to permit early termination of the study . two hospitals in the bronx , ny participated in the study , jacobi medical center and montefiore medical center . a total of 54 bmps ( 27 sbm and 27 obm ) were performed by 11 hematology fellows under the observation of 3 attending hematologists and 1 research technologist . the mean age of the 54 patients was 58.9 ( 15.1 ) years and 61.1% were male . the mean height and weight were 168.1 ( 11.0 ) cm and 77.9 ( 19.3 ) kg , respectively . for these demographic parameters , there was statistical homogeneity between the two groups ( table 1 ) . of the 54 patients in the study ( table 2 ) , 11 ( 20.4% ) had myeloma , the most frequently - occurring diagnosis . there was no significant difference in the frequency of diagnoses between the two groups ( p=0.563 ) . table 1patient demographics.demographicsbmobmpnumber of male / female16/1116/110.609mean age ( yearsstandard deviation)60.716.357.214.00.399mean height ( cmstandard deviation)167.410.3168.012.10.842mean weight ( kgstandard deviation)76.317.078.822.20.634body mass index27.25.627.76.10.755race / ethnicity : numbers of:0.730 black1410 hispanic912 asian23 white22 table 2patient diagnoses.diagnosissbmobmmyeloma74pancytopenia35lymphoma , non - hodgkin's33anemia25myeloproliferative disorder32monoclonal gammopathy of undetermined significance22myelodysplastic syndrome13thrombocytopenia21hodgkins lymphoma11acute myeloid leukemia10igm paraprotein10metastatic carcinoma10eosinophilia01 the primary study endpoint , the mean marrow biopsy length was significantly longer in the obm group ( 15.3 mm ) than in the sbm group ( 9.8 mm ) , p<0.003 ( table 3 ) . the mean procedure time , a secondary endpoint , was significantly shorter in the obm group ( 175 s ) compared to the sbm group ( 292 s ) , p<0.007 . table 3study results : meansstandard deviation.variableobmsbmpobjective device efficacy biopsy specimen length ( mm)15.37.39.86.70.003 * ( primary endpoint ) procedure time ( seconds)174.6105.1292.1210.00.007*subjective pain scores 010 perceived by patient4.72.85.92.80.11 reported by fellow3.22.24.92.70.002 * reported by attending2.91.74.52.40.008 * willingness by patient to repeat bmp1.02.22.93.50.03 * procedure ease / difficulty ( 010 ) reported by patient1.11.72.23.30.11 reported by fellow2.62.65.02.80.002 * reported by attending3.02.65.13.40.006 * perception of specimen adequacy ( 010 ) aspirate- fellow5.03.35.42.70.59 aspirate- attending4.93.65.63.10.47 core biopsy - fellow6.41.95.13.10.07 core biopsy - attending6.72.04.83.40.01 * number ( proportion ) of dry taps7.0 ( 25.0%)4.0 ( 15.4%)0.505*indicates statistical significance indicates statistical significance other secondary endpoints which significantly favored the obm group included the mean pain scores recorded by the fellows ( p<0.002 ) and by the attendings ( p<0.008 ) . in regard to the mean pain scores reported by the patients in the 2 study groups , a lower score was tabulated in the obm group ( 4.7 ) than in the sbm group ( 5.9 ) , but the difference was not significant ( p=0.11 ) . however , patients indicated a greater willingness to have a repeat obm ( 1.0 ) than a repeat sbm ( 2.9 ) , p<0.03 . both the fellows ( 2.6 vs. 5.0 , p<0.002 ) and attendings ( 3.0 vs 5.1 , p<0.006 ) perceived the obm to be easier to perform than the sbm . the attendings , but not the fellows , reported superior biopsy specimens in the obm vs. the sbm group . the superiority of the obm specimens was verified by objective blinded measurements in the pathology department . however , the fellows and the attendings scored the quality of the marrow aspirates as about equal . there were a greater number ( proportion ) of aspirates scored 0 ( dry tap ) in the obm group ( 7/25% ) than in the sbm group ( 4/15.4% ) , but the difference was not significant ( p=0.505 ) . there were no serious adverse events in either the obm or the sbm study groups . the needle had penetrated the cortex of the iliac bone , but the needle could not be detached from the driver in order to proceed with the aspiration and biopsy . a minor adverse event occurred in a 56 year old man hospitalized for gastrointestinal bleeding , cirrhosis with portal hypertension , thrombocytopenia and coagulation abnormalities . five days after an obm , there was a spontaneous local drainage of a soft tissue hematoma from the posterior iliac marrow biopsy site . this responded to local and systemic therapy ; the patient improved and was subsequently discharged . another minor adverse event occurred in a 50 year - old female undergoing an outpatient sbm , which was successfully completed . immediately after the procedure , the patient complained of numbness and weakness in the right lower extremity and was unable to stand . the patient was transferred via wheel chair to the emergency department , where an aortic sonogram and an echocardiogram were performed and reported normal . two hours after the sbm , the patient was examined by a neurologist who found that the patient had completely recovered . the consultant felt unable to distinguish between an excess of local anesthetic adjacent to the right sciatic nerve versus an anxiety reaction to a difficult procedure . since the introduction of the jamshidi needle for bmps 40 years ago , few technical advances have been made in the field . commercial introduction of the obm , a battery powered drill with attached needle for bone marrow aspirations and biopsies , followed fda approval of the device in 2007 . preliminary studies indicated that the obm was safe and yielded adequate aspirates in a short period of time . a prospective randomized study by berenson et al . , comparing obm and sbm , indicated that the obm yielded bone marrow biopsy specimens of significantly greater volume , in a shorter period of time , with less residual pain in adult patients . a prospective , randomized study of obm versus sbm by swords et al . , using experienced operators , indicated that significantly longer biopsy cores were obtained with the obm method . the mean biopsy lengths obtained in that study ( 13.1 mm obm and 8.2 mm sbm ) were very similar to those obtained in the present study ( 15.3 mm obm and 9.8 mm sbm ) , as shown in table 3 . thus , the primary endpoint in the present study confirmed the observation that the length of the marrow biopsy is significantly longer with the obm method than with the sbm method . the length of the marrow biopsy is widely regarded as a surrogate for biopsy quality . since the only reason for subjecting patients to this painful procedure is to obtain diagnostic information , the device which yields the most must be regarded as superior . the fact that the primary endpoint of the study , the length of the marrow core , was objectively determined in the pathology laboratory ( which had no involvement in the study and no information as to how individual specimens were obtained ) , underscores the objectivity and validity of the conclusion . an element of operator bias appeared unlikely , since neophyte hematologists are maximally motivated to obtain optimal marrow specimens . a pre - planned interim analysis detected significant differences , and , as a result , the study was terminated early . the present study is the first to be conducted in teaching hospitals utilizing inexperienced operators , i.e. hematologists - in - training . the fact that the results were similar whether the operators were experienced or not confirms the ease with which the obm technique is mastered . indeed , the majority of the participating fellows expressed a preference for the obm method which suggests that obm is a better training tool for teaching programs . the key secondary endpoint of the study was the duration of the procedure , skin - to - skin . the mean procedure time was significantly shorter with the obm ( 174.6 s ) than with the sbm ( 292.1 s ) . in other words , even with inexperienced operators , obm changed bmp from a 5 minute procedure to a 3 minute procedure , on an average . other comparative studies , employing experienced operators , have reported even faster obm procedure times , and all have demonstrated significantly shorter times compared to a sbm control group . according to kuball et al patients are generally willing to undergo the bmp and a reasonable level of pain , providing that the procedure time is relatively short . pain scores , as reported by the patients , the fellows and the attending hematologists/ research technologist were also secondary endpoints of the study . the patient - reported pain scores showed a trend favoring obm , but the difference was not significant ( p=0.11 ) . a similar result was reported by berenson et al who opined that the overall patient - reported pain score is largely dominated by the sharp pain of marrow aspiration . both the fellows ' and the attendings ' perceptions of patient pain were significantly less with obm compared to sbm , a result subject to observer bias . . these could include identifying patients at risk for significant pain during bone marrow procedures , discussing analgesia and even sedation options with patients including associated risks , and possibly re - dosing during the procedure , particularly if multiple punctures are required . interestingly , the obm patients expressed a significantly greater willingness to have a repeat bmp than the sbm patients ( p<0.03 ) . that result may be criticized as possibly subject to physician influence . however , another possible explanation for the higher level of patient willingness to have a repeat procedure may be the difference in degree of procedure difficulty between the two procedure types . as shown in table 3 , the fellows did have more difficulty with the sbm than with the obm procedure . other secondary endpoints included the scores assessing ease / difficulty of the procedure by patients , fellows and attendings . on a scale of 010 for procedure difficulty however , the difference was not statistically significant , owing to the wide standard deviation in both groups . on the other hand , the fellows and attendings rated the obm procedure as significantly less difficult than the sbm , a result subject to observer bias . the questionnaire scores regarding quality of marrow aspirates and biopsies from the fellows and the attendings / research technologist did not show notable differences between the obm and the sbm groups and were also subject to observer bias . refusal by patients to undergo bmps , especially in diseases like myeloma , leukemia and lymphoma , lead to delays in the diagnosis and treatment which may have fatal consequences . any device , such as obm , which promotes greater patient acceptance of a painful , but necessary , procedure may be anticipated to improve quality of care and to enhance favorable clinical outcomes . the data presented in this and other obm studies leave little question as to the superior effectiveness of obm when compared to sbm . in a study involving 767 patients , bishop et al reported that only 42% of bone marrow biopsy specimens were long enough for definitive diagnosis . first , the battery powered driver has a negligible cost since it can be used for about 500 procedures . second , the obm sterile disposable trays retail for about $ 40 more than many sbm trays . but the lower cost of sbm trays is offset by the inferior quality of the marrow specimens , delays in diagnosis and treatment , the necessity of repeating some bmps , to say nothing of the longer procedure time and the inferior patient acceptance with the sbm . the long term costs and consequences of training future hematologists with inferior devices are not readily calculable . as with other device studies , this study was limited by the absence of blinding of patients , operators or observers . the noise and the vibration of the obm driver limited ability to blind the study for the patients ; the operator or observer could not be blinded for obvious reasons . although the primary endpoint ( biopsy length ) and a secondary endpoint ( procedure time ) were objectively determined , other secondary endpoints derived from the various questionnaires could not be free of observer bias or physician influence . there was also potential operator bias in the primary endpoint since bone penetration by the operator might be influenced by the fellow 's personal preference for the sbm vs. obm . another limitation was including data from multiple marrows for different fellows , as opposed to one marrow of each type per fellow , which would have taken years to complete . there was also variability in the number of marrows performed by each fellow , however this was partially offset by each fellow alternating between the sbm and obm . some senior fellows also had greater prior experience with the sbm before they were certified for the study . however greater experience with the sbm would have shifted the results in favor of the sbm rather than obm . finally , a detailed , blinded comparison of the pathologic quality of obm versus sbm specimens was not carried out , owing to the omission of a specific consent phrase in the patient consent form . nonetheless , blinded observations of the obm and the sbm biopsy specimens by the hematopathologists did not reveal any notable differences in the amount of marginal necrosis ( as might be caused by heat denaturation ) , hemorrhage , aspiration artifact , or crush artifact . both obm and sbm biopsy specimens displayed variable aspiration artifact , which was expected since the study design mandated a one puncture / one needle approach . as with other device studies , this study was limited by the absence of blinding of patients , operators or observers . the noise and the vibration of the obm driver limited ability to blind the study for the patients ; the operator or observer could not be blinded for obvious reasons . although the primary endpoint ( biopsy length ) and a secondary endpoint ( procedure time ) were objectively determined , other secondary endpoints derived from the various questionnaires could not be free of observer bias or physician influence . there was also potential operator bias in the primary endpoint since bone penetration by the operator might be influenced by the fellow 's personal preference for the sbm vs. obm . another limitation was including data from multiple marrows for different fellows , as opposed to one marrow of each type per fellow , which would have taken years to complete . there was also variability in the number of marrows performed by each fellow , however this was partially offset by each fellow alternating between the sbm and obm . some senior fellows also had greater prior experience with the sbm before they were certified for the study . however greater experience with the sbm would have shifted the results in favor of the sbm rather than obm . finally , a detailed , blinded comparison of the pathologic quality of obm versus sbm specimens was not carried out , owing to the omission of a specific consent phrase in the patient consent form . nonetheless , blinded observations of the obm and the sbm biopsy specimens by the hematopathologists did not reveal any notable differences in the amount of marginal necrosis ( as might be caused by heat denaturation ) , hemorrhage , aspiration artifact , or crush artifact . both obm and sbm biopsy specimens displayed variable aspiration artifact , which was expected since the study design mandated a one puncture / one needle approach . the results of this first prospective , randomized trial in two teaching hospitals comparing bmps in obm and sbm patients , as performed by novice hematologists , indicate that significantly longer and better quality marrow biopsy cores may be obtained in a much shorter period of time , and with less patient pain when using the obm device . adverse events were inconsequential . the slightly greater expense of obm appears justifiable in balance . wider acceptance of obm as a preferred bmp device in teaching hospitals may lead to greater acceptance of bmp as a necessary procedure by patients , to improvements in the diagnosis and treatment of hematology/ oncology patients and better training for fellows .

noncommunicable diseases ( ncds ) pose a significant global burden in both developed and developing countries . hypertension ( htn ) is a significant cardiovascular disorder in sub - saharan africa and is raising epidemic representing a major public health concern . although hypertension is a growing health concern in uganda , and like many under resources environments , reliable data on the incidence and its prevalence are lacking . however , it is estimated that , by 2025 , 41.7% of males and 38.7% of females in sub - saharan africa will develop high blood pressure ( hbp ) . in uganda , a country of 33 million , with an overwhelming morbidity and mortality rate associated with infectious diseases such as hiv / aids , tuberculosis , and malaria , is now battling increasing rates of hypertension . the prevalence of hypertension in uganda has risen dramatically and is estimated to range from 22.5% to 30.5% in the adult population [ 3 , 4 ] . it is further estimated that nine out of ten people ( 90% ) are unaware of their hypertensive condition . undetected , poorly managed and uncontrolled hypertension is a major primary and public health care problem contributing to the ncd morbidity and mortality rates in uganda . high blood pressure is a known contributor to cardiovascular events such as cerebral vascular accidents , myocardial infarctions , congestive heart failure , peripheral vascular disease , and chronic renal failure [ 57 ] . if untreated or unmanaged , hypertension could lead to significant disability adjusted life years ( dalys ) , estimated at 8.1% in less developed countries such as uganda [ 8 , 9 ] . the disease burden of hypertension is also associated with poor quality of life and loss of productivity , consequently leading to low social economic status . this can potentially affect the social economic status of the whole country [ 7 , 10 ] . complicating this health crisis , uganda is also faced with a woefully shorthanded health work force with estimates of 1.2 physicians and 13.1 nurses and midwives per 10,000 patients [ 3 , 10 ] . nurses and midwives make up the largest part of the health workforce in uganda and play a major role in patient care including screening , taking vital observations , and patient teaching . moreover , the nurses ' educational role has been expanded to make a major contribution to improved patients ' lifestyle behaviors , physical activity , weight , stress relief , alcohol intake , medication adherence , and self - efficacy [ 5 , 8 , 1114 ] . using the johns hopkins nursing evidence - based practice ( jhnebp ) tools for appraising the strength of evidence for both research and nonresearch studies , a comprehensive and rigorous literature review was conducted on hypertension and effective nurse - led interventions [ 3 , 8 , 12 ] . ten randomized control trials ( rcts ) , 2 systematic reviews of rcts without meta - analysis , 3 systematic reviews of rcts with meta - analysis , 1 descriptive analysis , and 1 nationally recognized expert committee were reviewed . the majority of the systematic reviews ( level 1a - b ) in the review revealed that nurse - led care for blood pressure control was effective . it should be noted that education alone directed to patients or health professionals was unlikely to influence control of high blood pressure but rather a multifaceted intervention approach was determined to be beneficial . the literature further suggested that determining the correct nursing intervention and appropriate treatment plan to reduce blood pressure for individual patients depended on nurses ' basic understanding of the pathophysiology of hypertension , history taking , risk assessment , interpretation of lab results , and the cardiac system [ 1517 ] . the need to duly prepare nurses and physicians working with hypertensive patients has been heavily emphasized to improve care delivery in the developing world [ 16 , 18 , 19 ] . the world health organization ( who ) has developed comprehensive hypertension guidelines for low and middle income countries that focus on risk assessment , patient education on diet , weight gain , and other lifestyle changes . these guidelines have not been disseminated nor utilized by ugandan nurses during the provision of nursing care for hypertensive patients leading to significant variability in nursing practice and missed opportunities for identification and management of hbp . little is known about studies that address the capacity of the ugandan nurses in regard to early detection , risk assessment , and patient education on diet , physical activity , and other lifestyle changes for hypertensive patients . this paper draws on quantitative data collected in a nurse - led hypertension pilot project that aimed at enhancing nurses ' knowledge , attitude , and skills in early detection , risk assessment , and patient education using culturally adapted nursing hypertension management program based who - ish guidelines and protocols for low and middle income countries . this was a one group pre - post design using a convenience sample of nurses . the study protocol was approved by the mulago hospital research and ethics committee having been determined not to be human subjects ' research ( mrec : 248 ) . seven ( 7 ) nurses from mulago hospital 's medical outpatient clinic participated in the pilot study . this outpatient clinic is part of mulago hospital which is a national referral and teaching hospital with a bed capacity of 1500 beds . mulago hospital 's medical outpatient clinic is managed by seven ( 7 ) nurses , 4 physician assistants , and one consulting physician who attend to an average of 300 patients / day . of 300 patients , 83% have common infectious diseases such as malaria , respiratory , urinary tract infections , and skin diseases . the educative interventions were implemented over 3 months and consisted of a hypertension nursing educational program developed and adapted from the who - ish training manual for cardiovascular risk assessment and management ( 2009 ) for low and middle income countries . the evidence based interventions focused on improving nurses ' knowledge , skills , and attitudes in hypertension management . knowledge on hbp prevention , detection , risk assessment , patient education , and management was addressed by providing twenty - two ( 22 ) hours of didactic classroom sessions . self - directed learning was bolstered using a cd - rom on hypertension management previously developed and culturally adapted from the joint makerere university and johns hopkins university team . participants were also engaged in 1 : 1 clinic sessions that focused on using the who risk prediction charts , accurate manual bp measuring techniques , motivation interviewing for patient education on lifestyle changes , and use of the who - ish protocols . english translation to luganda ( local language ) was undertaken to ensure all the participating nurses were at the same level of understanding . participant knowledge , skills , and attitudes were measured using pre - post interventions tools . knowledge was measured using a 10-item multiple choice and multiple response instrument developed by the investigator . the instrument was developed specifically for this study , and asked the nurse participants to respond to questions related to a written case study of a hypertensive ugandan man with diabetes comorbidity . before its use , the pre - post knowledge intervention tool was sent to two hypertension experts for review ; corrections were incorporated into the final version of the tool and later piloted it with 5 nurses from another nearby hospital . based on the case study , the questionnaire was composed of questions related to hypertension diagnosis and classification , risk assessment , risk prediction , patient education , and management . in view of being comprehensive , accurate manual blood pressure measuring skills were measured with a 12-step standardized blood pressure measurement techniques skills checklist retrieved from tennessee primary care association ( tpca ) web page . nurses ' attitudes toward hypertension were measured by using an adapted version of the respondents ' attitude to assessment strategies for prevention of high blood pressure . the respondents ' attitude to assessment strategies for prevention of high blood pressure is a 10-item pre - post attitudes tool which assessed the respondents ' attitudes to assessment strategies for prevention of hbp . isioma of nigeria to measure nurses ' knowledge and attitudes toward prevention and management of high blood pressure in primary health care centers in delta state , nigeria , and was adapted with permission for use in this study . the tool is a 5-point likert scale type with responses ranging from strongly agree to strongly disagree . the attitude pre - post tool like the knowledge assessment tool was also sent to the same experts and same nurses before using it with the study participants . pre - post interventions data on knowledge , skills , and attitudes was entered into the statistical package for social sciences ( spss ) software version 16 . in this pilot study , all the seven nurses working in the outpatient clinic were eligible and invited to participate in the study . participants were informed of the content on evidence based hypertension management to be completed in 3 months . participants were also provided with an opportunity to opt out in case they were uncomfortable . all seven female nurses from the medical outpatient clinic agreed to participate in the pilot study . their ages ranged from 37 to 53 years with a mean age of 46.7 years . approximately 70% received their nursing education in uganda with at least a diploma level qualification . mean of years of experience was 2.8 4.1 and less than half ( 42.9% ) had attained a nursing officer ( no ) position , the highest nursing position at the outpatient clinic ( table 1 ) . there was a significant difference in the proportion of nurses who obtained satisfactory answers on the knowledge pre - post interventions test after the educative interventions . paired sample t - test indicated a 32% knowledge increase ( p < 0.009 ) . while the small sample size precluded analysis for each item , it was clear that there was a marked improvement in the nurses ' knowledge associated with bp classification , patient education that focused on therapeutic lifestyle changes such as limiting or stopping alcohol intake quitting tobacco use , 30 minutes exercises , losing weight , and eating 400 g of fruits . similarly , a marked increase in participants ' knowledge was observed on risk assessment that included taking blood for cholesterol and serum proteins levels as well as determining the recommended hypertension first line drug used in uganda . overall , there was an observed small change in the proportion of nurses who could use of the risk prediction chart at postintervention ( table 2 ) . there was a significant difference in the proportion of nurses who correctly performed the required skills as regards accurate manual bp measurement on the standardized bp measurement techniques skills checklist . mean test scores on the accurate bp measurement skills prior to the interventions were 62.7% and after interventions were 94.3% . paired sample t - test indicated a 50% bp skills increase ( p < 0.000 ) among participating nurses . due to small sample size , step by step analysis was not conducted ( tables 3 and 5 ) . there was a significant difference in the proportion of nurses with positive attitudes on the adapted respondents ' attitude to assessment strategies for prevention of high blood pressure tool . mean test scores on the attitude test prior to the interventions were 22.4 and after interventions were 39.4 . paired sample t - test conducted on the means indicated a 76% increase and improvement in attitudes about assessment strategies used to prevent and treat hbp ( p < 0.002 ) . similarly , while the small sample size precluded analysis for each item on the attitude pre - post test , it was clear that there was a marked improvement in nurses ' attitudes specifically in regard to patient education , assessment of alcohol and tobacco consumption , importance of documentation of htn patients ' medication , and the need for weight reduction ( tables 4 and 5 ) . in uganda , nursing education occurs at the certificate and diploma level with qualifications requiring two to three years of education . curriculum that focuses on the three competencies , namely , knowledge , attitudes , and skill acquisition , as regards hypertension diagnosis and management occurs during the first year of education . however , the results of the knowledge , skills , and attitudes pretest suggest that nursing curricula should be strengthened . particularly , this includes evidence based practices to prevent , detect , health - educate , and manage hbp . the concept of evidence based practice ( ebp ) and translation of best practices is novel to uganda and has not been incorporated into the nurses ' curricula . in this study , as the nurses were introduced to the concept of ebp with the help of the who - ish guidelines and protocols , they became more aware of the available strategies to lower hbp . the introduction of the hypertension risk prediction charts and risk assessment as an element of ebp were challenging to the participating nurses . assessing a 10-year risk for cardiovascular diseases using the risk prediction charts is vital and therefore extra effort is required to continue enhancing nurses ' knowledge and skills in the use of such evidence based tools . it was evident from the study that the participating nurses ' performance on the pre - post interventions test and specifically in regard to risk assessment appeared to be enhanced by use of a multimodal nursing education intervention . the world health organization ( who ) has developed hypertension guidelines for use in low and middle income countries . one of the challenges in changing nursing knowledge and skills is the development of standardized resources . the resources available from who are readily available , evidence based , and easily disseminated . when appropriately introduced and culturally adapted , this is an invaluable resource that can assist as standardized protocols for hypertension prevention , risk identification , and risk management in a given population . in this study and prior to the interventions , none of the participating nurses were aware of any existing guidelines or resources be it local , national , or international to facilitate clinical decisions related to hypertension drug treatment . this is because ugandan nurses lack accessibility and availability to resources related to guidelines and protocols for hypertension management . access to best care practices is critical to improve and support professionalism of ugandan nurses and is required for better patients ' outcomes . it was clearly observed in this study that the commitment and enthusiasm of the nurses ' ability to learn new approaches to care can not be overstated . antidotally , there was improvement in nurses ' team cohesion , motivation , and self - efficacy associated with the introduction of a multimodal education approach . therefore , the concept of team cohesion and team skill building among nurses caring for patients with chronic diseases should further be explored . this may be reflected in the significant improvement ( 76% ) of positive attitudes regarding evidence based interventions for hypertension management . additional areas to explore are nursing satisfaction with the new professional roles as well as self - efficacy . lastly , the central role of the patient in terms of satisfaction with care , motivation to engage in therapeutic lifestyle changes , and improved outcomes are important elements to assess in order to provide additional evidence necessary for improved nursing practice . the positive attitude change when used in the enhancement of patient education role forms part of the various evidence based strategies to reduce hbp [ 17 , 22 ] . it is evident that using a multimodal approach to address various nurse related practice gaps is feasible and could be beneficial when implemented for other chronic diseases with a strategy to regularly update the nurses ' knowledge , skills , and attitudes as regards translation of best evidence into their practice for better patients ' outcomes . findings from this pilot study highlight the importance of using an evidence based guideline to improve nurses ' knowledge , skills , and attitudes in blood pressure management . risk assessment knowledge and skills must be emphasized during any educational intervention and organized to accentuate short , medium , and long term benefits of hbp detection , prevention , and management . evidence based strategies that improve nurses ' capacity to manage hbp in a low resource primary setting are also imperative . rolling out of the study intervention could improve nurses ' practices especially those working in outpatient care settings . self - efficacy and barriers to gaining knowledge , skills , and attitudes among nurses need to be explored . future research could focus on other groups of nurses and not necessarily working in outpatient clinics to overcome sample size issues and compare hypertension knowledge , skills , and attitudes in other areas of specialization . similarly , there is need to identify and select valid and reliable instruments for use in assessing outcome measures . after 3 months of implementing the nurses ' hypertension management educative interventions program , knowledge , skills , and attitudes regarding prevention , detection , risk assessment , patient education , and hbp management increased significantly . the pilot study demonstrated the feasibility of implementing a multimodal evidence - based educational intervention in a low resource environment .

application of amplazer occluder has become an effective and conventional way in percutaneous treatment of congenital heart disease such as ventricular septal defect ( vsd ) . there are rare complications reported , although sometimes cardiac conduction abnormalities , for example , may occur . we met a case of congenital heart disease , got persistent complete left bundle branch block ( clbbb ) which leaded to increased left ventricle and decreased left ventricular systolic function after vsd treatment by amplazer occluder , and finally the patient got heart function restored by cardiac resynchronization therapy ( crt ) . a 54-year old female with perimembranous ventricular septal defect was hospitalized for transcatheter closure of the defect . echocardiogram showed left ventricular end - diastolic diameter ( lvedd ) of 53 mm , left atrium of 44 mm , left ventricular ejection fraction of 65% , congenital heart disease , and perimembranous ventricular septal defect of 4 mm with the formation of aneurysm of the membranous septum ( left to right shunts ) . venous methylprednisolone was administered to the patient for three days , and ecg showed that the clbbb disappeared and qrs wave width was normal . echocardiography showed lvedd of 53 mm , left atrium of 45 mm , and left ventricular ejection fraction of 70% . ecg at more than two months after the procedure showed sinus rhythm , normal qrs wave , and left electrical axis ( figure 1a ) . however , three months after the procedure , ecg showed clbbb and premature ventricular contraction ( figure 1b ) . after three months , ecg showed persistent clbbb ( qrs wave width , 176 ms ) , ( figure 1c ) . ( a ) : two months after closure of perimembranous ventricular septal defect showing normal sinus rhythm , normal qrs wave , and left electrical axis ; ( b ) : three months after closure of perimembranous ventricular septal defect showing sinus rhythm , complete left bundle branch block , occasional premature ventricular contraction , and left electrical axis ; and ( c ) : more than four months after closure of perimembranous ventricular septal defect showing sinus rhythm , complete left bundle branch block ( qrs wave width , 176 ms ) , and left electrical axis . nine months after persistent clbbb , the patient was admitted into our hospital because of her chest distress , palpitation , and sweating at daily activities . after admission , ecg showed sinus rhythm , clbbb ( qrs wave width , 174 ms ) , and left electrical axis . cardiac echo showed left atrium ( 45 mm ) and lvedd of 68 mm and left ventricular end - systolic diameter ( lvesd ) of 56 mm with reduced left ventricular ejection fraction of 37% ( figure 2 ) . the 6-min walk distance was 155 m , and the blood brain natriuretic peptide ( bnp ) level was 480 pg / ml . the patient was recommended to receive crt because she exhibited clbbb and reduced left ventricular ejection fraction . moreover , beta - blocker metoprolol and angiotensin - converting enzyme inhibitor benazepril were administered to the patient . coronary sinus angiography showed that the lateral cardiac vein was a good target to place the left ventricular lead . we employed attain starfix model 4195 ( medtronic , inc . , minneapolis , mn , usa ) , an active fixation electrode , with deployable lobes to fix the lateral vein . the starfix acute pacing threshold was 0.7 v , lead impedance was 554 ohms , and slope was 1.2 . was placed in the right ventricular apex , with a pacing threshold of 0.6 v , impedance of 868 ohms , r wave amplitude of 10.5 mv , and slope of 0.8 , without phrenic nerve stimulation ( pns ) at 10 v pacing . a j - shaped atrial lead ( capsure sense model 4574 , medtronic , inc . ) was placed at the right atrial appendage , with measured pacing threshold of 0.4 v , slope of 0.2 , p wave amplitude of 2.0 mv , and lead impedance of 512 ohms . the crt device was implanted with syncra model c2tr01 ( medtronic , inc . ) , ( figure 3 ) . ( a ) : left ventricular end - diastolic diameter of 68 mm with reduced left ventricular ejection fraction of 37% nine months after persistent complete left bundle branch block ; and ( b ) left ventricular end - systolic diameter of 56 mm nine months after persistent complete left bundle branch block . the patient 's symptoms on palpitations and chest distress after the procedure improved compared with those before the procedure . ecg showed double ventricle pacing rhythm and qrs wave width of 136 ms at 87 beats / min ( figure 4 ) . one week after crt , echocardiography showed an enlarged left atrium and left ventricle , as well as left ventricular ejection fraction of 41% . six months after crt , the patient was reviewed in our out - patient clinic . the echo showed left atrium of 44 mm , lvedd of 61 mm , lvesd of 45 mm , and left ventricular ejection fraction of 50% ( figure 5 ) . ten months after crt , the echo showed left atrium of 44 mm , lvedd of 61 mm , lvesd of 43 mm , and left ventricular ejection fraction of 53% . the 6 min walking distance was 325 m , and the blood bnp level was 81 pg / ml . conduction block after transcatheter closure of vsd may result in some different types of conduction blocks , including left bundle branch block . bundle branch block and atrioventricular block occurs in 3.5% to 8.6% of cases. most atrioventricular block and left bundle branch block are transient . left bundle branch block has been proven to induce left ventricular remodeling and cause heart failure . , for clbbb patients , the left ventricular motion shows significant delay and is uncoordinated . simultaneously , the ef value and ejection time of the left ventricle decrease , suggesting that the systolic function of the left ventricle is impaired . vaillant , et al . reported that heart failure may develop over a mean of 11.6 years of left bundle branch block . in our case of persistent clbbb for nine months , the patient exhibited the symptoms of heart failure and a significant decrease in the left ventricular enlargement with left ventricular ejection fraction after nine months . left bundle branch block - induced left ventricle enlargement is referred to as left bundle branch block - induced cardiomyopathy . ( a ) : posterior - anterior ; ( b ) : rao 30 degree ; and ( c ) : lao 45 degree . ( a ) : left ventricular end - diastolic diameter of 61 mm with left ventricular ejection fraction of 50% six months after cardiac resynchronization therapy ; ( b ) : left ventricular end - systolic diameter of 45 mm six months after cardiac resynchronization therapy . , multi - center randomized clinical research miracle and its follow - up study confirmed that both left ventricular end - systolic and end - diastolic volumes decrease significantly six months after crt , as demonstrated by doppler echocardiography . the effect of crt on cardiac structure and function can improve the symptoms of heart failure and survival rate . doppler echocardiography showed a decrease in lvedd and blood bnp levels , and increase in left ventricular ejection fraction and 6 min walking distance . this case demonstrated that persistent clbbb for nine months after transcatheter closure with vsd amplatzer occluder might lead to left ventricular enlargement and a significant decrease in left ventricular systolic function . crt corrected the left and right ventricular dyssynchrony complicated by clbbb , decreased lvedd , and increased left ventricular ejection fraction , thereby improving the patient 's heart function .

anthrax is a rare , potentially fatal zoonotic disease caused by the bacterium bacillus anthracis , which can infect both animals and humans . infection via inhalation of bacillus anthracis spores can result in a mortality rate of up to 96% . major routes of exposure and subsequent infection have been confirmed through skin contact with , or the inhalation or ingestion of , bacillus anthracis spores . although west africa has the highest incidence in the world , anthrax is also a significant problem in central america , romania , greece , and turkey [ 46 ] . cutaneous anthrax , the most common form of the disease , accounts for 95% of anthrax cases . the most common areas of exposure are the hands , arms , face , and neck . unfortunately , the diagnosis of cutaneous anthrax can be difficult , particularly in endemic regions , because of its variable clinical phenotype . adenosine deaminase ( ada ) , an enzyme present in red blood cells and blood vessel walls , catalyzes the irreversible hydrolytic deamination of adenosine to inosine and 2-deoxyadenosine to 2-deoxyinosine . inosine and 2-deoxyinosine are converted to hypoxanthine , xanthine and , finally , to uric acid . this enzyme is considered a suitable biochemical marker of cell - mediated immunity . ada has been accepted as an important enzyme in the proliferation and differentiation of t - lymphocytes , as well as for the maturation and function of blood monocytes and macrophages . its levels are 10 times higher in t - lymphocytes than in erythrocytes , and it is frequently considered a noninvasive diagnostic test for tuberculosis , with 90100% sensitivity and 89100% specificity . the aim of this study was to investigate serum ada activity and other inflammatory markers in patients diagnosed with cutaneous anthrax in the van region of turkey , where the disease is prevalent . all patients ( 5 males and 11 females ) were diagnosed with cutaneous anthrax at outpatient clinics of the department of infectious diseases and clinical microbiology ( medical faculty , yuzuncu yl university , van , turkey ) in a 2-month period ( 1 november31 december 2013 ) and 17 healthy controls ( 11 males and 6 females ) were without a history of chronic or recurrent disease and had normal physical examination results . all of the cutaneous anthrax cases were characterized by evolving skin lesions and had a history of animal contact . the diagnosis of cutaneous anthrax was based on dermatologic findings , including papules from the vesicular stage ; pruritic ulcers covered by characteristic black eschar ; and edema , erythema , or necrosis without ulceration . cases were confirmed by a positive smear or by isolation of bacillus anthracis in clinical specimens . bacillus anthracis isolates were identified on the basis of conventional methods , such as gram - positive bacilli with spores present in the smear , the presence of a capsule , the lack of motility , or catalase positivity . all subjects were informed about the study and written consent was obtained from each subject . the exclusion criteria included a history of alcohol abuse , habitual smoking , intravenous drug abuse , pregnancy , antioxidant supplement use , hypertension , diabetes mellitus , liver or renal disease , rheumatoid arthritis , pulmonary disease , and coronary artery disease . blood samples were collected in tubes at 8:00 a.m. and 11:00 a.m. after an overnight fasting period and were immediately stored at 4c . peripheral blood leukocyte , lymphocyte , neutrophil and monocyte counts , and erythrocyte sedimentation rates ( esr ) were measured . the serum was then separated from the cells by centrifugation at 3000 rpm for 10 min , and c reactive protein ( crp ) levels were measured . the serum samples were stored in plastic tubes at 80c and used to analyze ada activity . serum ada activity was measured with an enzymatic spectrophotometric method determined by giusti and galanti , which is based on the direct measurement of ammonia formation , a byproduct that forms when ada is in the presence of excess adenosine . the serum crp levels were determined by using commercially available assay kits ( roche , mannheim , germany ) with an autoanalyzer ( roche / hitachi modular p-800 , mannheim , germany ) . parameter comparisons of patients and healthy controls were performed using student s t - test . the results were considered to be statistically significant when the p - value was less than 0.05 . the exclusion criteria included a history of alcohol abuse , habitual smoking , intravenous drug abuse , pregnancy , antioxidant supplement use , hypertension , diabetes mellitus , liver or renal disease , rheumatoid arthritis , pulmonary disease , and coronary artery disease . blood samples were collected in tubes at 8:00 a.m. and 11:00 a.m. after an overnight fasting period and were immediately stored at 4c . peripheral blood leukocyte , lymphocyte , neutrophil and monocyte counts , and erythrocyte sedimentation rates ( esr ) were measured . the serum was then separated from the cells by centrifugation at 3000 rpm for 10 min , and c reactive protein ( crp ) levels were measured . the serum samples were stored in plastic tubes at 80c and used to analyze ada activity . serum ada activity was measured with an enzymatic spectrophotometric method determined by giusti and galanti , which is based on the direct measurement of ammonia formation , a byproduct that forms when ada is in the presence of excess adenosine . the serum crp levels were determined by using commercially available assay kits ( roche , mannheim , germany ) with an autoanalyzer ( roche / hitachi modular p-800 , mannheim , germany ) . parameter comparisons of patients and healthy controls were performed using student s t - test . the results were considered to be statistically significant when the p - value was less than 0.05 . the demographic characteristics of the patients with cutaneous anthrax and the controls are shown in table 1 . there were no significant differences between the groups with respect to age and sex ( p>0.05 ) ( table 1 ) . blood leukocyte counts were significantly higher in the patients with cutaneous anthrax compared with the controls ( p=0.037 ) . although lymphocyte and neutrophil counts were higher in the patient group , there were no statistically significant differences ( p>0.05 ) . esr and crp levels were significantly higher in the patients with cutaneous anthrax compared with the controls ( p<0.001 ) ( table 1 ) . serum ada activity was significantly higher in patients with cutaneous anthrax than in the control subjects ( p<0.001 ) ( table 1 ) . a positive correlation was observed between ada activity and lymphocyte counts ( r=0.589 , p=0.021 ) ; however , there was no correlation between ada and the other inflammatory markers investigated ( p>0.05 ) . we evaluated the serum ada activity in patients with cutaneous anthrax . in the diagnosis of cutaneous anthrax , the clinical presentation of the disease and a history of close contact with sick animals are very important . if a patient has a typical malignant pustule or malignant edema and has had a history of contact with animals , the diagnosis may be easy . the clinical laboratory diagnosis of cutaneous anthrax is generally established by conventional microbiological methods , such as bacterial cultures and directly gram staining smears of clinical specimens . however , the clinical presentation could be atypical and the patient s recollection of contact with animals may not be accurate , or the patient may neglect to supply this information . in these situations , the diagnosis of cutaneous anthrax might be difficult . pcr , immunohistochemistry , laboratory parameters ( e.g. , crp , esr , and white blood cell [ wbc ] ) , and anthracin skin tests may be helpful in the diagnosis of anthrax . inflammatory markers such as esr and wbc count were found to be high in some patients . however , the elevation of crp levels in patients with cutaneous anthrax , as we found , has not been reported previously . ada is a key enzyme in purine metabolism ; it converts adenosine and deoxyadenosine to inosine and deoxyinosine by irreversible deamination . ada is widely distributed in human tissues ( its highest activity being in lymphoid tissues ) , and it is primarily associated with t - lymphocyte proliferation . although ada has been considered a nonspecific marker of t - cell activation , the precise mechanisms by which serum ada activity is altered have not been clearly identified . high levels of serum ada have been reported in infectious diseases such as viral and bacterial pneumonia , hiv infection , extra - pulmonary and pulmonary tuberculosis , h. pylori , acute appendicitis , visceral leishmaniasis , and mononucleosis and might have diagnostic value . moreover , circulating levels of ada have been shown to increase in several inflammatory conditions , including behet s disease , systemic lupus erythematosus , rheumatoid arthritis , and certain malignancies , especially those of hematopoietic origin [ 2434 ] . although to our knowledge this is the first study to investigate serum ada enzyme activity in patients with cutaneous anthrax , it has a major limitation . our study design did not allow us to investigate whether ada activity is uniquely associated with cutaneous anthrax . ideally , a third group of sick patients , with elevated crp or esr , but without lymphocytes or ada levels , could have been included to highlight the issue . however , in the current study , we observed that serum ada enzyme activity was significantly higher in patients with cutaneous anthrax than in the healthy controls . moreover , we found that acute cutaneous anthrax patients had increased wbc , crp levels , and esr compared with the control group . these results suggest that ada contributes to the inflammation seen in cutaneous anthrax and might be used in the clinical setting .

cancer cell lines are extensively used as models to investigate the genetics and behaviour of specific tumours . more generally , they are an important resource for basic research on diverse aspects of cellular biology , differentiation and pathology . the widely studied sh - sy5y human neuroblastoma cell line provides a classic example of how a cancer cell line can be instrumental for discoveries of broad biological and medical significance . neuroblastoma is a paediatric malignancy of neuroectodermal origin , characterised by genetic heterogeneity and variable clinical progression . the sh - sy5y cell line is a third successive sub - clone of the sk - n - sh line , originally established from a bone marrow biopsy of a metastatic neuroblastoma patient . the sk - n - sh parental line comprises at least two morphologically and biochemically distinct phenotypes : neuroblastic ( n - type ) , that led to the sub - cloning of sh - sy5y ( neuroblast - like ) , and substrate adherent , non - neuronal form ( s - type ) , that led to the sub - cloning of sh - ep ( epithelial - like ) . different theories have been postulated with regard to the possible biological phenomenon behind the co - existence of those two different cellular phenotypes . trans - differentiation or the ability of neuroblastoma cells to interconvert bi - directionally , in vitro , from a neuroblast ( n ) to a non - neuronal ( s ) form was the initial explanation . , or the ability of one of the clones co - existing in the parental cell line to expand over the other , was advanced as an alternative explanation . unquestionably , the most important characteristic of the sh - sy5y cells is their ability to differentiate into a functionally mature neuronal phenotype in the presence of various agents , for example sequential exposure to retinoic acid and brain - derived neurotrophic factor in serum - free medium , and when cultured three - dimensionally . upon differentiation this property has conferred the sh - sy5y cell line with the potential to provide an alternative to the experimental limitations caused by the inability of primary neurons to propagate in vitro . consequently , the sh - sy5y cell line has been extensively used as a neuronal model since the early 1980s in experimental neurological studies , including analysis of neuronal function , growth and damage in response to insult , degeneration and differentiation . differentiated and undifferentiated sh - sy5y cells have become a popular in vitro cell model for parkinson disease as they possess many characteristics of dopaminergic neurons [ 69 ] . because upon differentiation the sh - sy5y cells have been found to express mature tau isoforms , this cell line has also gained a status as an in vitro model for research into alzheimer s disease . the repertoire of biological research that relies on the use of the sh - sy5y cells has been rapidly expanding and has recently included investigations on autism - spectrum neurodevelopmental disorders [ 11 , 12 ] , studies of mitochondrial metabolism and antioxidant defences upon neuronal differentiation and research of productive varicella - zoster virus infection of neuronal cells . neuroblastoma , like most human cancers , is characterised by non - random chromosomal abnormalities , to include large - scale chromosomal imbalances , with diagnostic and prognostic significance [ 1519 ] . the first cytogenetic analysis of the sh - sy5y cell line was performed by spengler and collaborators in 1983 and a revised g - banded karyotype subsequently published by the same authors in 2002 . those first classical cytogenetic studies succeeded in describing with certain accuracy some , but obviously not all , of the chromosomal abnormalities that were subsequently to be identified in the sh - sy5y cell line by means of higher - resolution molecular cytogenetic techniques . in 2001 , van roy and collaborators published a detailed description of genetic alterations in 16 neuroblastoma cell lines , to include the sh - sy5y parental cell line sk - n - sh . in 2003 , by applying fluorescence in situ hybridization ( fish ) with gene- and chromosome - specific probes , to include a sky multi - colour labelling kit for spectral karyotyping , cohen and collaborators published a comparative cytogenetic analysis of the parental sk - n - sh cell line and sh - ep and sh - sy5y , highlighting karyotypic similarities and differences between the three lines . further molecular cytogenetic insights specifically into the karyotype of the sh - sy5y cell line were gained by do and collaborators by means of comparative genomic hybridization on a custom - designed 4000 bacterial artificial chromosomes microarray , covering the whole human genome . that study allowed the identification of unbalanced chromosomal changes gains and losses at a resolution of 1 mb . more recently , kryh and collaborators published the first high - density single - nucleotide polymorphism ( snp ) array study on the sh - sy5y , revealing the presence of previously undetected allelic imbalances and copy - neutral loss of heterozygosity ( loh ) . in the present study , by means of a comprehensive molecular cytogenetic approach , including single - probe fish , multi - colour karyotyping by m - fish and mcm - banding , and microarray analysis for copy number variants ( cnv ) and loh , we carried out a detailed re - assessment of the chromosomal complement and genomic profile of the sh - sy5y . given the common use of this neuroblastoma cell line as an in vitro model for studies of neurodegenerative and neurodevelopmental disorders , our aims were to resolve previous inconsistencies and provide the definitive sh - sy5y karyotype description . the sh - sy5y cell line was purchased from the european collection of cell cultures , a health protection agency culture collection . chromosome preparations were obtained from unsynchronised cultures of early passage cells upon receipt from the repository following standard procedures . karyotyping by multiplex fluorescence in situ hybridization ( m - fish ) was performed as recommended by the 24xcyte mfish probe kit manufacturer ( metasystems , germany , http://www.metasystems-international.com ) . directly labelled chromosome - specific probes used for validation fish experiments were : aquarius whole chromosome painting probes for chromosome 17 ( fitc ) and chromosome 15 ( texas red ) ( cytocell ) , poseidon whole chromosome 22 probe ( blue , platinum bright 415 ) ( kreatech ) , classical 1qh satellite ( fitc ) and classical d9z3 satellite ( rhodamine ) ( qbiogene ) . fish and m - fish experiments were analysed on a cytovysion system ( genetix , uk , http://www.genetix.com ) , consisting of an olympus bx-51 epifluorescence microscope coupled to a jai cvm4 + ccd camera . multi - colour mband fish imaging ( mcm - banding ) and analysis were performed as recommended by the xcyte1 mband probe kit manufacturer ( metasystems , germany , http://www.metasystems-international.com ) , utilizing a carl zeiss axioimager.z2 epifluorescence microscope coupled to a metasystems coolcube camera and metasystems isis software . analysis of copy number ( cn ) changes and loh was performed on affymetrix cytogenetics whole - genome 2.7 m arrays . genomic dna from the sh - sy5y cell line was extracted from unsynchronysed cultures of early passage number cells upon receipt from the repository ( and in parallel with chromosome preparations ) using a qiagen blood & cell culture dna kit . after ethanol precipitation , the dna was resuspended in te buffer ( 10 mm tris , ph 8.0 , 1 mm edta ) at a final concentration of 33 ng/l . whole genome amplification , fragmentation and labelling were performed following the array manufacturer instructions . briefly , after incubation at 50 c in a genechip hybridization oven with rotation at 60 rpm for 19 h , the arrays were stained in the gene chip fluidic station 450 and scanned with affymetrix gene chip scanner 3000 . for data analysis , the cel files were imported into chromosome analysis suite software v.1.2.2 . array quality was evaluated as per manufacturer s instructions and only arrays with snp - qc > 1.1 and mapd < 0.27 were accepted for further data analysis . parameters were set at minimum filter values of 60 kb , 35 marker count and 0.85 confidence for both gains and losses , and minimum filter values of 100 kb and 35 marker count for mosaicism . all copy number changes observed were compared to cnvs catalogued in the database of genomic variants ( dgv ) ( http://projects.tcag.ca/variation/ ) and the ucsc genome browser ( http://genome.ucsc.edu/ ) . we have applied an all - inclusive cytogenomic approach to re - evaluate the karyotypic profile of the sh - sy5y , a neuroblastoma cell line widely used as an in vitro neuronal model . by combining fish - based techniques , which retain information on a per cell basis , with high - resolution microarray - based techniques , we were able to report thus far unnoticed karyotypic features of the sh - sy5y cell line . our findings are summarised in table 1 together with findings from previous investigations.table 1sh - sy5y : summary of karyotypic findingsauthors , techniqueschromosome 1chromosome 2chromosomes 4 and 5chromosome 7chromosomes 9 and 10chromosome 14chromosome 16chromosomes 15 , 17 and 22spengler et al . , g - bandingder(1)(1pter 1q25::1q25 1q11::1q44 1q25::1q25 1qter)+ 7der(9)(9pter 9q34::7q22 7qter)der(22)(22pter 22q13::17q21 17qter)cohen et al . , g - banding and skydup(1)(q12q25)+7 t(7;8)(q34;q24.2)der(9)t(2;9)(p15;q34)der(22)t(17;22)(q21.3;q13)do et al . , array - cgh ( ( 4000 ) bac clones)1q12 - 1q44 gain2p25.3 - 2p16.3 gain+714q21.1 - 14q21.3 loss17q21.32 - 17q25.3 gain ; 22q13.1 - 22q13.2 losskryh et al . , snp - array1q12 - 1q44 gain2pter-2p16.3 gain5q34.1 loss+79p21.2 - 9p21.1 gain14q cn - loh 14q loss16q22.2 - 16q22.3 loss17q gain 22q cn - lohthis study , snp - array , mc - banding , fish and m - fish1q12 - 1q44 der(1 ) ( 1q121q31::1q31 1q12::1q44 1q31::1q31 1q44)2p25.3 - 2p16.3 gain / mosaic4q28.3 loss 5q34 loss 5q14.3 loss+7 t(7;8)(q34;q24.2)9p21.2 - 9p21.1 gain ; der(9)t(2;9)(p15;q34)/mosaic ; 10q26.13 gain14q13.3 - 14q21.3 loss ; 14q21.3 - 14q32.33 cn - loh16q22.2 - 16q22.3 loss17q21.33 - 17q25.3 gain ; 22q12.3 loss ; 22q13.1 - 22q13.2 loss ; der(15)t(15;17;22 ) ; der ( 22)t(15;22)cgh comparative genomic hybridization , bac bacterial artificial chromosomeadditional information on cn data in table 2 sh - sy5y : summary of karyotypic findings cgh comparative genomic hybridization , bac bacterial artificial chromosome additional information on cn data in table 2 m - fish analysis confirmed the presence of a set of previously reported chromosomal abnormalities that in combination define the cytogenetic identity of the sh - sy5y cell line ( fig . 1 ) , consisting of chromosome 7 trisomy , a duplication of the entire q arm of chromosome 1 , a balanced translocation involving chromosomes 7 and 8 , a derivative 9 t(2;9 ) , and a derivative 22 believed to be the result a of a t(17;22 ) . another distinctive karyotypic trait of the sh - sy5y is the lack of amplification of the n - myc proto-oncogene.fig . the karyotypic identity of the sh - sy5y cell line is confirmed by multi - colour karyotyping representative example of m - fish analysis . the karyotypic identity of the sh - sy5y cell line is confirmed by multi - colour karyotyping in our study , the derivative chromosome 9 , resulting from a translocation of extra chromosome 2p material to chromosome 9 , appeared in only 50 % of metaphases analysed by m - fish and dual - colour fish analysis with chromosome - specific probes ( data not shown ) . this observation ties with the mosaic status of the detected imbalance ( gain ) on the short arm of chromosome 2 ( 2p25.3 - 2p16.3 ) , as shown by our cnv microarray data ( table 2 ) . a high incidence of unbalanced jumping translocations involving a gain of chromosome 2 short arm material with a minimum region of overlap 2pter-2p22 and various partner chromosomes had been previously observed in a panel of 18 neuroblastoma cell lines , including the parental sk - n - sh [ 21 , 24 ] . recent snp array studies had indicated the gain on chromosome 2p to be an aberration shared by the sk - n - sh parental line and its neuroblast - like sub - clone sh - sy5y , but not its epithelial sub - clone sh - ep . given that the three cell lines were found in general to be very similar , sharing many of the chromosome aberrations , it could be argued that most of the chromosomal alterations seen in the daughter cell lines were present in a mosaic form in the parental cell line ( and perhaps also in the original tumour ) , and that the karyotypes of the daughter cell lines are the result of the combined effect of the initial sub - cloning and subsequent cell line evolution over time.table 2sh - sy5y : gains and losses identified in this study by microarray analysischromosometypecn stateminmaxsize ( kbp)cytobandgenesother1gain3145,388.014247,906.738102,5191q21.1 - 1q44large number1q trisomy2gain3234.05248,165.78647,9322p25.3 - 2p16.3large numbermosaic4loss1134,926.438135,186.3912604q28.3pabpc4ldgv5loss1168,029.695168,226.3331975q34slit3 , mir218 - 2dgv / segm.dupl.5loss183,782.54183,907.6541255q14.3nonedgv / segm.dupl.7gain346.845159,118.443159,0717p22.3 - 7q36.3large number7 trisomy9gain326,628.22828,223.9801,5959p21.2 - 9p21.1c9orf82 , plaa , ift74 , lrrc19 , tek , ncrna00032 , c9orf11 , mobkl2b , ifnk , c9orf72 , lingo2dgv / segm.dupl.10gain3123,334.116123,541.04420710q26.13fgfr2 , ate1dgv14loss137,139.36249,563.02012,42414q13.3 - 14q21.3large numberdgv / segm.dupl.14copy - neutral loh248,216.230107,242.02759,02614q21.3 - 14q32.33large number14q cn - loh16loss172,558.13473,017.68446016q22.2 - 16q22.3zfhx3dgv / segm.dupl17gain343,825.91181,004.77037,17917q21.33 - 17q25.3large numberdgv / segm.dupl.22loss132,411.00732,472.9306222q12.3slc5a1no22loss138,337.61144,222.6935,88522q13.1 - 22q13.31large numberdgv / segm.dupl sh - sy5y : gains and losses identified in this study by microarray analysis an intriguing finding emerging from our investigations was the nature of the rearrangement(s ) involving chromosome 17 and 22 , which were revealed to be more complex than previously reported . structural abnormalities of chromosome 17 resulting in gain of material are the most frequent genetic abnormalities in neuroblastoma and powerful independent predictor of poor outcome , commonly found in primary tumours and cell lines . our snp array analysis showed in the sh - sy5y cell line a gain on chromosome 17q ( 17q21.33 - 17q25.3 ) and two distinct losses on chromosome 22q ( 22q12.3 and 22q13.1 - 22q13.31 ) . differently from what reported previously , our m - fish and validation fish experiments with chromosome - specific probes revealed the derivative 22 to be the result of a translocation involving not chromosome 17 , as initially thought , but chromosome 15 . we also identified a der(15)t(15 ; 17 ; 22 ) ( figs . 1 and 2).fig . 2three - colour fish shows a complex rearrangement involving chromosomes 15 , 17 and 22 . three - colour fish with directly labelled chromosome - specific paints for chromosome 15 ( red ) , 17 ( green ) and 22 ( blue ) on reverse - dapi banded chromosomes confirms the presence of a der(22)t(15;22 ) ( top arrow ) and a der(15)t(15;17;22 ) ( bottom arrow ) three - colour fish shows a complex rearrangement involving chromosomes 15 , 17 and 22 . three - colour fish with directly labelled chromosome - specific paints for chromosome 15 ( red ) , 17 ( green ) and 22 ( blue ) on reverse - dapi banded chromosomes confirms the presence of a der(22)t(15;22 ) ( top arrow ) and a der(15)t(15;17;22 ) ( bottom arrow ) the affymetrix cytogenetics whole - genome 2.7 m array used in this study provides high - density coverage across the genome , with 2.7 million markers , including 400,000 snps , spaced throughout at a median inter - marker distance of 1 kb . indeed , multiple areas of loh and copy number gain were identified in this analysis of the sh - sy5y cell line ( table 2 ) . although most of the gains and losses overlap with polymorphic cnvs documented in the dgv , it is nevertheless interesting that the list of genes concerned by the imbalances includes a number of transcription factors involved with cancer and neuronal differentiation , like zfhx3 on chromosome 16q , slit 3 on chromosome 5q and fgfr2 on chromosome 10q . loci of neuro - biological interest , also mapping on structurally re - arranged chromosomal regions in the sh - sh5y cell line , are the parkinsons - associated lingo2 gene on chromosome 9p , the mapt gene ( encoding tau ) on chromosome 17q , psen1 gene on 14q , and the 15q11 - 13 chromosomal region . however , we also observed extensive copy - neutral loh on chromosome 14q ( 14q21.3 - 14q32.33 ) . loh on the long arm of chromosome 14with a consensus region in 14q23-q32has been consistently observed in primary neuroblastomas , suggesting the deletion of this region to be a common abnormality in these tumours . upd or loh with neutral copy number of chromosome arm 14q was also reported in primary neuroblastoma tumours and derivative early - passage cell lines . our investigations by high - resolution multi - colour chromosome banding on the long arm of chromosome 1 suggest a complex rearrangement arising from a duplication of the entire chromosome arm as confirmed by our snp - array analysis followed by a paracentric inversion , resulting in a der(1)(1q12 1q31::1q31 1q12::1q44 1q31::1q31 1q44 ) ( fig . 3 ) . duplication of the juxtacentromeric heterochromatic area was confirmed by fish analysis with a d1z1 probe for the classical satellite dna ( data not shown).fig . 3high - resolution multi - colour chromosome banding provides new clues on complex 1q rearrangement . a duplication of the entire long arm appears to have been followed by a paracentric inversion high - resolution multi - colour chromosome banding provides new clues on complex 1q rearrangement . a duplication of the entire long arm appears to have been followed by a paracentric inversion in the light of what previously published and confirmed by us , and what newly identified by us , we would like to suggest an updated karyotypic description for the sh - sy5y cell line to imply the existence of two cytogenetically related subclones ( stemline and sideline ) , as follows : 47,xx , der(1)(1q12 1q31::1q31 1q12::1q44 1q31::1q31 1q44 ) , + 7 , der(7)t(7 ; 8)(q34 ; q24.2 ) , der(8)t(7 ; 8)(q34 ; q24.2 ) , der(15)t(15;17;22 ) , der(22)t(15;22)/47 , idem , der(9)t(2;9 ) . the number of cells for each sub - clone in our sample is given in square brackets . m - fish observations on sh - sy5y cells at a higher passage number ( data not shown ) would suggest the sideline karyotype to become prevalent , with most of the metaphases analysed presenting the der(9)t(2;9 ) as well as new chromosomal aberrations resulting from the extended culture . given the importance acquired by the sh - sy5y cell line as a neuronal in vitro model , a thorough understanding of its genetic background is of paramount importance . we believe that information on the sh - sy5 cytogenomic profile should be factored in when designing experimental studies based on these cells . in particular , the interpretation of the results should be passage number- and karyotype - informed , especially with regard to gene expression data and genome architecture of genetic loci linked to cancer or neurological diseases which coincidentally map to chromosomal regions that are highly re - arranged in this cell line . for instance , biomedical research relying on the use of the sh - sy5y cells for highly topical diseases like parkinson and alzheimer s and autism - spectrum developmental disorders , should take into account possible dosage effects or position effects on loci like the parkinsons - associated lingo2 gene on chromosome 9p , the mapt gene ( encoding tau ) on chromosome 17q , psen1 gene on 14q , and the 15q11 - 13 chromosomal region . our results demonstrate the impact of an integrated molecular cytogenetic approach on the ability to resolve complex karyotypes and underline the importance for the cytogenomic profiling of cancer cell lines to inform their use in research .

the natural course of hepatitis c virus ( hcv ) infection , which is transmitted via parenteral route , is modulated by both host and viral factors and typically tends to evolve towards chronicity , which will ensue in approximately 85% of cases following acute infection . given that this virus affects approximately 3% of the world 's population , hcv infection represents a leading cause of chronic hepatitis , cirrhosis , end - stage liver failure , and hepatocellular carcinoma . more recently , evidence suggests that hcv is capable of crossing the blood - brain barrier and that the brain serves as an important reservoir for subsequent viral replication [ 35 ] . there is evidence that chronic hcv infection results in neurocognitive dysfunction even in the absence of advanced liver disease [ 6 , 7 ] . in most cases , these observations sparked a number of investigations that sought to characterize neuropathological changes in patients with hcv with mild ( noncirrhotic ) liver disease . in vivo proton ( h ) magnetic resonance spectroscopy ( mrs ) is a noninvasive technique that gives information on brain metabolism at the end of a standard brain magnetic resonance imaging ( mri ) protocol . using proton mrs technique , forton and colleagues were among the first groups to report cerebral metabolite abnormalities suggestive of frontal - subcortical dysfunction in patients with mild chronic hcv infection [ 8 , 9 ] . used h mrs to study 30 hcv - infected patients with normal liver function who underwent cognitive testing . they found decreased n - acetylaspartate - to - creatine ratios ( naa / cr ) in the cerebral gray matter compared to healthy controls which can be attributed to either decreased naa or increased cr . naa is a neuronal marker and cr is a component of high - energy phosphate metabolism . there were no abnormalities in any other regions ( parietooccipital white matter , basal ganglia , or pons ) or any perturbations in choline containing compounds . among patients with noncirrhotic chronic hepatitis c elevated choline ( ch ) and myoinositol ( mi ) ratios have been found in the basal ganglia , and central and frontal white matter of hcv - infected patients [ 11 , 12 ] . mcandrews and colleagues performed h mrs analysis on their cohort of 37 hcv - infected patients with mild hepatitis . elevations in cerebral ch and reductions in naa were found in voxels that were localized to the central white matter . these findings are thought to reflect reduced neuronal integrity and inflammation or proliferation of glial cells . interestingly , the role of neurotransmitters such as gamma - aminobutyric acid ( gaba ) , an inhibitory neurotransmitter , and glutathione ( gsh ) , an antioxidant , has received less attention in the literature although gaba is essential for mental concentration and focus whereas gsh protects the brain against oxidative stress . it is plausible that gaba and gsh may play an important role in hcv that is not detectable using conventional one - dimensional ( 1d ) mrs spectral techniques . in 1d spectral representation , the overlap of metabolite peaks is caused by many features , including j - modulation leading to varying phase artifacts with echo time , te . 1d mr spectral editing techniques to unravel the overlapping resonances rely on j - coupled proton metabolites that have well - separated multiplets . a technique based on subtraction methodology is very sensitive to motion artifacts leading to subtraction errors . an additional drawback is that only one metabolite can be identified at a time . two - dimensional ( 2d ) localized correlated spectroscopy ( l - cosy ) overcomes this problem by adding a second frequency dimension to each spectrum by acquiring multiple 1d spectra with incrementally longer tes and applying double fourier transform on the set of spectra to produce a 2d spectrum . a hypothesis of this current study was that using a 2d mrs approach combined with profit quantitation would facilitate observing changes in cerebral metabolites such as naa , choline groups , mi , gsh , and gaba more accurately than the previously reported 1d mrs work demonstrating the underlying central nervous system involvement in hcv . the goal of the present study was to employ an emerging mrs approach2d l - cosy and combine that with a prior knowledge fitting ( profit ) algorithm [ 15 , 16 ] to better characterize and quantify cerebral metabolite abnormalities present in hcv+ patients versus healthy controls . fourteen patients with advanced hcv disease ( mean age of 56.2 years ) and fourteen healthy controls ( mean age of 46.6 years ) were recruited from infectious disease clinics for the mri / mrs study . all the scans were performed with a siemens 3 t trio - tim ( siemens medical solutions , erlangen , germany ) mri / mrs scanner using 12 channel head receive coil . the entire protocol was approved by the institutional review board ( irb ) , and informed consent was obtained from each human subject . a t1-weighted mri procedure was used to guide to select a volume of tissue from which the 2d mrs were acquired . a wet ( water suppression enhanced through t1 effects ) method with three frequency - selective radiofrequency ( rf ) the fast automatic shimming technique by mapping along projections ( fastestmap ) has been successfully used in order to get better line width . the water line width was ~15 hz obtained in gray / white matter in the left frontal . for the 2d mrs , a 2d l - cosy sequence consisting three slice - selective rf pulses was used similar to 1d press , but the last 180 pulse was replaced by 90 for the volume localization , with the last 90 rf pulse also enabling the coherence transfer necessary for 2d l - cosy . the spectral encoding for the second dimension was inserted between the second and third slice - selective pulses . 2d l - cosy spectra were recorded using the following parameters : effective echo time ( te ) = 30 ms , repetition time ( tr ) = 2000 ms , and the total number of scans of 800 ( 100 t1 increments and 8 averages per t1 ) , with voxel size of 3 3 3 cm corresponding to a total duration of approximately 26 min . the 2d raw matrix consisted of 2048 complex points along the first dimension and 100 points along the second dimension . the profit algorithm has been further optimized for the quantitation of 2d l - cosy . the profit algorithm was implemented using matlab ( mathworks , natick , ma , usa , version 7.3 ) and was executed on an intel 2.8 ghz with windows xp . the profit algorithm uses prior knowledge constraints and a combined linear and nonlinear optimization for fitting . it uses a prior knowledge basis set generated using the gamma library in combination with the chemical shift and j - coupling values reported in the literature . the profit algorithm quantified the following metabolites : creatine ( cr ) , n - acetylaspartate ( naa ) , glycerylphosphorylcholine ( gpc ) , phosphorylcholine ( pch ) , free choline ( ch ) , alanine ( ala ) , aspartate ( asp ) , gaba , glucose ( glc ) , glutamine ( gln ) , glutamate ( glu ) , glycine ( gly ) , glutathione ( gsh ) , lactate ( lac ) , myoinositol ( mi ) , n - acetylaspartylglutamate ( naag ) , phosphoethanolamine ( pe ) , taurine ( tau ) , scyllo - inositol ( scy ) , and ascorbate ( asc ) . 2d l - cosy spectra were then processed using the modified profit code , and the measurement accuracy was characterized using cramr - rao lower bound ( crlb ) . to avoid a false attribution of signals , five independent macromolecules ( valine , leucine , isoleucine , threonine , and lysine ) and three lipids ( palmitic acid , linoleic acid , and oleic acid ) were added with metabolites basis set in the profit algorithm . the metabolite differences between hcv+ and healthy controls were tested using a two - tailed t - test . figure 1 shows the mrs voxel location in the prefrontal white mater on the axial mri of a 64 years old hcv+ patient . figures 2 and 3 show 2d l - cosy spectra of a 64 years old hcv+ patient and a 54 years old healthy control , respectively , recorded in the same voxel location shown in figure 1 . table 1 shows the mean and standard deviation ( sd ) of selected metabolite concentrations with respect to cr of healthy controls and hcv+ patients . figure 4 shows the box and whisker plots of mi and gsh between healthy controls and hcv+ patients . figure 5 shows box and whisker plots of selected metabolites changes between healthy controls and hcv+ patients . significantly increased gsh ( p = 0.003 ) and mi ( p = 0.029 ) with respect to cr were observed in hcv+ patients compared to healthy controls as shown in figure 4 . there were no significant differences observed in any other metabolite ratios ( table 1 ) even though there was a trend of decreased total naa ( tnaa = naa + naag ) and gaba and increased total choline ( tch = gpc + pch + ch ) in the patients ' group . our results showed significant increase of mi and gsh in the hcv+ patient group compared to healthy controls and demonstrated that the 2d mrs spectra allowed us to visualize and characterize the role of gsh in cerebral metabolism . elevation of mi levels was also consistent with prior studies that have indicated mi as an important marker of inflammation and glial proliferation among patients with neuroinflammatory disorders . myoinositol is found only in glial cells and is also a constituent of membrane lipids . increased levels are believed to reflect glial cell activation and increased cell membrane turnover [ 24 , 25 ] . elevations in the mi / cr and decreased naa / cr ratios have been identified using h - mrs in hiv - infected patients [ 2628 ] . gsh is a tripeptide that serves as a major antioxidant and vital component of host defenses . its primary role is to protect tissues from free radical injury via detoxification and repair of injury . the concentration of gsh in human brain is in the range of 13 mm and can exist intracellularly in either an oxidized ( gssg ) or reduced ( gsh ) form . glutathione is an important antioxidant and plays a role in the detoxification of electrophilic compounds and peroxides via catalysis by glutathione - s - transferases ( gst ) and glutathione peroxidases ( gpx ) . maintaining optimal gsh : gssh ratios in the cell is critical to survival . oxidative stress generated by the production of reaction oxygen species ( ros ) appears to be connected with the loss of neurons during the progression of neurodegenerative diseases . evidence suggests that gsh plays an important role in the detoxification of ros in the brain . our results suggest that elevations in gsh are indicative of oxidative stress in those with hcv infection , which is consistent with the elevations in mi . considering that we did not observe statistically significant differences in naa between patients and controls , it is possible that gsh is an early marker of inflammation that precedes actual neuronal damage . elevations in gsh levels may occur relatively early after hcv crosses the blood - brain barrier . hence , gsh should be considered along with other metabolites as an important marker of inflammation . a trend of elevated ch was observed in the patient group compared to healthy controls . elevated levels of ch reflect increased cell membrane turnover and have also been reported in other neuroinflammatory conditions [ 22 , 31 ] . choline increase has been observed in the presence of macrophage infiltration of the brain , for example , in patients with hiv infection with and without aids dementia complex [ 32 , 33 ] and in other chronic infections such as the john cunningham ( jc ) virus infection or subacute sclerosing panencephalitis . choline and mi are also putative markers for glial cell inflammation , and activation and elevations are believed to reflect cellular proliferation due to infection or inflammation [ 9 , 11 ] . immune cell activation by macrophages and/or neuronal astrocytes has been shown to produce ch peaks in hiv patients . while there was a trend towards lower levels of naa and gaba in the hcv+ group , this was not statistically significant . changes in gaba metabolism may play an important role in the origin and spread of seizure activity [ 3739 ] . several reports suggest that gabaergic neurons are decreased in the epileptic neocortex [ 37 , 4042 ] . significant reductions in cerebrospinal fluid ( csf ) gaba concentration are seen in patients with various epileptic syndromes . our preliminary results showing increased mi and ch and decreased naa are in agreement with previous reports [ 810 ] . additional findings from this work include the quantitation of gsh , gaba , glu , scy , and asp using the 2d l - cosy spectra postprocessed by the profit algorithm . future studies should employ longitudinal methods to better characterize metabolic changes that occur as a result of hcv infection and determine whether elevations in gsh precede significant decreases in naa . understanding the attendant changes in cerebral metabolism will allow us to understand how hcv treatments alter the course of neurological functioning . the ability to detect early abnormal changes in cerebral metabolism among hcv patients is useful for healthcare providers working with hcv - infected patients , as they will be aware of the nature and course of cognitive changes that occur during the course of the disease . these preliminary results need to be reproduced in a larger cohort of hcv+ and hcv subjects .

diabetic nephropathy ( dn ) is an important diabetic microvascular complication and the major cause of disability and death . dn is characterized by albuminuria , glomerular hypertrophy , and progressive accumulation of glomerular matrix , culminating in glomerulosclerosis , tubulointerstitial fibrosis , and progressive loss of renal function [ 2 , 3 ] . many studies have confirmed that glomerular sclerosis and interstitial fibrosis are the main pathologic characteristics in dn , especially in the midanaphase of dn . deposition of extracellular matrix ( ecm ) such as collagens and fibronectin regulated by transforming growth factor 1 ( tgf-1 ) is the core mechanism of glomerular sclerosis and interstitial fibrosis . the mesangial cells play important roles in dn , being responsible for the accumulation of ecm and mesangial expansion . tgf-1 is the core cytokine leading to the synthesis of ecm , which is responsible for mesangial fibrosis and hypertrophy under diabetic conditions . the major components of the ecm proteins collagen types i iv and their synthesis and immoderate deposition are consistently observed in multifarious renal disease processes affecting humans and experimental animals [ 7 , 8 ] . combined with unphosphorylated akt , trb3 can prevent akt activity and negatively regulate the insulin signaling pathway . trb3 and its gene polymorphism are associated with insulin resistance , a vital pathophysiologic characteristic of type 2 diabetes . trb3 also serves as a scaffold protein and regulates the activation of the three classes of mitogen - activated protein kinases ( mapks ) . as a member of the mapk family , extracellular signal - regulated kinase 1/2 ( erk1/2 ) therefore , trb3 may be involved in dn through an erk pathway . in this study , we aimed to explore the role of trb3 in dn and the possible regulating mechanism between trb3 , erk , and tgf-1 in vivo and in vitro . we obtained antibodies for trb3 ( santa cruz biotechnology , santa cruz , ca ) , tgf-1 ( abcam biotechnology , ca ) , and pd98059 , phospho - erk1/2 , and total erk1/2 ( cell signaling technology , beverly , ma ) . trizol reagent and reagents for rt - pcr were from takara biotechnology ( dalian , china ) . elisa kits for collagen types i and iv were from r&d systems ( minneapolis , mn ) . db / db diabetic mice ( c57bl / ksj ) and their matched ( 12-week - old ) controls ( db / m ) were obtained from vital river laboratory animal technology ( beijing ) . all animals were maintained on a normal diet under standard animal house conditions at the cardiovascular remodeling laboratory animal center in qilu hospital of shandong university . animal experiments were conducted in accordance with guidelines established by the animal care and use committee of shandong university . animals were divided into 3 groups ( n = 5 ) and killed at 16 , 20 , and 25 weeks . blood glucose and body weight were randomly monitored weekly ; levels of urinary albumin excretion , serum creatinine , and blood urea nitrogen ( bun ) were detected before death once . the left kidney pieces fixed in 4% paraformaldehyde were embedded in paraffin , sectioned at 4 m thickness , and mounted on glass slides . the slides were used for morphological observation and immunohistochemical staining to detect the expression of trb3 ; the right kidney was snap - frozen in liquid nitrogen and stored at 80c for rt - pcr and western blot analysis . sections were stained with hematoxylin and eosin ( h&e ) , periodic acid schiff ( pas ) , and masson trichrome for investigating renal pathological changes . paraffin sections were dewaxed and washed in phosphate buffered saline ( pbs ) and then incubated in preheated 10 mmol / l sodium citrate buffer at 94c for 15 min . slides were washed and blocked with protein - blocking solution ( 10% normal goat serum ) for 30 min and then incubated with rabbit anti - mouse trb3 antibody ( 1 : 100 ) overnight at 4c , biotin - labeled secondary antibody working solution for 30 min at 37c , and then dab color . mycoplasma - free sv40 mes 13 cells ( murine mesangial cells , mmcs ) were purchased from china center for type culture collection . they were derived from glomerular explants of sv40 transgenic mice and showed both biochemical and morphological features of normal mesangial cells in culture . they were maintained in dmem - f12 ( 3 : 1 ) containing 6 mm glucose and supplemented with 14 mm hepes and 5% fetal bovine serum in 5% co2 and 95% humidified air at 37c as described previously [ 13 , 14 ] . cells were synchronized by culturing in serum - free medium for 24 h before testing and all experiments were performed with cells between passages 30 and 40 to minimize the effects of phenotypic variation in continuous culture . mesangial cells were divided by glucose concentration into 3 groups : ( 1 ) normal glucose ( ng , 5.6 mm glucose , and control ) ; ( 2 ) hg ( 25 mm glucose ) ; and ( 3 ) ng + high mannitol ( hm , ng plus 19.5 mm mannitol ) . to investigate the effects of glucose on the expression of trb3 and perk1/2 , cells were stimulated with hm or hg for 6 , 12 , 24 , and 48 h. at the end of each time , total rna and protein of the cells were extracted for trb3 and perk1/2 expression . to examine the effect of the mapk pathway on collagen expression by hg , a specific erk inhibitor ( pd98059 , 10 mol / l ) was added 1 h before stimulation . to examine the effect of trb3 on collagen expression by hg , trb3 small interfering rna ( sirna ) was transfected 6 h before stimulation and cultured for 48 h in ng or hg medium ; then , total rna and protein were extracted from cells for analysis of trb3 , tgf-1 , and perk1/2 expression and culture medium was collected for measurement of concentration of collagen types i and iv . cells were replated and transfected in 6-well plates with 150 l opti - mem ( invitrogen , ca ) and 1.5 l / well lipofectamine 2000 ( invitrogen , ca ) with 20 pmol sirna and its controls . the sense and antisense sequences of the primers were 5-gcacagaguacaccugcaatt-3 and 5-uugcagguguacucugugctt-3. as a negative control , we used randomly mixed sequences of trb3 sirna , 5-uucuccgaacgugucacgutt-3 , and 5-acgugacacguucggagaatt-3. the effect of sirna knockdown of trb3 on the expression of tgf-1 and collagen types i and iv was evaluated by western blot analysis or elisa at 48 h. all rnai experiments were repeated at least 3 times . g total rna was reverse - transcribed in a 20 ml volume containing 0.5 mg oligo - dt primer , 1 ml dntp mixture , 1.25 ml rnase inhibitor , and 4 u reverse transcriptase . real - time quantitative pcr involved the sybr - based method in a 20 ml reaction in a roche light - cycler . reaction specificity was confirmed by analyzing melting curves and by electrophoresis on 2.0% agarose gel analysis of products . the relative change in gene expression was analyzed by the 2 method and normalized to the expression of the housekeeping gene -actin . the protein extracts were separated on 10% sds - page then transferred to pvdf membranes , and blocked in tbst with 5% skim milk at room temperature for 2 h. the blots were incubated with antibodies for trb3 ( 1 : 200 ) , tgf-1 ( 1 : 200 ) , perk1/2 ( 1 : 2000 ) , erk1/2 ( 1 : 1000 ) , and -actin ( 1 : 1000 ) overnight at 4c , washed , and then incubated with goat anti - rabbit antibody ( 1 : 10000 ) at room temperature for 1 h. protein bands were analyzed by use of alphaview sa software . soluble collagen types i and iv proteins were determined by elisa kits according to the manufacturer 's protocol . the kits for mouse collagen types i and iv were species - specific and sensitive up to 1000 , 10 , and 20 ng / ml . all concentrations of proteins were normalized to the total protein amount . groups were compared by one - way anova and correlation analysis involved pearson correlation coefficient . statistical analysis involved spss v17.0 for windows ( spss inc . , chicago , il ) . the blood glucose results for control mice remained stable , whereas dn mice showed hyperglycemia . levels of uae , scr , and bun and body weight were higher for db / db mice than age - matched controls ( p < 0.05 , p < 0.01 ) ( table 2 ) . mesangial matrix expansion and mesangial area were wider for dn than control mice . at 25 weeks , dn mice showed diffuse and nodular mesangial sclerosis ( figure 1(a ) ) , and the area of mesangium matrix and basement membrane was increased ( figure 1(b ) ) as was the relative fibrosis area ( figure 1(c ) ) . trb3 was expressed mainly in the nucleus of intrinsic glomerular cells and tubular epithelial cells ( figure 2(a ) ) . the mrna and protein expression of trb3 and tgf-1 were higher in dn than control mice from 20 weeks and increased with time ( figures 2(b ) and 2(c ) ) . the protein level of trb3 was positively correlated with tgf-1 level ( r = 0.944 , p < 0.01 ) and renal interstitial fibrosis ( r = 0.857 , p < 0.05 in dn mice ) . to confirm the effect of glucose on the expression of trb3 , the mrna level of trb3 was increased within 12 h after hg stimulation and peaked at 48 h ( p < 0.01 , versus ng ; figure 3(a ) ) . trb3 protein level was increased under hg at 12 , 24 , and 48 h ( figure 3(b ) ) . this increase also peaked at 48 h ( p < 0.05 , versus ng ) . thus , hg can upregulate the expression of trb3 in mmcs . as compared with ng , hg time - dependently increased both the mrna and protein expression of tgf-1 and collagen type iv ( figure 4 ) within 48 h and 12 h , respectively , for mrna and for both within 48 h for protein ( p < 0.01 , versus ng ) . however , collagen type i expression did not change under any conditions within 48 h ( data not shown ) . therefore , hg increased tgf-1 and collagen type iv secreted from cultured mmc cells . to verify the effect of glucose on the activation of the erk1/2 mapk pathway in mmcs , cells were cultured in ng medium and then stimulated with hg or hm for various times . the level of perk1/2 increased during the first 6 h ( p < 0.01 , versus ng ) after hg stimulation and peaked at 24 h ( p < 0.01 , versus ng ) ( figure 5(a ) ) . therefore , hg can activate the erk1/2 pathway in mmcs . to confirm the effect of trb3 on this pathway , we transfected trb3 sirna into mmcs exposed to hg medium for 24 h and evaluated perk1/2 levels . expression of fam increased at 6 h and peaked at 48 h ( figure 6(a ) ) . to test the efficacy of the selected sirna sequence , we measured the protein level of trb3 after 48 h transfection with trb3 sirna . trb3 protein expression was lower in mmcs with trb3 sirna than in cells with control sirna ( p < 0.05 , versus ng + nc ) ( figure 6(b ) ) . perk1/2 expression was decreased in mmcs transfected with trb3 sirna ( p < 0.05 , versus hg + nc ; figure 5(b ) ) . the expression of perk1/2 was markedly reduced by blocking erk1/2 mapk signaling by a specific erk inhibitor ( pd98059 , 10 mol / l ) ( figure 5(b ) ) . the mrna and protein levels of tgf-1 and collagen type iv were decreased with the erk1/2 pathway blocked ( figures 7(a)7(d ) , p < 0.01 versus hg ) as was the perk1/2 level ( figure 5(b ) , p < 0.01 versus hg ) , which suggests that hg can regulate tgf-1 and collagen type iv through the erk1/2 mapk pathway . the protein expression of trb3 was markedly increased after blocking the erk1/2 mapk pathway ( figure 7(e ) , p < 0.01 versus hg ) . this finding indicates a possible feedback regulation between trb3 and some downstream cytokines of erk1/2 mapk . here , we found augmented expression of trb3 in kidneys of dn mice , which was positively related to tgf-1 expression and renal interstitial fibrosis . hg upregulated the expression of trb3 , tgf-1 , collagen type iv , and phosphorylated - erk1/2 mapk in mmcs . after inhibiting trb3 with trb3 sirna , the hg - induced level of perk1/2 mapk was attenuated and hg - induced expression of tgf-1 and collagen type iv decreased . moreover , the protein expression of trb3 was increased after blocking the erk1/2 mapk pathway . trb3 may be involved in dn by regulating the fibrosis cytokine tgf-1 and collagen type iv via erk1/2 mapk signaling . tribbles was first discovered to regulate drosophila embryogenesis , and it has the same location as the type 2 diabetes gene , so trb3 may have a natural link with diabetes . compared with wild - type mice , db / db diabetic mice were found to have increased trb3 mrna and protein expression in liver ; trb3 expression increased in the db / db mouse liver promoted blood glucose and increased glucose tolerance . we examined the expression of trb1 , trb2 , and trb3 in the dn mouse kidney and found only trb3 expressed differently between dn and control groups , so trb3 could be a potential cytokine to widen the current knowledge of dn . our further study revealed that the mrna and protein levels of trb3 were higher in the dn than normal kidney , which was also positively correlated with tgf-1 protein level and content of collagen . previous study showed that trb3 was positively correlated with kidney tissue fibrosis , which may play a role in promoting the progression of fibrosis by inducing the transformation between epithelial and mesenchymal tissue . therefore , trb3 may be involved in dn by inducing interstitial fibrosis , in which tgf-1 plays a key role . we wondered about the role of trb3 in the fibrosis of dn and the relationship between trb3 and tgf-1 . several studies supported that trb3 may function as a scaffold protein to control mapk activity [ 10 , 16 , 17 ] . among the several mapk signal pathways , the erk1/2 pathway is activated under hg in mesangial cells , followed by the complicated synthesis of tgf-1 . the activation of the erk1/2 pathway is necessary for hg - induced production of tgf-1 and connective tissue growth factor ( ctgf ) in mmcs . therefore , we focused on the erk mapk pathway . silencing trb3 decreased erk1/2 activation , followed by decreased mrna and protein levels of tgf-1 and collagen type iv in mmcs , so trb3 may participate in renal fibrosis of dn by upregulating tgf-1 and collagen type iv in mmcs via erk mapk signaling . to our knowledge , our study is the first to reveal the interaction between trb3 , erk1/2 , and tgf-1 in renal tissue of dn . recently , trb3 was found stimulated in diabetic kidneys , regulated by the er stress marker chop , and inhibited the podocyte expression of monocyte chemoattractant protein 1 , which first suggested that trb3 plays a protective role in diabetic kidney disease . these findings that differ from our results may be due to cell specificity ; we used mesangial cells , but the previous study used podocytes . numerous cytokines may interact through different pathways , and different signal pathways may have crosstalk . so , the role of trb3 in dn can also be studied from other signal pathways . trb3 plays a role in the pathogenesis of dn by participating in insulin resistance , functioning as a negative modulator of akt [ 9 , 22 , 23 ] . another study showed that trb3 may cause renal cell apoptosis by participating in the nf-b pathway and thus participate in the process of renal fibrosis in dn [ 24 , 25 ] . there must be some interactions between erk , akt , nf-b , smad3 , and other unknown pathways . more detailed and systemic studies are needed to establish the complete theory on trb3 involved in dn . in this study , we found that the expression of trb3 increased after blocking the erk1/2 mapk pathway , which may indicate a negative feedback regulation between trb3 and some downstream cytokines of erk1/2 mapk or special trb3 expression is upregulated in renal tissue of dn mice in vivo and mmcs in vitro . trb3 may be involved in dn by regulating the fibrosis cytokine tgf-1 and collagen type iv via erk1/2 mapk signaling .

for the investigation of hemorrhage after sci the rat can be considered to be a good model as the anatomy of arterial supply is almost an exact match to the human . in both rats and humans , the blood supply to the spinal cord is provided by three vessels that run along the spinal cord , namely the ventral ( anterior in a human ) spinal artery and the paired dorsal ( posterior in a human ) spinal arteries [ for an extended review , see for example ( bosmia et al . , 2013 ) ] . the ventral spinal artery arises from the confluence of two distal branches of the vertebral arteries close to the medulla oblongata and runs caudally along the midline of the ventral aspect of the spinal cord . this artery supplies about 75% of the blood needed by the spinal cord ( wan et al . , 2001 ) . the dorsal spinal arteries emerge from either the vertebral arteries or the dorsal inferior cerebellar arteries and run caudally on the dorsolateral side of the spinal cord adjacent to the entrance of the dorsal roots . in the rat , there are sometimes additional longitudinal arteries , namely a median dorsal spinal artery located at or near the dorsal septum , and two lateral spinal arteries located at equidistance between the entrance of the dorsal and ventral roots . the presence of these additional arteries is only frequent in the cervical part , and less in the more caudal parts of the spinal cord ( paxinos , 2004 ) . below the cervical level , the arterial supply in human and rats is strengthened by the radicular arteries , which location can vary , derived from segmental arteries that split into ventral and dorsal branches . the microcirculation into the spinal cord is either centrifugal ( directed outward from the center ) or centripetal ( directed toward the center ) . the centrifugal system is formed by branches of the ventral spinal artery called the sulcal arteries . these branches enter the ventral median fissure of the cord and provide blood to the greater part of the grey matter and the inside half of the white matter . the centripetal microcirculation is formed by the combination of other branches of the ventral spinal artery , named the pial arterial network , with branches of the dorsal arteries . this centrifugal system supplies the dorsal horns , most of the posterior white matter , and the outer portion of the ventrolateral white matter . it is important to note that the capillary network is much denser in the central grey matter than in the white matter of the spinal cord . thus in terms of gross spinal anatomy , the cord is the opposite of the brain in this respect where the cortex is highly vascularized . this is likely to have a bearing on outcome after injury to the brain or spinal cord and may contribute to cavitation , which is often observed in the cord after injury , but not in the brain . veins in the central sulcus , dorsal to the ventral spinal artery run the entire length of the spinal cord and damage to this vessel may result in damage that extends further rostrally and caudally in the cord . as a spinal trauma occurs , the integrity of the spinal arteries and those adjacent to the spinal column are often compromised . the deeper arteries are , fortunately , rarely affected as such damage leads to devastating neurological deficits . however , the spinal cord microcirculation system is commonly damaged by sci , giving rise to intraparenchymal or intramedullary hemorrhage . the presence of hemorrhage in the human spinal cord after sci has been connected with significantly decreased motor function ( flanders et al . , 1996 ) . while 21% of the patients in the flanders study with incomplete motor injuries presented with evidence of hemorrhagic lesions on their initial exams , 85% of the patients with complete motor deficits had evidence of spinal hemorrhage . numerous other clinical studies confirmed that the presence of hemorrhage in the cord after sci is associated with poor neurological outcome [ for example ( andreoli et al . secondary petechial hemorrhages follow the primary bleed around the primary injury site and are related to an enlargement of the initial lesion ( gerzanich et al . , 2009 ) . in the surrounding region of the hemorrhage if we solely consider the hemorrhagic component of sci , the reduction of flow could be , as suggested by data extracted from intracerebral hemorrhagic studies ( vespa , 2009 ) , a consequence of low rates of oxygen consumption due to the impairment of mitochondrial function in the surrounding tissue , and would not represent per se an ischemic state . this matter is still controversial among intracerebral hemorrhage ( ich ) researchers , but it is of interest to note that in a recent animal study , improving the blood flow in a sci contusion model did not reduce acute microvessel , motor neuron or oligodendrocyte loss and failed to lead to improved functional recovery ( muradov and hagg , 2013 ) . on the other hand , the same laboratory showed that rescuing the vasculature using angiopoietin-1 and v3 integrin peptide was protective after sci in mice ( han et al . , 2010 ) and had lasting effects when treated from 4 hours after injury and during a week . reducing ischemia could be one of the explainations for the improving the outcome , but the prevention of the leakage of blood / plasma into the spinal cord is also likely to play a key role . blood per se has been shown to be toxic to cns populations ( asano , 1980 ) and internal bleeding leads to elevated thrombin formation , increased extracellular glutamate level , red blood cell lysis , iron toxicity , inflammation and vasospasm ( hua et al . , 2006 ; wagner et al . , 2006 ; sinescu et al . , the presence of hemorrhage has also been related to edema formation via thrombin production and erythrocyte lysis . this is important as after sci as edema volume is directly proportional to a poorer neurological outcome . in a recent clinical study , it was shown that removing the clots in patients with ich significantly decreased the perihematomal edema ( mould et al . , 2013 ) . in this study , the percentage of the clot removed was also positively correlated to the perihematomal edema reduction . the potential benefit of clot removal after ich is only possible due to the recent advances and refinement of minimally invasive surgery . in the spinal cord , zhang et al . ( 2004 ) showed that clot removal , approximating the cord and closing the dura can lead to a significant improvement in outcome in a laceration sci animal model . in order to test the potential beneficial outcome of intraspinal clot removal following sci , further studies need to be performed using advanced minimally invasive surgery before moving to humans . in conventional animal models of sci it has always been difficult to explore the contribution of hemorrhage to the outcome of sci in the absence of the trauma . traumatic lesions invariably generate hemorrhage , but the amount of bleeding is often very variable . to isolate the impact of hemorrhage on the outcome of spinal cord injury , our laboratory has established a new animal model ( losey et al . , 2014a ) . with the microinjection of collagenase into the rat spinal cord ( figure 1 ) , we were able to disrupt the blood - spinal cord barrier and produce an ongoing intra - spinal bleeding in a controlled manner . collagenase digests type vi collagen of the blood vessels basal lamina and it has become accepted as a useful model of ich ( maclellan et al . , 2008 ) . however , as we have discussed above , the vascular anatomy of the spinal cord is very different from the brain . the principal technical advantage is that the collagenase is injected stereotaxically such that the position of the hemorrhage can be preselected and the amount of hemorrhage is very reproducible , which is very useful for preclinical trials . classical sci animal models usually affect the integrity of the dorsal ( posterior ) arteries , while in human sci , it is mostly the anterior ( ventral ) arteries microvasculature integrity that is compromised ( therom et al . , 1978 ) . the dose of collagenase ( 0.12u ) was chosen ( losey et al . , 2008 ) to generate hemorrhage in the absence of any immediate and direct neurotoxicity ( matsushita et al . , the skin overlying the thoracic spine is shaved and a small incision is made in the midline . the subcutaneous tissues and muscle layers are blunt - dissected and a partial laminectomy is performed at thoracic level 8 ( t8 ) to expose the underlying spinal cord without opening the dura mater . to minimize movement , the animal is suspended in the stereotactic frame by clamping of t7 and t9 spinous processes . the tip of a finely drawn calibrated glass capillary tube is stereotaxically inserted into the grey matter of the spinal cord , 0.4 mm right lateral of midline and at a depth of 1.6 mm . the injection site was selected to ensure penetration into the spinal cord parenchyma and for its proximity to the white matter of the lateral funiculus . 0.5 l of collagenase ( 0.24 u/l ) ( or saline vehicle ) is then injected over 2 minutes and the capillary is left in place for a further 3 minutes before being slowly withdrawn . the muscle layer is closed with 4/0 sutures and the skin approximated with wound clips . after surgery of this type , the animals recovered well and we did not observe evidence of overt distress or discomfort during the survival times of up to 7 days . using this model , we could showed that disrupting the intraspinal vasculature and produced ongoing bleeding that was sufficient to induce extended neuronal damage . damage to neurons was observed from six hours and neutrophils recruitment was also a feature at this time point , as well as the appearence of ed-1-positive macrophages and activated microglia . while the presence of recruited neutrophils into the spinal cord of collagenase - injected animals strongly diminished over a week , the number of ed-1 positive cells continued to increase over time for at least one week . importantly , axonal damage , that was observable at six hours after the injection of collagenase continued for seven days . this was at a time - point well beyond the period of blood - spinal cord barrier breakdown . we know that the staining technique used is only sensitive to the presence of relatively newly generated axonal end bulbs ( newman et al . , 2001 ) . from a behavioral perspective , the rats exhibited locomotor deficits during the first few days , but recovered then quite well . the plasticity of the rat nervous system is unlikely to explain this rapid recovery ( darian - smith , 2009 ) , and thus some of the dysfunction we observed is likely to be owing to axonal conduction block caused by pressure induced by the blood occupying space and the edema in the spinal cord following the injection of collagenase . interestingly , another group recently published a study using a similar model to ours by injecting a different type of collagenase into the spinal cord and they also showed that bleeding affected the integrity of the white matter ( sahinkaya et al . , 2014 ) . in conclusion , the spinal cord - microinjected collagenase model is a simple and very reproducible model that confirmed the important role of hemorrhage after spinal cord injury . we expect that this model will allow a better understanding of the molecules and mechanisms involved and will so have an important impact on the development of new spinal cord injury treatment strategies .

hollow structure materials exhibit usually extraordinary adsorbing capacities to a wide range of species ( i.e. , metal ions , organic molecules , and biomolecules ) and have found practical applications in catalysis , water treatment , and drug delivery . the hollow nanospheres , because of their unique physical and chemical properties , have attracted more significant interest during the last few years [ 5 - 9 ] . up to now , several synthetic strategies have been developed , and a range of hollow nanospheres , especially metal oxides and sulfides , have been fabricated [ 3,6,8,10 - 12 ] , but it is still challenging to develop simple and reliable synthetic methods for hollow nanospheres with diverse chemical compositions , desired chemical / physical stabilities , and controlled size and shell structures ( shell thickness and porosity ) , which are critical for their practical applications . sodium tungsten bronzes ( naxwo3 , 0 < x 1 ) , besides their unique electronic / electric properties that vary greatly with their compositions [ 13 - 17 ] , have inert chemical properties , such as insolubility in water and resistance to most acids except hydrofluoric , which make naxwo3 promising for use in many extreme chemical cases . nanosized naxwo3 , predictably , should have more enriched properties differing from that of the corresponding bulk materials and might find more novel applications , but have barely been explored . we report herein a facile strategy for the fabrication of hollow nanospheres of sodium tungsten bronzes , naxwo3 , and their potential applications in water treatment . the fabrication , including the control on sizes of the spheres and hollow feature of the hollow naxwo3 nanospheres , was achieved through reduction of aqueous sodium tungstate ( na2wo4 ) solution by sodium borohydride ( nabh4 ) powder under well - controlled ph and temperature . the chemical composition , crystalline state , size , and morphology of the as - prepared hollow naxwo3 nanospheres were characterized complementarily using scanning electron microscopy ( sem ) , transmission electron microscopy ( tem , including hrtem ) , energy dispersive spectrum ( eds ) , x - ray photoelectron spectroscopy ( xps ) , and x - ray powder diffraction ( xrd ) . their application in the removal of organic molecules from water was illustrated using different molecules , such as coomassie brilliant blue , albumin bovine , and lysozyme . sodium tungstate , sodium borohydride , hydrochloric acid ( 37% ) , and ethanol were purchased from sinopharm chemical reagent co. , ltd . coomassie brilliant blue , lysozyme , and albumin bovine were from sino - american biotechnology co. ( shanghai , china ) . pure water ( electric resistance of 18.2 m cm ) was produced through an hf super nw water purification system ( heal force co. shanghai , china ) . a typical procedure for the preparation of hollow na0.15wo3nanospheres is as follows : 40 ml of 0.25 m na2wo4aqueous solution was put in a 250 ml flask and the ph of the solution was adjusted to 6.8 using concentrated hcl ( 37% ) . then , 0.025 mol of nabh4powder was added gradually into the na2wo4solution , and the mixture was stirred at room temperature ( ~25 c ) for 2 h. after the reaction , the brown precipitate was separated from the reaction system by centrifugation , washed three times with pure water and two times with ethanol , and finally dried at 80 c under a vacuum . solid na0.15wo3nanospheres were prepared under almost the same conditions used above except that the reaction temperature was 100 c and that the nabh4powders must be added step - by - step because the reaction at 100 c takes place vigorously . the na0.15wo3was first dispersed into water or buffer ; the stock solutions of coomassie brilliant blue or proteins were then added to the na0.15wo3suspension and incubated on the shaker . uv vis absorption spectra of coomassie brilliant blue and proteins in the supernatant were recorded at different time intervals to follow the adsorption process . the gel electrophoresis was run on a dyy-6c electrophoresis system ( liuyi electrophoresis co. , beijing , china ) . the standard 15% sds polyacrylamide gel was used and was run under constant voltage of 50 mv . scanning electron microscopy images were acquired on a sirion 200 field emission scanning electron microscope ( fei company , usa ) . tem images and energy dispersive spectra ( eds ) were taken on a jsm-2010 transmission electron microscope ( jeol ltd . , the powders of na0.15wo3nanospheres were first suspended in water and then transferred on to silicon substrates or copper tem grids for the sem and tem measurements , respectively . xrd patterns were recorded on a d / max 2200/pc diffractometer ( rigaku corporation , japan ) using cu k radiation , = 1.54 . xps measurement was performed on an axis ultra dld instrument ( kratos analytical , uk ) using a monochromatized al ( k ) source . vis absorption spectra were recorded on a uv-2550 spectrometer ( shimadzu corporation , japan ) . the brunauer emment teller ( bet ) specific area was measured on asap 2010 m / c surface area and porosimetry analyzer ( micromeritics instrument corporation , usa ) based on n2adsorption . in general , the bulk sodium tungsten bronzes can be prepared through the following chemical reaction [ 20 - 23 ] : in the reaction , the hydrogen generated from the hydrolysis of nabh4 under acidic reaction condition was partially consumed to reduce tungstate to tungsten , and the rest was released from the reaction system to the air . therefore , in practice , to prevent a rapid loss of hydrogen and to enhance the reduction ability of nabh4 , the aqueous solutions of na2wo4 and nabh4 were mixed first , and the initial ph of mixture solution was maintained at 11 or above . the na2wo4 reduction was initiated subsequently by adjusting the ph of the mixture down below 7 by adding acid , such as hcl . thus , there were not too many hydrogen gas bubbles accumulated in the reaction system , the loss of the hydrogen gas could be suppressed , and powder of bulk sodium tungsten bronzes was obtained finally . in this work , instead of mixing two pre - prepared solutions , the reaction was conducted by adding the nabh4 powder directly into the na2wo4 aqueous solutions . however , we found that when the ph of the na2wo4 aqueous solution is above 10 , the reaction took place very slow ; under the acidic condition , ph < 6 , the nabh4 was hydrolyzed rapidly and the as - generated hydrogen bubbles escaped from the reaction system severely . hence , in a typical procedure of preparing naxwo4 nanospheres in the work , na2wo4 aqueous solutions with ph near to neutral ( typically , 6.97.2 ) were prepared first , and nabh4 powder was then added gradually into the na2wo4 solutions under moderate stirring at room temperature ( ~25 c ) . the total amount of nabh4 added was usually three times of na2wo4 ( molar ratio ) to ensure the reduction of tungstate to tungsten . after completion of the reaction , the solid product was collected by centrifugation and was washed thoroughly using pure water and ethanol , and finally dried at 80 c under a vacuum ( 0.01 torr ) . scanning electron microscopy image , in fig . 1a , shows that the solid products are nanospheres with sizes ranging from a few 10 to 200 nm in diameter . as pointed out with arrows in fig . 1a , some broken nanospheres have a vacant interior structure , and the shell thickness of the broken nanospheres is about 25 nm . this provides us with a hint that the as - obtained nanospheres might have a hollow structure . to confirm this assumption , the nanospheres were subjected to tem measurement . as depicted in fig . 1b , the tem image of each nanosphere possesses the dark edge and bright center illustrating unambiguously their hollow nature . the averaged shell thickness of hollow spheres measured from the tem images is ~25 nm . this is in full agreement with the data ( ~25 nm ) measured on sem images of the broken nanospheres ( indicated via the dark arrows in fig . 1a ) , circular nanoholes ( ~2040 nm in diameter ) were observed on the shells of some nanospheres implying the formation of the open - shell hollow structures . it is impossible to take the images of the hollow nanospheres from all the directions at the same time , so the distribution of the nanoholes is unknown at the moment for us . thearrowsindicate the broken hollow nanospheres from which the thickness , ~25 nm , of the shell of the hollow nanospheres was estimated.btem image of the hollow na0.15wo3nanospheres . thedark edgeandbright centercharacter of the tem image of the nanospheres reveal the formation of the hollow structure the chemical compositions and crystallinity of the as - synthesized hollow naxwo3 nanospheres were characterized complementarily using xrd , hrtem , xps , and eds . as illustrated in figure s1 , supplementary material 1 , the xrd patterns demonstrated that the hollow naxwo3 nanospheres are amorphous . this was verified independently by the hrtem image ( see figure s2 , supplementary material 1 ) on which there is no crystal lattice observed . figure 2 shows the xps spectrum of w in the hollow naxwo3 nanospheres . the two major w 4f7/2 and 4f5/2 peaks centered at 35.75 and 37.58 ev are assigned to the w bound to oxygen . the corresponding binding energies of two relatively weaker w 4f7/2 and 4f5/2 peaks , 33.75 and 35.95 ev , are in agreement with the expected values for w bound to oxygen . the ratio of w to w estimated from the integrated areas of the aforementioned w 4f xps peaks is about 0.18 [ means w/(w + w ) = 0.18/(0.18 + 1 ) 0.15 ] . the eds results acquired from the same hollow nanospheres were depicted in figure s3 . the as - determined na content is of ~0.15 ( atomic ratio to w ) , which is in full agreement with the xps result . xps spectra of w ( 4f7/2and 4f5/2 ) in the hollow na0.15wo3nanospheres several mechanisms have been proposed for the formation of the nanosized hollow structures . the kirkendall effect ( simply be interpreted as an interfacial solid - state chemical reaction ) has been widely used to explain the formation of hollow structures via solid substance as the reactant as well as the hard template . more recently , a gas liquid interface aggregation mechanism was introduced to interpret the formation of hollow nanostructures with the gas bubble as a soft template . the gas liquid interface aggregation mechanism consists typically of three steps : the nanoparticle formation , diffusion , and aggregation . differently , in our case , we believe that the hydrogen gas bubbles accumulated in the reaction system play dual roles : reducing chemically the tungstate to tungsten and guiding the formation of hollow na0.15wo3 nanospheres . during the reaction , na2wo4 was reduced to na0.15wo3 at the interfaces of hydrogen gas bubbles and reaction solution , and the formed na0.15wo3 condensed in situ at the interface forming the hollow structure . liquid interface aggregation procedure , but more similar to kirkendall effect . to confirm the indispensability of the hydrogen gas bubbles as templates for the formation of hollow structure , the temperature for na2wo4 reduction with nabh4 was raised from 25 to 60 , 80 , and 100 c while other reaction conditions were kept the same . generally , high temperature accelerates the gas release from the reaction solution , thus would affect the amount of the hydrogen gas bubbles accumulated in the reaction solutions . as expected , the percentage of solid sodium tungsten bronzes nanoparticles in the product was increased with the increase in temperature . at 100 c , additionally , during the course of the reaction , some hydrogen gas bubbles in the reaction solution unavoidably escaped from the solution before they were fully covered by na0.15wo3 , which results in the formation of the holes on the hollow shells , see fig . afesem andbtem images of solid na0.15wo3nanospheres the metal oxide hollow nanoparticles , such as and fe2o3 , fe3o4 , mno2 , and tio2 , have been used as absorbents for removing the pollutants from water [ 1 - 4 ] , however , due to their reactions with acids , most of them can not be stable in acidic water . thus , the removal of pollutants from water using the metal oxides was usually performed under neutral or weak basic condition . differently , the as - prepared na0.15wo3 nanospheres are resistance to most acids . we found that after being immersed in water with ph = 2 for 2 days , the size and the hollow structure of the na0.15wo3 nanospheres were still preserved well ( figure s5 ) . nitrogen adsorption isotherm showed that the bet specific area of hollow na0.15wo3 nanospheres ( fig . 1 ) is 33.8 m g , which is much larger than that ( 9.3 m g ) of the same size solid na0.15wo3 nanospheres ( fig . the resistance to acids and large specific area of the as - obtained hollow na0.15wo3 nanospheres suggest that the hollow na0.15wo3 nanospheres might be an optimal adsorbent to remove organic pollutants from acidic waste water . to test this assumption , in a typical experiment , 100 mg of hollow na0.15wo3 nanospheres was suspended in 2 ml , 60 g / ml of coomassie brilliant blue ( a common dye ) aqueous solution with ph = 2 . the concentration variation of the coomassie brilliant blue in the supernatant as a function of adsorption time was followed using uv vis spectroscopy . as shown in fig . 4 , 87% of the coomassie brilliant blue was adsorbed within 300 min by the hollow na0.15wo3 nanospheres at room temperature . for comparison , a similar experiment was performed with the solid sodium tungsten bronzes nanoparticles as adsorbent . as depicted in fig . 4 , after 300 min , only 50% of the coomassie brilliant blue was adsorbed by the solid sodium tungsten bronzes nanoparticles . considering that the specific area of the hollow na0.15wo3 nanospheres is almost three times of that of the solid na0.15wo3 nanospheres , we , thus , believe that the surface absorption should play main roles for the removal of the dye molecules from water . in order to investigate the effects of ph value of waste water on the removal capacity of the hollow na0.15wo3 nanospheres , the ph values of the coomassie brilliant blue aqueous solutions adsorption abilities of the hollow and solid na0.15wo3nanospheres to coomassie brilliant blue.yaxis is the percentage of coomassie brilliant blue adsorbed at the corresponding incubation time hollow na0.15wo3nanospheres could also be used to remove biomacromolecules from water . the adsorption abilities of the hollow na0.15wo3nanospheres to albumin bovine ( mw , 66 kda ) and lysozyme ( mw , 14.3 kda ) were determined using gel electrophoresis and uv vis spectroscopy . figure 5a presents the images of sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds page ) of mixture ( albumin bovine to lysozyme is 1:3 in weight ) of two proteins before and after incubation with the hollow na0.15wo3nanospheres for 5 and 15 min , respectively . lane 2 and 3 show the supernatants after incubation with the hollow na0.15wo3nanospheres for 5 and 15 min , respectively . as seen from the intensities of the protein lanes , after 15 min adsorption , ~50% of albumin bovine and ~95% of lysozyme were adsorbed . the protein concentration of each samples , before and after the adsorption , were also precisely determined using uv vis spectroscopy . 95% of lysozyme was adsorbed , while only 50% of albumin bovine was adsorbed by the same amount of the hollow na0.15wo3nanospheres . . such adsorption ability difference suggested that the large size protein could mainly be adsorbed on the outer surface of the hollow na0.15wo3nanospheres , while the small size protein might be adsorbed on both the outer and inner surfaces of the hollow nanospheres . additionally , the different adsorption abilities to the proteins with different sizes could also be caused by the surface charge and structure difference of the proteins themselves . nevertheless , the facts that coomassie brilliant blue and proteins with different sizes could be adsorbed by the hollow na0.15wo3nanospheres suggest that the hollow na0.15wo3nanospheres should be potentially useful in water treatment . athe image of gel electrophoresis of albumin bovine and lysozyme.lane 1 , the mixture ( 1:3 in weight ) of the two proteins;lane 2and3 , the mixture ( 1:3 in weight ) of the two proteins after 5 min and 15 min incubation with hollow na0.15wo3nanospheres , respectively.buvvis spectra of albumin bovine and lysozyme before and after incubation with hollow na0.15wo3nanospheres for 15 min the hollow sodium tungsten bronze , na0.15wo3 , nanospheres have been successfully fabricated using the hydrogen gas bubbles as reactant to reduce the tungstate to tungsten and as template to direct the hollow structure formation as well . this , to our best knowledge , is the first example of using hydrogen gas bubbles as reactant and template at the same time to prepare nanosized hollow materials , and should provide a general means for preparing other inorganic nanosized hollow materials . the resistance to most acids and the pronounced removal capacity of the as - synthesized hollow na0.15wo3nanospheres to small organic molecules and proteins from acidic waste water should find widespread applications in water treatment . further studies on tailoring the surface chemistry and the shell porosity of the hollow na0.15wo3nanospheres would be essential to their practical applications and are under current investigation . the online version of this article ( doi:10.1007/s11671 - 009 - 9383-x ) contains supplementary material , which is available to authorized users . this work was supported by the national basic research program ( 973 program ) of china ( no . 2007cb936000 ) , the national high technology research and development program ( 863 program ) of china ( no . 2006aa04z309 ) , and the shanghai pujiang scholarship program ( nos . 06pj14025 , 06pj14030 ) .

in summary , our investigations reveal a close interplay between the glycopeptide geometry and the antifreeze properties of afgp . both afgp8 and s - afgp4 can adopt amphipathic structures wherein the alignment of the carbohydrates onto one face is governed by the proline content , which imparts rigidity to the afgp template and enhances the ppii helical content . this is important , as recent studies on larger afgp synthetic derivatives revealed lower thermal hysteresis gaps in afgp lacking prolines , which may be due to folded structures in these larger systems . the three key structural elements identified by synthetic and mutation studies(i ) an n - acetyl group in galactosamine , ( ii ) an -glycosidic linkage , and ( iii ) the thr -methyl group mainly restrict the conformational space available to the glycopeptide . in fact , the presence of the n - acetyl group in galactosamine locks the conformation of the sugar relative to the peptide bond via strong h - bonds and bridge waters , as observed in cold - spray ionization time - of - flight mass spectroscopy experiments . the antifreeze activity of afgp however is closely related to the modulation of the surrounding solvent h - bond network due to the glycopeptides . radial distribution functions , tetrahedral order parameters , and water water h - bond autocorrelation functions reveal the importance of free hydroxyl groups on gal modulating the water h - bond network . the formation of water bridges on the surface of the glycopeptide by gal affects the local tetrahedral order of the water molecules in the first solvation shell . this effect causes disorder in the h - bond network , which propagates to the remaining solvation shells at low temperatures , as evidenced by the water dynamics in 10.012.0 water shells around both afgp8 and s - afgp4 . this observation is in accordance with both the suppression of antifreeze activity on the addition of borate and retardation of long - range hydration dynamics observed in terahertz spectroscopy experiments . it must also be noted that upon the removal of terminal gal monosaccharide afgps exhibit very weak antifreeze activity , which agrees with our observation of the importance of gal in restructuring the surrounding solvent . the direct dependence of the antifreeze activity on the solvent restructuring by gal also addresses the observance of similar antifreeze activity for afgp8 and s - afgp4 even though the latter glycopeptide exibits enhanced conformational flexibility upon the loss of proline rigidity . interestingly , the proposed long - range effect on water dynamics at 250 k does not occur at 300 k. this suggests that the long - range disordering effect only manifests itself in the more ordered water environment at lower temperature , again in agreement with terahertz studies . such increased order is proposed to allow the local perturbation of water structure in the first hydration layer of the glycopeptide to be communicated to regions 10 or more from the solute , thereby leading to the antifreeze activity . the detailed structure mechanism correlation provided in this study could lead to the design of better synthetic afgp variants . on the basis of these results , it would be worthwhile to investigate modifications of the carbohydrate that facilitate the formation of bridged waters , thereby modulating h - bond dynamics and affecting the overall antifreeze activity . one such modification could be the incorporation of 1,2,3,4,5,6-cyclohexanehexol derivatives in place of the terminal gal monosaccharide . since the synthetic variants utilize chemical polymerization for the synthesis of oligomeric afgp , it might be difficult to incorporate proline residues at select locations . however , attempts could be made to increase the hydrophobic content of the amino acids , i.e. , at*v or vt*v , which may impart rigidity to afgp akin to the presence of proline . additionally , these results can be used to describe the antifreeze activity of the c - linked afgps and other synthetic variants like the afgp diastereomers . md simulations were performed with the charmm program . the charmm22 protein force field with cmap ( dihedral correction map ) , the charmm carbohydrate force field , and the modified tip5p or tip4p-2005 water models were used to represent the afgp systems in solution . the initial geometries of afgps were constructed in accordance with the available nmr data . for the carbohydrates , the h c2h2 dihedral in the n - acetyl side chain in galnac was set to the anti ( 180 ) configuration , while the o1ccn dihedral in the o - linkage was set to the gauche+ ( 60 ) configuration . for the peptides , the dihedral was set to 150 for all the amino acids , while the dihedral was set to 145 for thr and 75 for the remaining amino acids . the rest of the geometry was constructed from the topology information present in the force field . these initial geometries were subjected to a 1000-step steepest descent ( sd ) minimization followed by an adopted basis newton raphson ( abnr ) minimization to a force gradient tolerance of 10 kcal / mol / . the minimized geometries were then immersed in a pre - equilibrated cubic water box of size 65 65 65 . the size of the water box was selected on the basis of the condition that it extended at least 15 beyond the non - hydrogen atoms of afgp . water molecules with the oxygen overlapping with the non - hydrogen solute atoms within a distance of 2.8 were deleted . for all of the subsequent minimizations and md simulations , periodic boundary conditions were employed using the crystal module implemented in the charmm program . system equilibration was initiated with a 50-step sd and 50-step abnr minimization followed by a 100 ps simulation in the nvt ensemble at 300 k during which mass - weighted harmonic restraints of 1.0 kcal / mol / were applied on the non - hydrogen atoms of afgp . a 200 ps npt simulation at 1 atm and 300 k followed the nvt simulation , wherein all the previous restraints were removed . in the npt simulation , the center of mass of the afgp was restrained near the origin by applying a harmonic restraint of 1.0 kcal / mol / using the mmfp module in charmm . the long - range electrostatic interactions were treated via the particle mesh ewald method with a real - space cutoff of 12 , a kappa value of 0.34 , and a sixth - order spline.nonbond interaction lists were updated heuristically out to 16 with a force switch smoothing function from 10 to 12 used for the lennard - jones interactions . the leapfrog integrator employing an integration time step of 1 fs was used in conjunction with the shake algorithm to constrain all covalent bonds involving hydrogen atoms . a constant pressure of 1 atm was controlled using the langevin piston with a mass calculated using the equation pmass = integer(system mass/50.0 ) . hrex md production simulations were performed using the repdstr module of a modified version of charmm c36a2 . the hrex simulations were started from the equilibrated coordinates obtained after the 200 ps unbiased npt simulation at 1 atm and 300 k. the same harmonic restraints used in the npt runs were utilized in the hrex runs to constrain the afgp at the center of the simulation box . an exchange between neighboring replicas was attempted every 1000 md steps , and the coordinates were saved every 1 ps . each replica was simulated for 10.5 ns , thereby amounting to a cumulative simulation time of 84.0 ns ( 10.5 ns 8) , and the trajectories from the first replica ( unbiased , ground state replica ) were used for subsequent analysis . a combination of the two - dimensional ( 2d ) dihedral grid - based energy correction map ( cmap ) extension of the charmm force field and a saxon woods potential was used as the biasing potential across the different replicas . two cmap biasing potentials were used , corresponding to the sugar s/s and protein / dihedral pairs to sample the conformational space of afgp . woods potential was used to enhance the conformational sampling about the sugar s dihedral in the thr side chain.where h = ( n 0.75 ) kcal / mol , with n going from 0 to 7 for replicas 18 , p1 = 0.1 , p2 = 0.3 , and ref = 90. the biasing potential cmaps were obtained using an established protocol for glycopeptide o - linkages that has been reported in detail earlier and successfully applied in studying o - linked glycopeptides . to study the influence of afgp on the structure of the surrounding solvent at a subfreezing temperature , additional hrex simulations were performed at 250 k using both the tip5p and tip4p-2005 water models . these simulations were initiated from representative structures from the largest clusters obtained by clustering the 10.5 ns unbiased 300 k hrex simulation trajectory using the peptide / dihedrals . the same protocol described for the 300 k simulations was used to set up and run the 250 k simulations . the major differences were that the representative structures from the 300 k simulation selected on the basis of rmsd clustering ( see figures 2 and 3 ) were immersed in a water box ( either tip5p or tip4p-2005 water model ) pre - equilibrated at 250 k and the temperature in the remainder of the simulations was maintained at 250 k. for afgp8 , two hrex simulations were performed for 3 ns each amounting to a cumulative simulation time of 48.0 ns ( 3.0 ns 2 8) , while , for s - afgp4 , three hrex simulations were performed for 3 ns each amounting to a cumulative simulation time of 72 ns ( 3.0 ns 3 8) . the use of multiple simulations allows the solvent to reorganize around the different conformations of the glycopeptides , as initial calculations at 250 k showed the glycopeptides to maintain their starting conformations . the representative structures were also used to initiate 4 ns md simulations at 250 and 300 k ( two simulations for afgp8 and three simulations for s - afgp4 for both water models ) , which were used to calculate the h - bond autocorrelation function in figure 7 and figure s5 ( supporting information ) and the diffusion coefficient ( table s1 , supporting information ) . to delineate the influence of carbohydrates with respect to protein , hrex simulations at 300 and 250 k were also performed on the two peptides : aataatpataatpa and ataataataata . the protocol described to set up the 300 k afgp simulations was used to set up the 300 k protein simulations . this included retaining the restraints on the protein / to set up the initial protein geometry . following equilibration , hrex md simulations were performed for 2.0 ns , a cumulative sampling time of 16.0 ns ( 2.0 ns 8) at 300 k. the 250 k hrex simulations ( using both the tip5p and tip4p-2005 water models ) were set up using the top representative structure obtained by clustering the first 1.0 ns of the 300 k unbiased replica simulation trajectory using the / dihedrals . simulations at 250 k were run for 1.0 ns , leading to a cumulative sampling time of 8.0 ns ( 1.0 ns 8) . 3d probability distributions of the selected bridge water oxygens ( figure 4 ) were constructed from snapshots output every 20 ps from the 300 k hrex trajectory of the unbiased ground state replica . these coordinates were binned into 1 1 1 cubic volume elements ( voxels ) of a grid spanning the entire system .

it provides cis - elements , such as promoters and binding motifs , and trans - elements , such as transcriptional factors ( tfs ) . the promoters of target genes came from two sources : experimental determination and sequence - based computational prediction . hand curation was applied as a crucial part of the data collection to ensure data accuracy . based on the reliability of the supporting evidence for each promoter , a quality level was assigned . one key feature of tred is the easy access to interaction data between tfs and the promoters of their target genes , including binding motifs reported by the previous studies . although part of the data was obtained from the existing gene regulation resources , most of the data came from our exhaustive literature curation . the tf - binding motifs were mapped on to the promoter sequences of their target genes . along with the binding motifs , a binding quality level was assigned based on definitiveness of the binding evidence , which was determined by the experimental approaches employed to demonstrate the binding and data interpretation from experts . known as the binding quality level to a binding that has been proved by gel - shift competition , dnase i footprinting , etc . in order to provide users with more complete information of the genes , cross - references to other well - known database such as pubmed , genbank , genecards ( 1 ) and transfac ( 2 ) were established in tred . a comprehensive description of the content and the structure of tred has been published earlier ( 3 ) . in addition , many on - the - fly tools were implemented for the analysis of sequences retrieved from tred as well as imported from other resources . the user interface and software functionality were also described in the previous report ( 3 ) . upon the emergence of high - throughput technologies , a huge amount of large - scale gene expression and regulation profiling data have been made available by microarray and chromatin immunoprecipitation ( chip - chip ) studies . to uncover grns among the identified genes we used our promoter prediction program firstef ( 4 ) to predict promoters in the genomes of human , mouse and rat . these promoters were then combined with the known promoters extracted from epd ( 5 ) , dbtss ( 6 ) , genbank , etc and were deposited in our database cshlmpd ( 7 ) . it should be noted that cshlmpd also contains genes without any promoter . in this version , the number of genes with promoter(s ) in each genome assembly is lower than that of the previous version after removal of the redundancy . however , the number of known promoters and their related genes are close in both versions . there are more promoters than genes in each species due to alternative transcription start sites in many genes ( 7 ) . table 1 gives the statistics of promoters and genes in each quality category . number of promoters and genes in tred , with gene numbers in parentheses promoter qualities are ranked from high to low : 1 , known , curated promoters ; 2 , known , pipeline collected promoters ; 3 , predicted promoters with refseq evidence and putative promoters taking 5 ends of refseq as tss ; 4 , predicted promoters with mrna ( other than refseq and est ) evidence ; 5 , predicted promoters with est evidence ; 6 , predicted promoters supported only by gene prediction . promoters included in a higher ranking are automatically excluded from the lower ranking categories . the human genome codes for 1850 tfs , which account for 6.0% of its estimated total number of genes ( 8) . it is a daunting task to collect and curate comprehensive and precise interaction data between the tfs and their target genes . since cancer is one of the greatest threats to human health and has been a field under extensive study , including a broad interest in understanding cell cycle regulatory networks , we started out by focusing on target genes of cancer - related tfs . previously , tred contained mainly the target genes and promoters for tf families e2f and myc ( 3 ) . in this new release , we expanded it to 34 new tf families that have been implicated in cancer pathways , including p53 , ap1 , er and nfb / rel . they are involved in many cellular processes , such as proliferation , differentiation , cell motility and apoptosis . there are totally 9308 newly collected target genes ( 5365 in human , 2526 in mouse and 1417 in rat ) and 10 251 target promoters ( 5956 in human , 2736 in mouse and 1559 in rat ) for these tf families . the detailed distribution of the target promoters and the target genes is listed in table 2 . number of curated target promoters / genes for the 36 tf families although transfac also provides factor - site interaction data , it contains less information for the tfs and their target genes available in tred . its latest version ( version 7.0 ) has collected 1040 factors and 608 genes for human and 765 factors and 417 genes for mouse ( 2 ) . therefore , on average each factor has less than one target gene . in contrast , the number of target genes per tf in tred is much higher . for example , there are > 200 target genes on average for each tf family for human in tred ( table 2 ) . this can provide fairly resolved gene regulatory networks ( grns ) for the 36 tf families involved in cancer pathways . in addition to this , tred contains relatively complete genome - wide promoter annotation for human , mouse and rat . moreover , the binding sites in tred were also mapped on to the assembled chromosomes . these absolute genomic positions make it ready to associate tred with other genomic data for various studies . however , it should be noted that transfac also collects factor site interaction data of species other than human , mouse and rat . therefore , although tred and transfac overlap to certain extent , they complement each other at some aspects . the accurate and comprehensive knowledge of transcriptional regulatory elements in tred allows one to construct the grns for a given tf family by bringing all tf target gene pairs together . in our initial analysis , we found that some tfs are the target genes of other tfs and often more than one tfs control the expression of the same gene . furthermore , some target genes affect the expression or stability of tfs by feedback loop . as an example , figure 1 shows a simplified grn for tf family gli ( glioma - associated oncogene homolog ) . for the tf families with hundreds of target genes , such as ap1 , cebp and ets there are cross talks between the networks of different tf families through the same target genes or through direct interactions between the members of different tf families . the experimental evidence for each interaction between a tf and its target gene is available through the references provided in tred . this is an advantage over other networks computationally predicted from expression and/or phylogenetic profiles . in this release , grns for the tf families have been generated from the collected interaction data and statically stored in tred . the dynamic links to grns will be provided in the query result in the future . taken together , tred can facilitate to decipher the grns and help researchers to better understand the gene regulatory mechanisms . sample pages showing access to the gene regulatory network of tf family gli ( glioma - associated oncogene homolog ) in human . upon the emergence of high - throughput technologies , a huge amount of large - scale gene expression and regulation profiling data have been made available by microarray and chromatin immunoprecipitation ( chip - chip ) studies . to uncover grns among the identified genes we used our promoter prediction program firstef ( 4 ) to predict promoters in the genomes of human , mouse and rat . these promoters were then combined with the known promoters extracted from epd ( 5 ) , dbtss ( 6 ) , genbank , etc and were deposited in our database cshlmpd ( 7 ) . it should be noted that cshlmpd also contains genes without any promoter . in this version , the number of genes with promoter(s ) in each genome assembly is lower than that of the previous version after removal of the redundancy . however , the number of known promoters and their related genes are close in both versions . there are more promoters than genes in each species due to alternative transcription start sites in many genes ( 7 ) . table 1 gives the statistics of promoters and genes in each quality category . number of promoters and genes in tred , with gene numbers in parentheses promoter qualities are ranked from high to low : 1 , known , curated promoters ; 2 , known , pipeline collected promoters ; 3 , predicted promoters with refseq evidence and putative promoters taking 5 ends of refseq as tss ; 4 , predicted promoters with mrna ( other than refseq and est ) evidence ; 5 , predicted promoters with est evidence ; 6 , predicted promoters supported only by gene prediction . promoters included in a higher ranking are automatically excluded from the lower ranking categories . the human genome codes for 1850 tfs , which account for 6.0% of its estimated total number of genes ( 8) . it is a daunting task to collect and curate comprehensive and precise interaction data between the tfs and their target genes . since cancer is one of the greatest threats to human health and has been a field under extensive study , including a broad interest in understanding cell cycle regulatory networks , we started out by focusing on target genes of cancer - related tfs . previously , tred contained mainly the target genes and promoters for tf families e2f and myc ( 3 ) . in this new release , we expanded it to 34 new tf families that have been implicated in cancer pathways , including p53 , ap1 , er and nfb / rel . they are involved in many cellular processes , such as proliferation , differentiation , cell motility and apoptosis . there are totally 9308 newly collected target genes ( 5365 in human , 2526 in mouse and 1417 in rat ) and 10 251 target promoters ( 5956 in human , 2736 in mouse and 1559 in rat ) for these tf families . the detailed distribution of the target promoters and the target genes is listed in table 2 . number of curated target promoters / genes for the 36 tf families although transfac also provides factor - site interaction data , it contains less information for the tfs and their target genes available in tred . its latest version ( version 7.0 ) has collected 1040 factors and 608 genes for human and 765 factors and 417 genes for mouse ( 2 ) . therefore , on average each factor has less than one target gene . in contrast , the number of target genes per tf in tred is much higher . for example , there are > 200 target genes on average for each tf family for human in tred ( table 2 ) . this can provide fairly resolved gene regulatory networks ( grns ) for the 36 tf families involved in cancer pathways . in addition to this , tred contains relatively complete genome - wide promoter annotation for human , mouse and rat . moreover , the binding sites in tred were also mapped on to the assembled chromosomes . these absolute genomic positions make it ready to associate tred with other genomic data for various studies . however , it should be noted that transfac also collects factor site interaction data of species other than human , mouse and rat . therefore , although tred and transfac overlap to certain extent , they complement each other at some aspects . the accurate and comprehensive knowledge of transcriptional regulatory elements in tred allows one to construct the grns for a given tf family by bringing all tf target gene pairs together . in our initial analysis , we found that some tfs are the target genes of other tfs and often more than one tfs control the expression of the same gene . furthermore , some target genes affect the expression or stability of tfs by feedback loop . as an example , figure 1 shows a simplified grn for tf family gli ( glioma - associated oncogene homolog ) . for the tf families with hundreds of target genes , such as ap1 , cebp and ets there are cross talks between the networks of different tf families through the same target genes or through direct interactions between the members of different tf families . the experimental evidence for each interaction between a tf and its target gene is available through the references provided in tred . this is an advantage over other networks computationally predicted from expression and/or phylogenetic profiles . in this release , grns for the tf families have been generated from the collected interaction data and statically stored in tred . the dynamic links to grns will be provided in the query result in the future . taken together , tred can facilitate to decipher the grns and help researchers to better understand the gene regulatory mechanisms . sample pages showing access to the gene regulatory network of tf family gli ( glioma - associated oncogene homolog ) in human . the website ( ) offers the following services : easy access to tred entries through text - based query interface ; search for the target genes of a given tf ; retrieval of the promoter sequences and the tf - binding motifs ; further analysis of the retrieved sequences of promoters and motifs . tred homepage also provides the access to the grns of the tf families in human , mouse and rat , which were constructed from its collection of the interaction data between the tfs and their target genes .

the term of stem cells came up to us via histologists in the nineteenth century , who introduced it as a general , abstract term for cells specifically involved in repair or regeneration . with the discovery in the 1950s that bone marrow cells could reconstitute the hematopoietic systems of irradiated individuals , the modern stem cell concept began to crystallize around the experimental procedures of transplantation and reconstitution [ 1 , 2 ] . the definition for tissue stem cells proposed by potten and loeffler was undifferentiated cells ( relative to a functional tissue ) , capable of proliferation and production of a large number of differentiated functional progeny ; they have the ability of self - maintenance of their population and for regeneration of the tissue after injury . this means that stem cells are defined by virtue of their functional attributes and not by an explicit directly observable characteristic . this functional definition is relative to the stem cell role linked to the functional tissue regeneration feature . but this definition does n't give us any characteristic to identify morphologically the stemness . another point is the fact that it is assumed that stem cells are undifferenctiated and they come from the earlier stages of the development , this means , in a tissue , we can find various types of stem cells , or a stem cell at different points of maturation . ( so , gives a possible way to classify descendent transit and mature cells ) . most over could be that there are specific differentiation markers which would enable a distinction of stem cells in relation to each other and in relation to the functional cells they are eventually producing . flexibility is a key aspect we should include in the definition of stem cells . it may be possible for a stem cell to cease proliferation , that is , become quiescent , in which case it does not act as an actual stem cell , but since it can reenter the cycle it has the potential to act as a stem cell . likewise a transit cell may not normally self - maintain , but may do so under special circumstances , thereby representing a potential stem cell . the recent discovery that stem cell behaviors can be acquired by ordinary cells following the introduction of a small number of genes has intensified its interest . with this background , all the stem cells are the same type of cell , but as we refer before there are different groups of them depending on the differentiation state ; in this sense the three main groups included : toti or pluripotents , multipotents , and commitment cells . the first group are embryonic cells who have the ability to create any kind of tissue . second group are cells more differentiated , still stem cells , which can create any kind of tissue derived from one of the embrionary layers , endoderm , mesoderm , or ectoderm , for example , mesenchymal stem cells ( mscs ) . finally the third group are those who can generate two or more lineages in a tissue , for example , cardiac stem cells ( cscs ) . focusing on adult stem cells , as we referred before , they distribute in the different adult tissues , but it 's very curious they do n't have an aleatory distribution . in fact they use to localize in the most protect areas of the tissue , that is , in heart ; they are more abundant in the atria and in the apex , the two localizations were the pressure that supports the tissue is minor or in the ventricular area in nervous system . we can find lonely stem cells , but generally they distribute in niches ( figure 1 ) . we define a niche like type of cells and extracellular substrates that can indefinitely house one or more stem cells ( scs ) and control their self - reproduction and production of their progeny in vivo . so this means they are specific anatomic locations that regulate how they participate in tissue generation , maintenance , and repair . the niche saves stem cells from depletion , while protecting the host from overexuberant stem cell proliferation . it constitutes a basic unit of tissue physiology , integrating signals that mediate the balanced response of stem cells to the needs of organisms . so functions of niches included : spatial organization , filtration of signals ( proliferative , apoptotic ) , provided supporting cells , specific unions like cadherins , and determined type division ( symmetric or asymmetric ) . the niche may also induce pathologies by imposing aberrant function on stem cells or other targets . the interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues and for the ultimate design of stem cell therapeutics . an important aspect of the niche is that determining type division of the stem cells . these cells are very special in this aspect too , because usually when a cell enters in cell cycle producing two daughter cells similar to it . in the case of stem cells this can change , so there are two options as follows.symmetrical division : the cell gives two daughter cells similar to her , like a regular cell type or two commitment cells.asymmetrical : as the result of a stem cell division we will get two different daughter cells , one similar to the mother and another one that is a commitment cell , so she will be a mature cell ( figure 2 ) . symmetrical division : the cell gives two daughter cells similar to her , like a regular cell type or two commitment cells . asymmetrical : as the result of a stem cell division we will get two different daughter cells , one similar to the mother and another one that is a commitment cell , so she will be a mature cell ( figure 2 ) . once again the regulator of this cellular function is the niche , and this receives the name hypothesis of the free niche . this hypothesis tells us that if there is space for only one stem cell more in the niche , the division will be asymmetrical , but if there is space for two , the division will be symmetrical . when the proteins are homogenously distributed in the cell , the division will be symmetrical ; instead if protein concentrates in one cellular extreme , division will be asymmetrical . they are affected for senescence too , as demonstrated by the anversa 's work [ 7 , 8 ] . telomerase shortening , increase in ros products , and increase in levels of p53 , p16 and p66 were demonstrated in stem cells , all of them shared facts with senescent cells . many efforts are being made in the study of the mechanism implicated in stem cells , with therapeutic purposes . fields like degenerative lesions , tisular necrosis , had all their witness in the application of stem cells . in fact , there are several clinical trials focusing on the substitutive therapy with stem cells , in cardiology , neurology , and orthopedic , but still the results are not as good as researchers and clinicians expect . recently new discovers can be applied to the stem cells knowledge in the oncology field . nowadays the theory of the origin of the tumors is in the stem cells is more and more accepted , and every day there are more researchers focusing their efforts implicated in this aspect . cancer stem cells ( cascs ) is best defined functionally , as a subpopulation of tumor cells that can enrich for tumorigenic property and can regenerate the heterogeneity of the original tumor in immunocompromised mice . the existence of cascs was hypothesized in the 60s , and experimentally isolated in the last decades , first in acute myeloid leukemia and later in solid cancers , such as , breast cancer . importantly , cascs were shown to be resistant to conventional therapies , such as , chemotherapy and radiation . therefore , the prospective isolation , molecular characterization , and therapeutic targeting of cascs in cancer will possibly mark major advances in understanding their pathogenesis . at the same time , the finding that only a small fraction of the cells within malignant tumors can initiate new tumors upon transplantation has led many cancer biologists to embrace the notion that stem cells are the driving force behind malignancies and to advocate redirecting cancer therapy toward controlling or eradicating stem cells . clearly , we live in an era of biology when ideas and theories about stem cells are a major part of the intellectual landscape . the hypothesis of cancer is a stem cell disease includes that in a tumour we have , at least , two types of cell population : adult tumour cells and stem cell like , as well as in normal tissue . it has been suggested that cancer is due to an alteration in the normal homeostasis of stem cells . the abundance of cancer stem cells is derived for their symmetric division , and this would be the point to eradicate for cancer treatment . the tumor stem cell hypothesis indicates that this type of cell has all the characteristics of the stem cell : capability of self - renewal , unlimited proliferation potential , multiline differentiation , formation of new adult cells , and asymmetric division , and they are originated of the formation of metastasis , meaning that all the adult tumor cells are coming from this kind of cell ( figure 3 ) . for these properties they are called initiating tumor cells too , and probably , are the responsible for tumors refractoriness and recurrence . another concept is the circulating tumor cells ( ctcs ) that are cells that have detached from a primary tumor and circulate in the bloodstream . ctcs may constitute seeds for subsequent growth of additional tumors ( metastasis ) in different tissues . cells capable of metastasis also acquire the ability to invade another tissue . for epithelial cancers emt involves epithelial cells losing their epithelial characteristics and acquiring a more mesenchymal phenotype which occurs as a result of cytoskeletal changes within the cells . these changes allow the cell to acquire a more migratory phenotype [ 10 , 12 ] , increasing the probability of tumour cells entering the blood and lymphatic systems . this process is influenced by chemokines and their receptors which are thought to play an important role in tumour development by influencing tumour transformation , survival , proliferation , invasion , and metastasis and also regulation of angiogenesis and tumour - leukocyte interactions . despite this , the majority of circulating tumour cells appear to be destroyed . those that persist may acquire the ability to metastasize and once inside the target organ may undergo mesenchymal - epithelial transition ( met ) , proliferate and if the environment is conducive the disseminated cells may grow to establish a new tumour thus completing the metastatic process . first evidence indicates that ctcs markers applied in human medicine are conserved in other species . five of the more common markers including ck19 are also useful to detect ctcs in the blood of dogs with malignant mammary tumors . standard procedure for isolating circulating stem cells ( ctcs ) involves cell sorting of a subpopulation on the basis of cell surface markers . the detection of ctcs may have important prognostic and therapeutic implications but because their numbers can be very small , these cells are not easily detected . circulating tumor cells are found in different frequencies per ml of whole blood in patients with metastatic disease . to date , a variety of research methods have been developed to isolate and enumerate ctcs . the only usa food and drug administration ( fda ) cleared methodology for enumeration of ctcs in whole blood is the cellsearch system . extensive clinical testing done using this method shows that presence of ctcs is a strong prognostic factor for overall survival in patients with metastatic breast , colorectal , or prostate cancer . morphological appearance is judged by human operators and is therefore subjected to large interoperator variation . several ctcs enumeration methods exist which use morphological appearance to identify ctcs , which may also apply different morphological criteria . a study in prostate cancer showed that many different morphological definitions of circulating tumor cells have similar prognostic value , even though the absolute number of cells found in patients and normal donors varied by more than a decade between different morphological definitions . the behavior of the cells in cancer and metastasis developing will be better known if we will be able to follow these cells . over the last decade , advances in molecular imaging have allowed a deeper understanding of the in vivo behavior of stem cells and have proven to be indispensable in preclinical and clinical studies . there are two main classes of molecular imaging techniques : direct cell labeling and reporter - gene imaging ( figure 4 ) . the former employs contrast agents , such as , magnetic particles , luminescent nanoparticles , or radionuclides to directly label the cell , whereas the latter genetically alters the cell to transcribe and translate a reporter protein . while direct labeling is both straightforward to implement and is commonly used , the contrast signal is diluted with each cellular division and the technique can not distinguish viable cells from dead cells . reporter genes , on the other hand , are only expressed by live cells and the signal is propagated by daughter cells . however , reporter gene imaging requires transfection of genetic material using plasmids , retroviral , or viral vectors , which raises the concern of insertional mutagenesis and may necessitate the use of apoptosis - inducing suicide genes before possible future use in the clinic [ 21 , 22 ] . as one of the first imaging modalities for monitoring pluripotent stem cells in vivo , magnetic resonance imaging ( mri ) offers high spatial and temporal resolution to obtain detailed morphological and functional information . it requires the uptake of a contrast agent by the stem cell , the most common of which are superparamagnetic iron oxide ( spio ) nanoparticles . spios can induce changes in t2 relaxivity at nanomolar concentrations [ 23 , 24 ] . there are two main methods by which stem cells can be directly labeled by spios . one method is magneto operation which involves the coating of anionic spios with cationic transfection agents , such as , protamine sulfate or poly - l - lysine . although many studies have shown that magneto oporation does not affect cell viability or function at low doses [ 2628 ] , there is evidence that high doses can inhibit mesenchymal stem cell ( msc ) migration and colony formation ability . several groups have shown the use of spios for noninvasive mri of neural stem cell migration , engraftment , and morphological differentiation [ 30 , 31 ] . the contrast signals in these studies were detected for up to six weeks and the stem cells retained the ability to proliferate and differentiate . other groups have shown that mri can be used to track mesenchymal stem cells ( mscs ) in cardiac repair after myocardial infarction . here however , one disadvantage inherent to both spio - labeling methods is their inability to distinguish viable cells from dead cells or from scavenging macrophages . the advantages of radioscintigraphic techniques include their picomolar sensitivity , good tissue penetration , and translation to clinical applications . in fact , radionuclide imaging is the sole direct labeling technique used thus far in human studies , involving both autologous bone marrow - derived stem cells and peripheral hematopoietic stem cells [ 3436 ] . there are two main techniques for radionuclide imaging : positron emission tomography ( pet ) and single photon emission computed tomography ( spect ) . spect tracers directly emit a gamma ray in one direction , in contrast to pet tracers , which send two gamma rays in opposite directions and thus possess coincidence detection with a higher spatial resolution . however , spect is generally less expensive due to its longer - lived and more readily available radioisotopes . the most widely used pet isotopes are fluorine-18 ( 18f ) , which has a half life of 110 minutes . cu can also be bound to a lipophilic redox - active carrier molecule , pyruvaldehydebis(n4-methylthiosemicarbazone)(ptsm ) . cu - ptsm has been used to image hescs differentiated towards renal lineages in fetal rhesus monkeys and has been shown to lack adverse cellular effects . the most widely used spect radionuclides are indium-111 ( in ) , with a half life of 67 hours and the metastable technetium-99 m ( mtc ) , with a half - life of 6 hours . while in provides a longer time window for cell imaging , mtc can be used in higher doses to improve short - term imaging resolution . several groups have used in to image in vivo trafficking and biodistribution of mscs around sites of myocardial injury in the canine [ 40 , 41 ] and porcine animal models . human clinical studies have also used in - oxine [ 35 , 43 , 44 ] to assess stem cell trafficking in acute and chronic myocardial infarction . although both pet and spect offer great sensitivity , there are several disadvantages to both techniques , including the leakage of radionuclides into nontarget cells , limited time window for imaging due to half - life decay , lower spatial resolution as compared to mri , and the emission of ionizing radiation that may impair stem cell proliferation and survival . stem cells share many properties in common with cancer cells , including self - renewal , rapid proliferation , lack of contact inhibition , and high telomerase activity [ 46 , 47 ] . furthermore , cellular manipulations , such as , the reprogramming of somatic cells into induced pluripotent stem cells ( ipscs ) , transfection of reporter genes , and overexpression of survival genes , can have unintended tumorigenic side effects . teratoma formation is another concern , along with its potential to degenerate into malignant teratocarcinomas . given these risks , understanding stem cell tumorigenicity is of paramount importance for future clinical applications . one study using a double fusion reporter containing enzyme firefly luciferase ( fluc ) and gfp showed that teratoma formation is dependent upon cell number . assessed over a period of eight weeks , a minimum of 1 105 intramyocardially furthermore , a lower threshold of 1 104 cells following hind limb injection was required to form teratomas , providing insight into niche dependency . since angiogenesis is known to play a major role in tumor growth and development , it is important to investigate not only the tumor itself but also its supporting stroma . one study used sodium iodide symporter ( nis ) reporter imaging to show that mscs actively home in on growing tumors , where they differentiate into vasculature and supporting structures . the upregulation of v3 integrin is also known to play a role in tumor angiogenesis . one study used direct pet imaging with cu - dota - rgd4 to target v3 integrin , which successfully visualized in vivo hesc teratoma formation in the mouse model . . moreover , pet imaging may have promising clinical applicability for monitoring tumorigenicity in humans because bli lacks the ability to penetrate deep tissues . due to the risks of teratoma formation , having a reporter gene that serves as both an imaging modality as well as one study used hsv1-tk pet reporter imaging to selectively destroy emerging teratomas with the administration of ganciclovir . future directions for mitigating the risks of tumorigenicity include not only the use of reporter - suicide genes , but also vector - and transgene - free reprogramming of somatic cells into ipscs and long - term multimodality imaging capable of observing both emerging tumor cells and their supporting stroma . although a great deal of information is already known about the survival , biodistribution , tumorigenicity , and immunogenicity of pluripotent stem cells , significant gaps in knowledge remain . molecular imaging will continue to play a pivotal role in answering crucial questions about clinical applications as well as in helping us understand the underlying mechanisms of stem cell biology . in fact , a new imaging agent , radio - labelled hedgehog , detects cancer stem cells , potentially allowing for imaging of stem cell - like cancer cells by positron emission tomography ( pet ) in patients with breast cancer , according to results of a pilot study , presented at the american association for cancer research 101st annual meeting 2010 . jennifer sims - mourtada , ph.d . , director of molecular research and development at radiomedix , inc . , houston , texas and colleagues tested the ability to detect breast cancer stem - cell - like populations using a protein , sonic hedgehog that was radiolabeled with the positron emitting isotope gallium-68 . increased activation of the hedgehog pathway binding of the radio - labelled hedgehog to the patched-1 hedgehog receptor on the surface of breast cancer cells occurred , suggesting potential for molecular imaging of breast cancer by pet . a significant increase in binding more in vivo molecular imaging studies must be conducted to confirm long - term survival of these cells . the safety of stem cell therapy in terms of tumorigenicity and immune rejection must also be thoroughly examined . to that end , molecular imaging studies capable of evaluating the risk of cancer formation long - term or assessing methods of immune suppression for viable engraftment are highly valuable .

this paper is based on the topic evaluation of the effects of iranian phase changed material cold vest on the physiological indices in hot and dry temperature in climatic chamber , appropriate for those who are interested to understand the body central temperature in which increase in those people who work in hot climates is natural phenomena , instead of wearing of suitable cloth , could be lead to thermal strain . however , the symptoms of expose to high temperature fatigue , thermal weakness , thermal syncope , thermal trauma , and ultimately death are also explained . in the usa , annually , around 510 million workers expose to heat stresses . every year , only in hong kong , in military and training operations , two percent , and in britain more than 80 service staff get to the hospital . therefore , controlling this physical factor , can prevent these health problems and can increase the staff efficiency and the quality of products . according to the statistic of iran central health and work environment , in the year of 2010 , the overall number of industrial workshops was 625,000 which have 2,500,000 workers , of which 10% of these workshops and 9% of the workers are exposed to these heat and humid harmful conditions . personal cooling systems include active systems ( e.g. , air or liquid cooling garment ) and passive systems ( e.g. , ice vest , gel - ice vest , and phase change materials [ pcms ] ) . active systems require a source of power to operate while passive systems require material supplies such as cold water and ice . although many physiology studies have demonstrated that personal cooling systems reduce heat strain , the actual use of personal cooling systems in practice is limited . the main barriers are that personal cooling systems require logistic supply such as ice , power source , and cold water , and add an extra load of several kilograms to users . organic materials can be further classified into paraffin and non - paraffin 's such as esters , fatty acids , alcohols , and glycols . pcms have been put into use for several innovative applications such as cooling of electronic devices , transporting sensitive medications , and cooling vest for athletes . thermal energy storage with pcms is one of the most efficient ways of storing available energy because of its advantages such as providing higher heat storage capacity , lower storage temperature , isothermal operation , and less storage space . pcm is a substance with a high heat of fusion , in which , melting and solidifying at a certain temperature , is capable of storing and releasing large amounts of energy . heat is absorbed or released when the material changes from solid to liquid and vice versa ; thus , pcm are classified as latent heat storage units . jovanovic et al . , during the study of heat stress activity test on 10 male soldier volunteers , which consist of walking on treadmill with a speed of 5.5 km / h in 40c temperature in one climatic chamber , and by wearing of nuclear , biological , and chemical protecting suit was done and the results indicated that with using of cooling vest , temperature of ear and skin , compared to without using of cooling vest , was significantly lower . in another study from jovanovic et al . , with a goal to evaluation of one kind of changed phase material with paraffin wax that consist of n - hexadecane that have been done on 10 male soldier volunteers in a condition of 40c temperature on treadmill with 5.5 km / h , indicated that dressing of cooling vest on changed phase material basis with combat uniform m10 , relation to control group that they have only combat uniform , has caused reduction of physiological strain ( average of skin and ear temperature ) . , during study by the title of personal cooling with pcms in a very hot environment , indicated that pcm ( sodium sulfate type ) cooling vest , has more effect on torso skin , in which , this area was covered by vest . because this vest reduced the chest skin temperature around 1c to 3c , the reduction of rectal temperature was 0.2c . also , gao et al . , in their previous studies indicated that phase change material cooling vest ( mostly sulfate sodium with melting point 28c ) either on thermal manikin or persons , were effective , although in another study from kuklane with the goal to investigate personal cooling vest in kind of phase change material on improving of thermal comfortable in one simulated office , with temperature of 34c , with study on 8 male volunteers in climatic chamber indicated that torso skin temperature has decreased around 23c and remained in about 33.3c , and the findings indicate that the personal cooling with pcm can be used as an option to improve thermal comfort for office workers without air conditioning . because that there was no research have done about the function of iranian cooling vest , the purpose of the present study was an investigation of the effectiveness of pcm cooling vest on thermal strain indices . the results of this study could be useful in working environment that have heating sources to prevent diseases caused by heat . this research is an intervention from the kind of experimental ; the participants in this examination were the male students of isfahan university of medical sciences ( this was due to this fact that the experiment had to be done in climate chamber , there was no possibility to choose workers to participate ) . the test was performed on 12 male student volunteers with the average age : 23.66 2.83 years , the average weight : 66.09 11.43 kg , and the average of vo2 max ( maximum oxygen consumption ) of 2.53 l / min . the criteria for choosing the volunteers were normal body mass index ( 18.525 kg / m ) and the absence of precedent of cordial , pulmonary , nervous , skeleton - muscular , epilepsy , paroxysm , diabetes diseases , disusing of blood pressure and heartthrob drugs , and disusing of coffee , caffeine , and alcohol before 12 h to initiate the test . any time that heartbeat of volunteers , increases from 180 beats / min and body core temperature increases from 39c , test become stopped . all information that mentioned above was collected by the questioner , and a written consent has been taken from participants to perform the test . all volunteers have been visited by a doctor before commencing the test . in this research , cooling vest from the type of phase change material has been used . the quality of vest that contains change phase material was from cotton 70/30 including 30% of cotton and 70% of polyethylene material . inside of the vest , there are eight pockets for phase change material packs . the phase change material is liquid in its normal form , for using of pcm packs , it has to be charged ( become solid ) , and for this reason , they have to be placed inside the freezer for 2 h , and then will be placed inside the pockets that is designed in the vest , and be usable for 34 h. this type of vest has been used for middle - heavy activities . in order to prevent the effects of different clothes of volunteers on thermal strain and equality of the vest using condition , all of the volunteers wear the same boiler suite that it has separate blouse and trouser and it is made up of 30.2% cotton and 69.8% polyester . moreover , the effect of vest on physiological indices : skin temperature , oral temperature , heartbeat , thermal sense , and vest comfortable were tested . because here to fore that there was no similar study on iranian persons has been performed and volunteers were university students in this study , participants fulfilled the test by activities of intensity of 2.4 and 4.8 km / h , and 0% incline by using treadmill ( kettler model ) in a climatic chamber , they considered the weather condition which has the capability of adjusting temperature and humidity with and without vest . during the test average , dry temperature , wet temperature , radiant temperature , relative humidity , and wet - bulb globe temperature have been measured in the following order 38.83 1.29c , 25.30 1.34c , 39.33 1.32c , 32.85 2.26c , and 29.84 0.9c , respectively , in warm and dry conditions of the climatic chamber . the test duration for each was 30 min , the number of all activities was 4 with 1 per day , once they were running at a speed of 2.4 km / h without vest and once with vest for 30 min on treadmill , this has been done for 4.8 km / h , as well . during the activity , physiologic indices , such as oral temperature ( with the use of emerson digital thermometer ) , skin temperature which consists of two sensors for measuring skin temperature which refer to chest and shoulder , heart rate ( with usage of polar sport tester ) , vo2 max ( maximum oxygen consumption ) with the use of a strand nomogram and thermal sense , were measured and registered for both with and without vest . heart rate and skin temperature were checked and registered every minute . for collecting thermal sense data , heat strain score index , or hssi questionnaire is used , this questioner consist of 17 questions from environment weather , the question 1 to 12 is answered through asking , questions 13 to 17 through observing , and the score of each option ( which is inserted beside of each option ) was multiplied by effect factor ( which is inserted in parenthesis beside of each question ) and the result is inserted in a square beside of each question , and finally the numbers in squares are added to get to the final result and if the overall numbers is < 13.5 , shows that the person is out of thermal strain ( level one thermal strain risk or green zone ) and if it is between 13.6 and 18 , shows that the person probably has the thermal strain and a more precise evaluation is needed ( level two thermal strain risk or yellow zone ) and if it is more than 18.1 , shows that the person has thermal strain and it is necessary to take suitable control actions for minimizing strain ( level three thermal strain risk or red zone ) . at the end of the test for the condition of vest usage , vest comfortable rate check list this consists of 9 questions from vest comfortable rate such as being tight , comfortable , flexibility , wearing and easy taking off , movement limitation , and adjustability with body size and design which can be expressed by option namely strongly disagree , disagree , normal , agree , and strongly agree . thermal sense questioner was filled with and without vest , but check list for vest comfortable rate was completed in a situation while using the vest . for data analyzation , with attending to the possibility of correlation between the dependent variables of study and the consideration of correlation that should be made , by their values for these correlation , the independent t - test for comparing the differences between dependent variables before and after the interference has been used . comparison of changes in groups also with the assistance of repeated measurement and all analyses with the help of spss 20 ( ibm spss statistics for windows , version 20.0 . armonk , ny : ibm corp ) have been made , the level of significance has been considered as 0.05 . this research is an intervention from the kind of experimental ; the participants in this examination were the male students of isfahan university of medical sciences ( this was due to this fact that the experiment had to be done in climate chamber , there was no possibility to choose workers to participate ) . the test was performed on 12 male student volunteers with the average age : 23.66 2.83 years , the average weight : 66.09 11.43 kg , and the average of vo2 max ( maximum oxygen consumption ) of 2.53 l / min . the criteria for choosing the volunteers were normal body mass index ( 18.525 kg / m ) and the absence of precedent of cordial , pulmonary , nervous , skeleton - muscular , epilepsy , paroxysm , diabetes diseases , disusing of blood pressure and heartthrob drugs , and disusing of coffee , caffeine , and alcohol before 12 h to initiate the test . any time that heartbeat of volunteers , increases from 180 beats / min and body core temperature increases from 39c , test become stopped . all information that mentioned above was collected by the questioner , and a written consent has been taken from participants to perform the test . in this research , cooling vest from the type of phase change material has been used . the quality of vest that contains change phase material was from cotton 70/30 including 30% of cotton and 70% of polyethylene material . inside of the vest the phase change material is liquid in its normal form , for using of pcm packs , it has to be charged ( become solid ) , and for this reason , they have to be placed inside the freezer for 2 h , and then will be placed inside the pockets that is designed in the vest , and be usable for 34 h. this type of vest has been used for middle - heavy activities . in order to prevent the effects of different clothes of volunteers on thermal strain and equality of the vest using condition , all of the volunteers wear the same boiler suite that it has separate blouse and trouser and it is made up of 30.2% cotton and 69.8% polyester . this experiment was done on a volunteer with and without cooling vest . moreover , the effect of vest on physiological indices : skin temperature , oral temperature , heartbeat , thermal sense , and vest comfortable were tested . because here to fore that there was no similar study on iranian persons has been performed and volunteers were university students in this study , participants fulfilled the test by activities of intensity of 2.4 and 4.8 km / h , and 0% incline by using treadmill ( kettler model ) in a climatic chamber , they considered the weather condition which has the capability of adjusting temperature and humidity with and without vest . during the test average , dry temperature , wet temperature , radiant temperature , relative humidity , and wet - bulb globe temperature have been measured in the following order 38.83 1.29c , 25.30 1.34c , 39.33 1.32c , 32.85 2.26c , and 29.84 0.9c , respectively , in warm and dry conditions of the climatic chamber . the test duration for each was 30 min , the number of all activities was 4 with 1 per day , once they were running at a speed of 2.4 km / h without vest and once with vest for 30 min on treadmill , this has been done for 4.8 km / h , as well . during the activity , physiologic indices , such as oral temperature ( with the use of emerson digital thermometer ) , skin temperature which consists of two sensors for measuring skin temperature which refer to chest and shoulder , heart rate ( with usage of polar sport tester ) , vo2 max ( maximum oxygen consumption ) with the use of a strand nomogram and thermal sense , were measured and registered for both with and without vest . heart rate and skin temperature were checked and registered every minute . for collecting thermal sense data , heat strain score index , or hssi questionnaire is used , this questioner consist of 17 questions from environment weather , the question 1 to 12 is answered through asking , questions 13 to 17 through observing , and the score of each option ( which is inserted beside of each option ) was multiplied by effect factor ( which is inserted in parenthesis beside of each question ) and the result is inserted in a square beside of each question , and finally the numbers in squares are added to get to the final result and if the overall numbers is < 13.5 , shows that the person is out of thermal strain ( level one thermal strain risk or green zone ) and if it is between 13.6 and 18 , shows that the person probably has the thermal strain and a more precise evaluation is needed ( level two thermal strain risk or yellow zone ) and if it is more than 18.1 , shows that the person has thermal strain and it is necessary to take suitable control actions for minimizing strain ( level three thermal strain risk or red zone ) . at the end of the test for the condition of vest usage , vest comfortable rate check list this consists of 9 questions from vest comfortable rate such as being tight , comfortable , flexibility , wearing and easy taking off , movement limitation , and adjustability with body size and design which can be expressed by option namely strongly disagree , disagree , normal , agree , and strongly agree . thermal sense questioner was filled with and without vest , but check list for vest comfortable rate was completed in a situation while using the vest . for data analyzation , with attending to the possibility of correlation between the dependent variables of study and the consideration of correlation that should be made , by their values for these correlation , the independent t - test for comparing the differences between dependent variables before and after the interference has been used . comparison of changes in groups also with the assistance of repeated measurement and all analyses with the help of spss 20 ( ibm spss statistics for windows , version 20.0 . armonk , ny : ibm corp ) have been made , the level of significance has been considered as 0.05 . during the test , none of the volunteers have showed the sickness from heat and in all stages of test , the heartbeat for all persons was 180 beats / min and core temperature at 39c as well , the result of study showed that pcm cooling vest has much lowered the physiologic indices in the activity with the intensity of 2/4 km / h rather than 4/8 km / h [ figure 1 ] . the start of the test until the final moments of the test heart rate was increased due to the heat and run operation , in the condition of with the usage of cooling vest , expected that the results of heart rate in this condition are less than no vest , and unexpectedly , there is no relationship between the increase or decrease in this variable . average of shoulder , chest skin temperature , and heat strain score index with a speed of 2.4 km / h ( a , b , and e ) and with a speed of 4.8 km / h ( c , d , and f ) the result of comparison changes in both groups during the time of study which is obtained by the usage of repeated measurement is shown in table 1 . the results of repeated measurement test for physiological indicators changes with and without the use of cooling vest the results of table 1 shows that the changes of both conditions with and without the usage of cooling vest during the test on heartbeat variable in both speeds , oral temperature , and heat strain score was not significant at speed 4/8 , but the changes of both conditions with and without the usage of cooling vest during the test on skin temperature in both speeds , oral temperature , and heat strain score was significant at 2.4 km / h . table 2 shows that the results of independent t - test , different values of the skin temperature variable in both conditions with and without the use of cooling vest between 15 and 30 min of test at both speeds 2.4 and 4.8 km / h , was significant , whereas the variables such as oral temperature , heat strain score , and heartbeat were not significant in their different values . most of the people were agreed on the comfortable rate and vest design in both activities [ figures 2 and 3 ] . moreover , wilcoxon test showed that people 's view about the comfortable rate and vest design were the same in both activities . ( the comfortable rate for all of the option in check list was obtained at p > 0.05 ) . the results of independent t - test for physiological indicators changes with and without the use of cooling vest volunteer 's opinions about the comfortable rate of cooling vest at 2.4 km / h people 's comment about the comfortable rate of cooling vest at 4.8 km / h the finding showed that during a 30 min test , pcm type cooling vests in hot and dry weather condition has affected the rate of decline in skin temperature , oral temperature , and heat strain score but not on the decrease or increase of heartbeat , while during 15 and 30 min of test , the skin temperature had decreased but oral temperature , heat strain score , and heartbeat in both conditions , with and without the use of cooling vest , did not have a significant difference . thermal energy storage with the usage of phase changing material is one of the most efficient ways for saving the accessible energy , because of many advantages that it has such as high thermal storage capacity and lower temperature storage . as the body temperature reaches the skin surface by the blood flow circulatory system it is absorbed by pcm pockets and results in skin temperature reduction and thermal strain score , therefore , pcm is able to absorb , store , and release large amount of energy in the form of hidden heat to higher than a define temperature value during phase transfer between solid and liquid . pcm is able to store and release a high amount of energy and while the materials transform from liquid to solid and vice versa , heat is absorbed or released . in a study , which is done by jovanovic et al . on two different types of pcm cooling vest , they showed that both cooling vests have affected on skin temperature reduction which is compatible with the result of this study , and the results of thermography from pcm during their study showed that the heat which is made inside the body , is the main factor of pcm melting time as well . in this study , the thermography has been used for the verification of pcm cooling effects . until now , lots of studies in the field of pcm applications with different goals by different people have been done , which , in many of them , the thermal manikin have been used instead of real people ( 7 and 8) . moreover , on the other hand , there have been a few researches in relation to temperature change effects on pcm cooling system applications on individuals . also in a study , under the title personal cooling with pcms in a very hot environment , showed that in the condition of hot weather , pcm cooling vest ( sulfate sodium ) has the highest effect on the torso part which is covered by vest , as the vest reduces the temperature of chest skin about 1 to 3c but the rate of rectal temperature decrease was 0.2c , which showed the compatibility between the results of this study and trivial oral temperature difference in both conditions with and without the usage of cooling vest , whereas in kuklane study , with the goal of the evaluation of pcm personal cooling on person thermal comfortable improvement which is simulated in a 34c office with eight male volunteers in the climatic chamber showed that temperature of torso skin has decreased about 23c and remained in about 33.3c , which the results of skin temperature obtained in this study is approximately close to our study 's results . on the other hand , the different value of heartbeat diversity during the test and the different values of heat strain score , oral temperature , and heartbeat variables on 15 and 30 min of test were not significant because of the consideration of few people participation , which was compatible with the results of study that has been done by smolander et al . in sweden , with the goal for evaluation of the influence of ice vest , which has been committed on four firefighters , and because of few samples , none of the statistical tests were significant . investigation of volunteer 's opinions about vest comfortable rate showed that most persons were agreed about comfortable rate , wearing and taking off , and vest design as well , but the comments were ordering about the flexibility , as it could be related to the pcm 's size and being solid , so it reduces the movements , which is totally adaptable to kopias results that they mention disadvantage is the reduction of movements in pcm cooling vests . because the pcm package started to melt , in the second 15 min of test , the subjects felt more heavily than in the first 15 min , to resolve this problem , we suggest that pcm packs were smaller , more , and used pcm with a high melting point . also , to prevent skin frostbite , caused by contact with pcm package , in addition , coverage of pcm packages , the subjects were asked to use thicker underwear . since the lack of awareness of workers and employers workshops in the tropics , especially in the south of the country , his study could be useful in order to increase their information about the cooling vest . the findings of study showed that in hot and dry conditions , iranian cooling vest ( pcm type ) was effective in reduction of skin temperature , oral temperature , and hssi in light activity ( speed of 2.4 km / h ) but has no influence on person heart beat rate ) . moreover , in medium activity ( speed of 4.8 km / h ) , cooling vest was lower effective to the reduction of oral temperature , hssi , and heart beat compared to light activity ( 2.4 km / h ) .

changes in the composition of cell surface glycolipids that take place during malignant transformation have been extensively described . gangliosides are glycosphingolipids containing sialic acid engaged in a wide variety of biological events that occur at vertebrate 's cell membrane . they are widely distributed in both normal and tumoral human tissues of neuroectodermal origin [ 3 , 4 ] . the most abundant sialic acid variants in mammals are n - acetylneuraminic acid ( neuac ) and n - glycolylneuraminic acid ( neugc ) . neuac acid is the predominant sialic acid species expressed in mammalian brain gangliosides . whereas , neugc is a predominant sialic acid species expressed in gangliosides from nonneural tissues of most nonhuman species [ 5 , 6 ] . in contrast to neuac , the expression of the neugc forming the structure of gangliosides and/or other glycoconjugates ( hanganutziu - deicher antigen ) in human has been considered as a tumor - associated antigen . the only structural difference between neuac and neugc is a single oxygen atom at the c-5 position of neugc catalyzed by the cytidine monophospho - n - acetylneuraminic acid hydroxylase ( cmp - neuac hydroxylase ) . this minor difference is able to induce an immune response as well as to develop specific antibodies raised against n - glycolylated gangliosides [ 10 , 11 ] . the aberrant expression of the neugc residues in humans has been considered to be associated with the altered metabolism of malignant cells [ 9 , 12 , 13 ] . normal human cells are incapable of synthesizing neugc due to a specific inactivating mutation in the cmp - neuac hydroxylase gene . however , some authors have suggested an alternative pathway to the neugc synthesis from other intermediates of cellular metabolism in some human tumors . recently , some reports of 14f7 mab ( a highly specific mab raised against n - glycolyl gm3 ganglioside ) reactivity in formalin - fixed and paraffin - embedded tissues have been published . nevertheless , epithelial - derived tumors have been mostly evaluated [ 1519 ] . in this way , the analysis of neugcgm3 expression in different human neoplasms could be useful as a better basis for understanding the molecular pathogenesis of malignancies as well as to extend the assessment of this molecule as target for cancer immunotherapy . for these reasons , in this work was evaluated the recognition of 14f7 mab in a serie of neuroectodermal , mesodermal , and epithelial derived tumors . samples of fetal , normal , and reactive astrocytosis were also included in the study . we used the 14f7 mab ( igg1 ) a highly specific anti - neugcgm3 ganglioside antibody . this mab was generated by immunization of balb / c mice with neugcgm3 hydrophobically conjugated with human very low - density lipoproteins ( vldls ) adjuvated with complete freud adjuvant ( cfa ) . afterward , 14f7 mab was obtained by the hybridoma resulting in the fusion of spleen cells with mouse myeloma cell line p3x63ag653 as described in . routinely processed , formalin - fixed , and paraffin - embedded archival samples with diagnosis of fetal tissues ( 3 ) , normal adult tissues ( 10 ) , reactive astrocytosis of the brain ( 3 ) , pediatric brain tumors ( 35 ) , sarcomas ( 30 ) , and thyroid carcinomas ( 25 ) as well as frozen adult tissues ( 84 normal and 11 tumoral ) were received from the pathology departments of ramn gonzlez coro gyneco - obstetric hospital , juan manuel mrquez pediatric hospital , the legal - medicine department at amalia simoni provincial hospital of camagey , the national institute of neurology and neuropathology , and the national institute of oncology and radiobiology . fetal tissues were obtained from 19-week - old fetus aborted by rivanol , and normal tissues were removed at autopsy of healthy persons having suffered clinical death or by conventional intraoperatory biopsy . all samples were used after obtaining an approved consent by the institutional ethical committees . for fresh samples , then , five micrometres sections were obtained in a cryostat and slides were stored at 20c until they were used . all samples were washed in tap water and rehydrated in distilled water for 10 minutes and tbs for 5 minutes . slides were incubated with biotin - blocking system ( x0560 , dako , denmark a / s ) , according to manufacture instructions . afterward , tissue samples were washed with tbs during 10 min . for formalin - fixed and paraffin - embedded tissues , five micrometer serial sections from each block were obtained , and the slides were processed as it was previously described . briefly , the samples were incubated with 14f7 mab followed by a peroxidase avidin - biotin system . colonic adenocarcinoma and a breast infiltrating carcinoma were taken as positive control for both paraffin - embedded and frozen tissues , respectively . enzymatic activity was visualized with a dab ( k3465 , dako , denmark a / s ) solution , and slides were counterstained with mayer 's hematoxylin ( s2020 , dako , denmark a / s ) . a semiquantitative scoring system was used to define levels of reactivity . according to the staining pattern , the tumor sample was assigned to 1 of 4 scores : 0 , no staining ; 1 , weak staining ; 2 , moderate staining ; and 3 , strong staining of malignant cells . no reaction was observed with the 14f7 mab in fetal tissues , except for a weak - to - moderate reactivity in less than 25% of brain neurons in 1/3 cases ( table 1 ) . no staining was evidenced neither in formalin - fixed and paraffin - embedded ( 0/10 ) nor frozen ( 1/84 ) normal tissues , except for a weak reaction of mucous cells from small intestine ( 1/3 ) ( table 2 ) . an intense homogeneous and finely granular pattern of recognition was detected in 2/2 anaplastic astrocytomas , 1/2 oligoastrocytic tumors , while the glioblastoma ( 0/1 ) showed no reaction with 14f7 mab . the reaction was mainly located on the plasmatic membrane and in more than 50% of malignant astrocytes . no staining was observed in oligodendrogliomas ( 0/3 ) and meningiomas ( 0/3 ) ( table 3 ) . in contrast , 3/5 ( 60.00% ) , 5/8 ( 62.50% ) of diffuse and anaplastic astrocytomas ; and 2/2 of glioblastomas were moderate - to - intense reactive with 14f7 mab in more than 50% of malignant cells ( table 4 , figure 1 ) . one case of anaplastic astrocytoma and the oligodendroglioma showed a weak - to - moderate and finely granular reactivity mainly located in plasmatic membrane and also in the cytoplasm of less than 25% of malignant cells . no immunorecognition was observed in ependymomas , while 14f7 mab was moderate - to - intense reactive at less than 25% of malignant cells in 1/5 anaplastic ependymomas . in the other hand , neuroblastomas ( 2/3 ) and ganglioneuroblastomas ( 2/2 ) exhibited a moderate - to - intense reaction in both the plasmatic membrane and the cytoplasm of more than 25% of malignant cells . concerning to medulloblastomas ( 0/2 ) and ependymoblastomas ( 0/2 ) no immunostaining was evidenced . the 14f7 mab reactivity was detected in 25/30 ( 83.3% ) of all studied soft tissue and bone sarcomas depending without the histopathological subtype . considering the different histological subtypes of sarcomas , 8/8 muscular sarcomas , 3/3 vascular sarcomas , 2/2 peripheral nerve sheath tumours , 7/10 other fusocellular sarcomas , 2/3 liposarcomas , and 3/3 osteosarcomas were recognized by 14f7 mab . no immunoreaction was evidenced in the synovial sarcoma ( table 5 ) . in general , the staining with 14f7 was observed as a finely granular reaction mainly located in the cell membrane but also in the cytoplasm of more than 50% of malignant cells ( figure 2 ) . almost all sarcomas were moderate - to - intense reactive with 14f7 although a weak intensity of staining was observed in a low - grade leiomyosarcoma . a moderate - to - intense reactivity in 23/25 ( 92.0% ) of thyroid carcinoma was detected ( table 6 ) . the reaction with 14f7 showed a finely granular pattern localized in both the plasmatic membrane and the cytoplasm of neoplastic cells ( figure 3 ) . unusual glycosylated or sialylated gangliosides have been identified with monoclonal antibodies generated against tumor - associated antigens , and they were considered as targets for use in passive and active immunotherapy of some malignant neoplasms [ 10 , 11 ] . between them , the expression of a nonhuman sialic acid ( n - glycolylneuraminic ) forming the structure of gangliosides and/or other glycoconjugates has been considered one of the most important antigens [ 1 , 20 ] . the structural difference between n - acetylneuraminic ( normal constituent of human tissues ) and n - glycolylneuraminic ( tumor - associated antigen ) is crucial in many aspects of the cellular behavior [ 12 , 21 ] and has permitted the development of specific antibodies raised against the hanganutziu - deicher ( hd ) antigen or n - glycolylated gangliosides as well as their immunohistochemical evaluation using both frozen and formalin - fixed and paraffin - embedded tissues [ 9 , 22 ] . therefore , hd is classified as a heterophile antigen and chemically defined as a glycolipid and/or glycoprotein ( glycoconjugates ) which contains neugc . this antigen has been reported to be almost absent in normal human tissues but can be expressed on a variety of human malignant cells . in our study , both frozen and formalin - fixed and paraffin - embedded nontumoral human tissues were not reactive with 14f7 mab ( igg1 highly specific against n - glycolyl gm3 ganglioside ) , except for a weak - to - moderate staining of some neurons in fetal tissues . additionally , we observed an intense staining of 14f7 in mucous cells from small intestine . in previous studies , normal eukaryotic cells were able to take in a portion of ingested neugc and process it for their own glycoconjugates [ 13 , 20 ] . in line with this , small levels of expression of neugc have been found in some normal human tissues ( e.g. , epithelial cells and their secretions ) . the limited reactivity of 14f7 mab in nontumoral tissues confirmed that 14f7 mab is able to distinguish between the n - glycolyl and the n - acetyl functions of the gm3 ganglioside . furthermore , the limited recognition of 14f7 mab in other normal tissues has been also reported by our group [ 1619 ] . in contrast , our group reported the expression of neugcgm3 in breast tumors using both thin layer chromatography ( tlc ) immunostaining and fast atom bombardment mass spectroscopy ( fab / ms ) analysis . in addition , we published the immunohistochemical recognition of the 14f7 mab in breast infiltrating ductal carcinoma and melanoma by immunohistochemistry using frozen tissues fixed in 4% paraformaldehyde . this finding suggested that the structure recognized in breast tumors could be the oligosaccharide core of neugcgm3 present in glycoconjugates . the in vivo tissular expression of neugcgm3 was also confirmed by the radioimmunoscintigrafic technique using 14f7 mab labelled with 99mtc . afterward , we reported the immunohistochemical reactivity of 14f7 in a variety of formalin - fixed and paraffin - embedded tumor tissues , despite the extraction of glycolipids during the routine histological procedures [ 1619 ] . recently , scursoni et al . reported the recognition of 14f7 mab in pediatric neuroblastoma using formalin - fixed and paraffin - embedded tissues and suggested that the expression of neugcgm3 ganglioside is preserved in the more aggressive tumors . moreover , a clinical trial with racotumomab ( anti - idiotypic vaccine ) in pediatric neuroectodermal tumors has been suggested in . in this study , we describe the reactivity of the 14f7 in tumors of the central nervous system using both frozen tissues after 4% paraformaldehyde fixation and formalin - fixed and paraffin - embedded tissues . malignancies with astrocytic differentiation and among them : diffuse and anaplastic astrocytomas , glioblastomas , an neuroblastomas were mainly recognized by 14f7 mab . our data seems to be also in agreement with the preferential reactivity of 14f7 mab in more aggressive types of human astrocytoma . in line with this , the progression of malignant brain tumors has been associated with altered gangliosides composition and distribution . on the other hand , a preliminary study about the reactivity of 14f7 in ewing sarcoma has been previously described suggesting the potential use of neugcgm3 for cancer immunotherapy . here , we obtained the reactivity of 14f7 mab in almost all soft and nonsoft tissues sarcomas without taking into account the histopathological classification . at present , a lot of studies are focused to better understand the molecular pathogenesis of sarcomas as well as the identification of reliable molecular markers and possible therapeutic targets . authors have reported increased amount of serum total sialic acid as well as the detection of n - glycolylneuraminic acid antibody in patient bearing sarcomas . interestingly , our group has evidence about the occurrence of higher levels of antibodies raised against neugcgm3 in patients bearing sarcomas ( carr a , unpublished data ) . neoplastic transformation of the thyroid gland has been reported to be accompanied by changes in cellular sialylation . a limited or absent expression of sialic acid in the surface of follicular cells in normal thyroid glands , adenomas , and goiters has been demonstrated . in contrast , a weak - to - intense positivity for sialic acid was found in thyroid carcinomas . in this study , we reported the immunohistochemical recognition of 14f7 mab in the majority of thyroid carcinomas but not in their normal counterpart . in this way , our data permit to consider the potential use of neugcgm3 as recognized by 14f7 in both the distinction of malignant from benign thyroid lesions and in being a potential target for active and passive immunotherapy in persistent and recurrent thyroid carcinomas . finally , intratumoral hypoxia ( low oxygen tension ) has been associated with aggressive disease , poor prognosis , and resistance to conventional therapies of malignant brain tumors , sarcomas , and thyroid carcinomas [ 3032 ] . tumor hypoxia has been considered responsible of neugcgm2 ganglioside expression in human cancer cells through the incorporation of neugc . the effect of hypoxia could be to expedite sialic acid transport from the external medium , in relation to the increment of sialin expression ( a sialic acid molecule transporter ) . the role of neugcgm3 in tumoral progression as well as its suppressor properties has been previously reported [ 34 , 35 ] . in this way , studies focused on the evaluation of intratumoral hypoxia and neugcgm3 relations are being planned in our lab . the expression of neugcgm3 in neuroectodermal , mesodermal , and epithelial derived tumors but not in normal sections suggests that the expression of this ganglioside could be related to the aggressive behavior of malignant cells , without depending on the tumor origin . our data could support the possible use of neugcgm3 as a target for both active and passive immunotherapies of malignancies expressing this molecule .

balcetis and dunning ( 2010 ) reported five experiments with evidence that desirable objects were judged closer than other objects ; an effect they termed wishful seeing . every experiment rejected the null hypothesis , thereby indicating evidence of the effect , and such replication across experiments is often taken as evidence that an effect is robust . however , this interpretation is valid only if the experiments have high statistical power , which is the probability that an experiment will reject the null hypothesis . if all experiments reject the null hypothesis despite having relatively low power , then the correct interpretation is that there was a publication bias that over - reports positive findings ( ioannidis and trikalinos 2007 ; francis 2012 in press ) . table 1 lists the sample sizes , standardized effect size , and power of each experimental finding in balcetis and dunning ( 2010 ) that investigated wishful seeing . the application of a meta - analytic method ( hedges and olkin 1985 ) , which pools the effect sizes across the experiments , reveals that the best estimate of the effect of wishful seeing is g = 0.537 . the last column of table 1 shows the power of each experiment to detect this pooled effect size . it is noteworthy that the two studies with the smallest samples sizes have power values less than one half . the sum of the power values ( 3.11 ) is the expected number of times these experiments should reject the null hypothesis . the probability that all five experiments would reject the null hypothesis is the product of the power values ( 0.076 ) , which is below the 0.1 threshold that is frequently used to indicate evidence of publication bias ( begg and mazumdar 1994 ; ioannidis and trikalinos 2007 ) . another way to describe this finding is that the reported experiments are not self - consistent . given the reported effect and sample sizes , it is not believable that there would be so many rejections of the null hypothesis if the experiments were run properly and reported fully . the proper interpretation of the experimental findings is that they are non - scientific or anecdotal . it might be tempting to argue that the probability of the balcetis and dunning ( 2010 ) experiments is not much below the criterion , so maybe there is hope that future experiments could make the findings more believable . although it is mathematically possible , such a situation is unlikely because ioannidis ( 2008 ) notes that most experiments overestimate the true effect size , so the above analysis probably overestimates the true power of the experiments . even if this were not true , new experiments are unlikely to change the conclusion of publication bias . if a new experiment rejects the null hypothesis with a similar effect size , then the product of the power values for all experiments can only be less than the product for the experiments in table 1 . if a new experiment fails to reject the null hypothesis , it will usually have a smaller effect size than what is shown in table 1 . the pooled effect size across experiments will be smaller , which will reduce the power of all experiments . there are two broad explanations of how publication bias could have contaminated the findings in balcetis and dunning ( 2010 ) . first , they may have run , but not reported , additional experiments that did not reject the null hypothesis . this type of file drawer problem could happen because the authors deliberately suppressed some findings or because reviewers or the editor insisted that the null / negative findings be removed from the manuscript . something similar to the file drawer problem can also occur for experiments that have multiple measures but report data from only a subset of the measures . the second broad explanation is that the experiments in balcetis and dunning ( 2010 ) were run improperly in a way that caused an elevated rejection rate for the null hypothesis . one invalid approach is to start with a relatively small set of subjects and run a hypothesis test . if the null hypothesis is not rejected , additional subjects are recruited and the test is repeated . this procedure is continued until the null hypothesis is rejected or the experimenter gives up . it may seem like good scientific practice to gather data until a research question is settled , but analyzing such data sets as if they were gathered with a fixed sample size leads to a dramatic increase in the rejection of the null hypothesis , regardless of whether it is true or false ( strube 2006 ) . there are several other experimental methods that also produce too many rejections of the null hypothesis . simons , nelson and simonsohn ( 2011 ) describe how some of these techniques can ensure that almost every experiment rejects the null hypothesis , regardless of whether it is true or false . there is no way of telling which of these broad approaches , and it could be both , were used by balcetis and dunning ( 2010 ) . in a similar way , now that the data are known to be contaminated with publication bias , there is no way to determine whether the null hypothesis is true or false . researchers interested in wishful seeing are advised to ignore the findings in balcetis and dunning ( 2010 ) and run new experiments without bias . a third possible explanation of the pattern of data in table 1 is that the studies measured different effect sizes , in which case the meta - analytic pooling is improper . for example , some experiments measured estimates of distance , while experiment 3a measured accuracy of distance related actions ( tossing a beanbag to a target ) . the effect of wishful seeing as expressed by the accuracy of tosses might alter the reported effect size . the action of tossing a beanbag might scale the overall magnitude of the measurement variable , but it will also introduce an additional noise term to the experimental measurements , which will increase the standard deviation . a change in scale will not alter the standardized effect size , but a larger standard deviation will decrease the effect size . based on this analysis , one might expect that experiment 3a will have a smaller standard deviation than the other experiments , but table 1 shows that experiment 3a has the largest effect size of all of the experiments . the study of balcetis and dunning ( 2010 ) is one of several new studies ( eg , balcetis and lassiter 2010 ) that have revived ideas of the new look theorists from the 1950s . the new look approach argued that an observer 's motivations and desires could alter perceptual experience , but the new look findings were ultimately rejected because of poor methodology . if the publication bias in balcetis and dunning ( 2010 ) is present in similar studies , then the empirical efforts to revive the ideas of the new look theory may suffer from variations of the methodological problems of the past .

major progress has been made over the past 20 years in the understanding of the cognitive consequences of critical illness . in order to expand the knowledge how disease states such as sepsis have a causal impact on the central nervous system and cognition , experimental animal models are certainly required . in a previous issue of critical care , tuon and colleagues reported a study in which they developed such a model in order to simulate the cognitive and behavioral effects of septic illness on memory functioning . they further provide evidence that this memory impairment can be attenuated by the administration adrenergic agents , which suggests that this mnemonic pathway may be mediated by adrenoceptors . the methodology employed by tuon and colleagues has been used in behavioral neuroscience and comparative psychology since the inception of classical conditioning . it is a well validated methodology that elicits a clear link between stimulus encoding and behavioral output . other recent research has provided important insights into the nature of aversive memory formation . as the understanding of memory and other cognitive processes has expanded , so too have the ties between these cognitive functions and the underlying anatomy and physiology supporting these abilities . memory is a multifaceted ability that is supported by disparate and distinct circuits in the brain , so much so that ablating structures in one mnemonic pathway may have little or no effect on the functioning of another mnemonic ability . in the classic neuropsychological evaluation of patient ' hm ' , even with profound anterograde amnesia that developed after removal of a major section of the medial temporal lobe , he was still able to form classically conditioned memories , specifically to aversive events . in recent years several methodologies have been developed that extend the ability to address questions regarding complex cognitive processes in animal models . for example , jonathan crystal at the university of georgia has demonstrated that it is possible to test not only episodic memory in animals but also attention and orientation to time and place . although each of these abilities involves a component of memory , these cognitive faculties differ in important ways from aversive classical conditioning . not only do these mnemonic processes rely on fundamentally different neurological substrates , but they are also homologous to the memory and attentional deficits that are observed in survivors of critical illness . by incorporating paradigms such as those developed by crystal and colleagues , future animal models have the potential to answer important questions regarding the nature of higher level cognitive deficits experienced by patients who survive critical illness in the intensive care unit ( icu ) . it may then be possible to begin to trace specific circuits related to icu - acquired neurocognitive injury . this could lead to an improved understanding of the sometimes subtle nature of attentional , declarative , and executive dysfunction observed in patients after critical illness . the hope is that these may help bridge the gap between bench research and bedside care . experimental investigations that incorporate the ability to assess subtle changes in animal cognition offer great promise for advancing our understanding icu acquired long - term cognitive impairment .

due to their power efficiency , brightness , and larger color gamut extremes lasers are ideal light sources for projector applications . a fully coherent source provides an image along the optical axis without the concern of losing focus . speckle , the granular intensity pattern resulting from constructive and destructive interference , can be observed from laser - illuminated surfaces . the resultant speckle in the projected image strains the eye , resulting in observer s headaches . speckle contrast associated with the overall intensity pattern is calculated by equation ( 1 ) , where is the standard deviation and iavg is the average irradiance across the image as described by equation ( 2).(1 ) c=iavg ( 2 ) c=(e[i2]-e[i]2)/iavg diffusers can be used to offset the original coherence in the laser light prior to reflection from a screen or surface . the degraded coherent optical field results in a change of the image irradiance levels , thus reducing the speckle contrast . a commonly used speckle reduction technique is temporal averaging of the irradiance pattern using a diffractive diffuser . a stationary diffuser introduces a single independent phase change . by moving the diffuser either though rotation or vibration , k rotation of a pseudo - random pattern diffuser determines the speckle reduction as a function of the number of patterns averaged . c=1k in order to temporal averaging to completely negate speckle for the human eye , sample rate of 20 hz , a diffuser would have to rotate a number approaching infinity all within 0.05 s. a rotating or vibrating diffuser would also be susceptible to mechanical failures , reducing product lifespan . rotational or vibrational methods require additional supporting components , increasing implementation difficulty in pico - projectors which have severely limited spatial allocation . a diffuser that reduces speckle without the need for physical manipulation is ideal for laser pico - projection systems . previous work on non - rotating diffusers have had limited success . a more recent article utilized an mems device for very small motion of the diffuser where speckle reduction of 0.48 was achieved . another method used piezoelectric benders to reduce speckle while limiting actual diffuser motion with a contrast of 0.16 . the piezoelectric method , while non - rotational , requires two mechanically vibrating parts making the design susceptible to failure . various contrast reduction methods [ 3,79 ] have resulted in significant speckle contrast , but all require motion . additionally , the majority of speckle reduction methods require large setups or devices that would need to be miniaturized for pico - projector application . we present a novel non - rotational , non - moving diffractive diffuser that is easily integrated into laser pico - projectors , significantly reducing speckle contrast . a binary diffuser commonly used in speckle reduction techniques has an associated phase shift of either 0 or for each individual phase element of the structure . a hadamard matrix is a square matrix used for performing hadamard transforms ( ht ) that is composed entirely of + 1 and 1 values . a special property of the hadamard matrix is the relationship between the matrix and its transpose as described in equation ( 4 ) . this definition requires all hadamard matrices to be square with orders n = 2 , 4 , 8 , 16 , where h1=1 . multiple versions of the hadamard matrix exist , each with a unique binary pattern.(4 ) hnhnt = hnthn = nin the first type of hadamard matrix is the sylvester matrix and is described in equation ( 5 ) with the condition that h1=1 . an order 16 sylvester matrix is shown in figure 1 where the dark area denotes + 1 and the white space denotes 1.(5 ) h2n = h2n-1h2n-1h2n-1-h2n-1 order 16 sylvester matrix . the other hadamard matrix types are known as the walsh paley and the walsh matrix , and can be constructed through recursion . while each matrix still follows the basic definition , each has an exclusive pattern creating different propagated irradiance patterns when observed . the 1 elements of the matrix can be replaced with 0 and a binary diffuser can be developed from the pattern . the original design requires 150200 rpms for rotating diffusers in order to achieve speckle contrast at or below 0.20 as discussed previously regarding temporal averaging of a diffuser . variations in the hadamard matrix pattern types will reduce speckle contrast without the need for temporal averaging . using two stationary statistically correlated hadamard diffusers with varying phase patterns in tangent can reduce the speckle more than two generic uncorrelated binary diffusers . a similar experiment was conducted in 2012 in which a single optimized binary diffuser was manipulated into two separate diffuser structures . while stationary diffuser speckle was reduced , the method did not lower the contrast more than previously demonstrated using single rotating diffuser contrast values . a single scattering , fourier model is used for initial analysis of the proposed dual hadamard diffuser reduction technique and proceeds with a full analysis of optical system simulation . a 16-order matrix is created as an individual cell and duplicated to fill the total size of the array dependent on the analysis of the entire propagation system . for any speckle reduction technique , hadamard matrices included , a decrease by ( m ) can be achieved where m is the degree of freedom . the degree of freedom represents the number of independent diffraction patterns that are created by a stationary diffuser . a 16-order hadamard matrix pattern can be expected to have a contrast measurement of 0.707 . placing two differing hadamard patterns in series along the optical path , within the fresnel zone of the primary diffuser allows for a speckle value of 0.343 . due to the correlation between the two patterns , one - half of the contrast reduction associated with the fully developed speckle can be assumed . calculation of the expected contrast value requires an extension of equation ( 2 ) , which can be done by assuming n independent pixels for the image irradiance . the standard deviation of the irradiance can now be expanded given by equation ( 6).(6 ) c=n=1nii - iavg2n-1iavg the image irradiance level can be found using fourier propagation with the assumption that the illuminated diffuser speckle pattern will propagate onto a projection lens prior to observation onto a screen . this layout is illustrated in figure 2 where z 1 and z 2 represent the propagation distances between the diffuser , lens , and observation screen and f represents the focal length of the lens . initial analysis will involve a single diffuser to verify the approach to satisfy the previously expected contrast values . the initial propagation prior to incidence on the diffuser as coherent monochromatic gaussian light is defined by equation ( 7).(7 ) ugx , y = agex2+y2w02 ag represents the amplitude of the beam and w0 is the initial waist of the beam . the diffuser can be modeled as a single scattering phase screen with the hadamard matrix structure defined in equation ( 6 ) . this matrix can be replicated to represent the whole of the diffuser as described in equation ( 8).(8 ) tax , y = ej/2h16 ... h16 ......... h16 ... h16 the 2 in the exponential is the phase shift that separates the two levels of the diffusers . the incident illumination can be multiplied by the diffuser within the fourier domain since it is modeled as a phase screen . it is assumed that the gaussian illumination is a negligibly small distance from the incident diffuser which allows direct convolution of the incident beam and the diffuser . the initial optical field immediately after the diffuser is defined in equation ( 9).(9 ) uinc(inc,inc)=ug(xinc , yinc)ta(xinc , yinc ) propagation after the diffuser can be accomplished through the transfer matrix approach , taking into account the distance of propagation is still within the near - field region . equation ( 10 ) shows the result of propagation after the diffuser.(10 ) udxd , yd = ejkzjzuinc,ejk2zx-2+y-2dd due to the very small size of the diffuser elements in x and y , the phase propagated pattern size will increase rapidly along the z axis causing the lens to be well within the focal length and thus , within the fresnel region of the diffuser output . this satisfies the condition for fresnel approximation given by equation ( 11 ) .(11 ) z34x-2+y-2max2 an important element in measuring the speckle is to ensure that the lens does not decrease the size of the speckle spots so much that the individual pixels of the camera used for measurement do not cause averaging , independent of the rest of the system . this matching of lens to speckle screen was used extensively for measuring laser beam size effects and diffuser speed while maintaining the individual speckle spot size , effectively comparing the speckle contrast across the surface . a lens , l , can be described as a phase function applied to the incoming wave . the transmittance function of the projection lens is described by equation ( 12 ) .(12 ) tlxl , yl = pxl , yle - jkfxl2+yl2 for a lens function , it can be shown that the distance d is replaced by the focal length of the lens f. the aperture function p can be simply described as a circle . propagation of the lens can occur using the fraunhofer propagation equation and replacing the distance parameter z with the focal length f of the lens . substituting z for the focal length f and inserting equations ( 10 ) and ( 12 ) results in equation ( 14).(13 ) ufxf , yf = ejkzjzejk2zx2+y2u1,e - j2zx+ydd ( 14 ) ufxf , yf = ejkfjfejk2fxf2+yf2udxd , ydpxf , yfe - j2fxfxd+yfyddxddyd uf represents the amplitude of the wave of the resultant propagation at the focal plane of the lens l. after the focal plane of the lens , propagation to the observation plane can be evaluated using the fresnel propagation equation described previously by equation ( 11 ) . the final amplitude output of the system uobs can be described by equation ( 15 ) where equation ( 14 ) can be viewed as the initial amplitude field and is propagated using the fresnel approximation to the final observation screen.(15 ) uobsxobs , yobs = ejkz2jz2ejkfjfejk2fxf2+yf2udxd , ydpxf , yfe - j2fxfxd+yfyddxddydejk2zx-2+y-2dd the distance from the focal plane of the lens to the final observation plane is greater than that previously described for the diffuser to the lens . this distance z 2 is still well within the near - field distance requirement and satisfies equation ( 12 ) . far - field propagation will need to be considered for projection onto surfaces for image viewing ( actual projector system ) , but is not applicable for the detection measurement . contrast measurements for the output speckle images can now be found from the previously discussed contrast equation involving speckle image intensity as described in equation ( 7 ) . the intensity of the observation measurement from the optical field in the observation plane can be broken into pixel elements as shown in equation ( 16).(16 ) iobsxobs , yobs = uobsxobs , yobs2 thus , the overall contrast created by the hadamard diffuser can be attributed to taking multiple fourier transforms of the initial phase screen ta . a second phase screen can now also be included by evaluating equation ( 8) again with all elements the same except for the phase screen . the analysis for both diffusers will need to maintain the same z value associated with the diffusers prior to the lens to maintain fresnel region propagation . a simulation will be used further to the previous analysis and provide an accurate accounting of the contrast reduction provided by the diffuser . modeling of the z value to match the phase elements of both diffusers will be discussed in section 3 . simulation of the primary diffuser is accomplished first to ensure the proper contrast evaluation prior to a two - diffuser simulation . fourier analysis requires that the discrete values associated with the sampling of the arrays be properly related to the linear size of each array . in this situation , the two primary physical objects that require extended examination are the diffuser , or diffusers , and the lens . a positive lens is used in imaging the aperture of the lens and will determine the final speckle spot size in the observation plane . an important part of measuring the speckle contrast is ensuring that the speckle spots will not be averaged together in a single pixel of the observation screen . this will be fixed by ensuring a large array for the lens that will project the pattern propagated from the diffuser . an 8000 8000 array group will be used for each screen , i.e. gaussian input , diffuser , and lens . this means a sampling size of 3.89 per pixel given a 54.8 cm diameter lens and a 25.4 cm linear , square diffuser . the most advanced photolithography machines are capable of incrementing diffuser size of around 1 . this physical size limitation is well within the current standards for producing an actual diffuser and our simulated sampling size stands . the final output for the single hadamard diffuser propagated through a projection lens is shown below in figure 3 . the output directly after the diffuser and a smaller cropped version of the final output image are shown as well . ( a ) intermediate image screen ; ( b ) final image screen ; ( c ) final image normalized . the contrast provided by the final image is 0.67 0.02 comparable to the theoretical value of 0.707 for a single hadamard diffuser . the distance between the two diffusers was optimized for lowest possible speckle and is currently set at 8 mm . ( a ) image directly after second diffuser ; ( b ) final image output from dual diffuser system ; ( c ) close up image output from dual diffuser system . hadamard diffuser designs were manufactured by the microoptics division of jenoptik optical systems , inc ( huntsville ) . speckle contrast results were measured for the diffusers using a 532.8 nm dpss laser with pseudo - random linear polarization . a single diffuser was measured with a measured speckle contrast minimum of 0.62 ( 0.02 ) using a 25 mm focal length projection lens . the image was captured using a ccd camera with an array size of 480 640 pixels and a pixel size of 7 m giving a total detector size of 3.36 4.48 mm . the image results from the single physical hadamard diffuser matches the output results of a zoomed - in version of the simulation output in figure 3(c ) . two diffusers were aligned along a common optical axis and the image contrast was measured . final contrast results for dual hadamard diffusers were slightly more difficult to compare with simulated values due to mitigating factors such as spatial alignment , separation distance between diffusers , and speckle spot size . each of these factors can arguably effects the speckle output , so all conditions of testing have been fully analyzed for each contrast result . a standard separation distance between the diffuser of approximately 5 mm ( 0.5 mm ) was set up . it is noted that a significant variance in the speckle measurements was observed with an 812% change in the contrast resulting from the lack of optimization with the separation distance between the two diffusers . simulation results confirm that separation distance between the diffusers can have a significant impact on the resulting speckle contrast value which was confirmed with the physical measurements fluctuations . further research is underway to optimize the separation distance to enhance the speckle reduction capacity of the hadamard matrix diffuser set . the minimum value achieved was within 23% of expected simulation results although still greater than the 6% that was theoretically predicted . it is hypothesized that a possible difference in contrast is due to larger than anticipated gaussian beam effects and pixel to speckle spot size matching . while the simulation attempts to overcome pixel size mismatch by utilizing large array sizes , the camera array size , and pixel size this is still of minor importance as differences in theoretical vs. physical measurements are within the variance caused by the distance discrepancies of the two diffusers . however , this change is within error parameters caused by the physical setup of the diffuser so it is of interest only in situations where the laser is completely polarized . a static , dual in - line hadamard diffuser optical system has been demonstrated for speckle reduction in pico - projection applications . hadamard diffusers were fabricated and measured with good agreement between experimental contrast measurements and simulated values . a minimum contrast of 0.40 ( 0.08 ) was achieved without using rotation or vibrational movements . using this type of structured binary matrix maintains partial coherence of the laser source , unlike static pseudo - random grayscale - style diffusers , necessary to preserve the perceived brightness of the laser . this static design ensures no mechanical failure modes and no vibration that could result in optical component alignment issues within a small enclosed system . the in - line hadamard diffuser notion allows for a greater reduction in speckle than a single diffuser while still maintaining partial coherence desired for integrated laser pico - projector sources .

tea tree oil ( tto ) is derived from the paper bark tea tree , which belongs to the family of myrtaceae ; it belongs to two genera , leptospermum and melaleuca . tto medications have been used for thousands of years by australian aborigines both internally and externally for alleviation of pain and promotion of wound healing , cure colds , and influenza . although tree tea oil is used around the world in a small number of cosmetic and medicinal products , its use as an ingredient of oral health care products remains limited . 1.8-cineole and terpinen-4-ol are the main active ingredients of tto.2 , 3 , 4 tto is a uniquely defined combination of monoterpenes , sesquiterpenes , and terpene alcohol with outstanding therapeutic properties . it is recognized as having broad - spectrum antibacterial , antiviral ( carson et al 2005 ) , antimycoplasmal , and antiprotozoal activities ( furneri et al , 2006 ) , as well as anti - inflammatory ( finlay - jones et al 2001 ) and anticancer properties ( greay et al 2010).9 , 10 it acts by damaging the microorganisms ' cellular membrane and subsequently denaturing the cell contents . anecdotally , tto is known as an excellent treatment for fungal infections , in particular , vaginal candidiasis and dermatophytoses , but relative data showing its oral antifungal property is rather sparse.11 , 12 , 13 the potential usefulness of tto in oral health care products has not been assessed , and hence , through this study , our aim was to assess and compare the efficacy of tto as an alternative form of medicine with clotrimazole ( i.e. , allopathy ) and a conservative form of management in the treatment of oral fungal infection . our objective was to create directly comparable and quantitative antifungal data for tto , for which little literature data exist and which can be a useful addition to the current range of oral antifungal drugs . the maharaj vinayak global university ( mvgu ) research ethics committee gave ethical approval for the study . patients visiting the oral medicine department of jaipur dental college , kukas , india were screened for oral fungal infection . fungal infection included : ( 1 ) leukoplakia superimposed candidiasis , ( 2 ) denture - induced candidiasis , and ( 3 ) pseudomembranous candidiasis . the most common presenting clinical features of the above - mentioned fungal infections included erythema ( sign ) , burning sensation ( symptom ) , inflammation ( sign ) , and fungal hyphae ( pathological ) as suggested in the literature.8 , 9 , 10 the efficiency of the three groups was compared on the basis of four parameter indices that were selected based on the most common clinical features of fungal infection mentioned so far in the literature . the inclusion criteria called for male and female participants who were : ( 1 ) willing to cooperate , ( 2 ) available for regular follow - up , and ( 3 ) within the age group of 2060 years . the exclusion criteria included patients who were : ( 1 ) uncooperative , ( 2 ) on antifungal medication , ( 3 ) hiv - positive , ( 4 ) known to have critical diseases ( e.g. , leukemia and lymphoma ) , ( 5 ) undergoing radiation therapy , and those ( 6 ) who discontinued the medication and follow - up and ( 7 ) have reported side effects from tto . histopathological smear of the lesion and the prosthesis used were taken to confirm the diagnosis of fungal infection . patients diagnosed with fungal infection were randomly divided into ( 1 ) tto group , ( 2 ) allopathy group ( i.e. , clotramizole ) , and ( 3 ) conservative group , based on the treatment to be carried out . evaluation of lesion was done on the basis of four parameter indices : ( 1 ) erythema ; ( 2 ) visual analog scale , burning sensation ; ( 3 ) inflammation ; and ( 4 ) fungal hyphae pretreatment . complete medical , drug , and any allergy history were noted for selected patients , and any subjective and objective symptoms associated with the lesion were also recorded . prestructured performa was filled with all the above details and with specific emphasis on the frequency of use of prosthesis and its maintenance . after the confirmation of the diagnosis , treatment was started , and those patients who were willing to undergo treatment signed a consent form . during their first visit , participants selected for treatment with group i were instructed to perform on - spot rinse with tto and were placed on a 24-hour observation for any toxic effect from the essential oil . the participants were handed a pamphlet that outlined the use of tto , in the form of a rinse ( diluting 5 ml oil/50 ml water concentration 0.10% ) to be done thrice daily until the first follow - up ( after a meal ) . patients selected for treatment under group ii ( allopathy ) had to apply clotrimazole ointment thrice daily after meals until the first follow - up . under group iii ( conservative management ) , participants were asked to carry out regular cleaning and washing of the prosthesis on a daily basis and removal of prosthesis during the night in case fungal infection turned out to be denture - induced . the participants were asked to refrain from using proprietary mouthwashes during the study , and no auxiliary cleaning aids were allowed to be used during the course of the study . medication was distributed to the participants in labeled ( a , tto ; b , allopathy ) dispensing bottles . follow - up was carried out after the 1 week , 2 week , and 3 week to evaluate the subsequent changes in the lesion on the basis of the four parameter indices ( fig . on the recall visit , any changes in clinical features associated with lesions were noted . patient performa posttreatment was filled up again , and the medication was continued if any symptoms and signs of the lesion persisted . the data for each participant were added , averaged , and weighted depending on the positive ( improvement ) or negative ( no improvement ) changes in the lesion in subsequent recalls . the efficiency , compliance , and safety index of the two drugs were evaluated and compared using a relative percentage chart for both groups . the efficiency of all three treatment methods was compared on a bar graph on the scale of 1 . a total of 36 participants , consisting of 11 females and 25 males ( table 1 ) , were recruited for the study . those not included in the study were either dropouts or were omitted based on the exclusion criteria . overall , there were 13 patients in group i , 13 patients in group ii , and 10 patients in group iii . a percentage analysis for all three groups was done , and a comparative chart in terms of percentage for all four parameter indices ( table 2 ) was drawn up . the following results were deduced ( table 2 ) . ( 1 ) the tto - treated group showed an 89% reduction in erythema and inflammation compared to the other two groups . thus , tto proved to be a better alternative to clotrimazole in reducing the two main clinical features associated with oral fungal infection . ( 2 ) in the tto group , there was a 100% reduction in visual analog scale burning sensation score . clotrimazole proved to be 100% successful in the reduction of clinical and histopathological features in this study . 100% reduction in burning sensation was achieved in subjects treated under group i & group ii , but only 50% reduction was achieved in group iii . from the above findings , it was preliminarily concluded that overall both group i and group ii are at par in giving symptomatic relief to patients suffering from oral fungal infection . as the study was directed toward comparing allopathy and alternative medicine in the treatment of oral fungal infection , we further filtered the results and compared all three modalities through a bar graph on the scale of 1 ( fig . ( 1 ) tto ( group i ) was the most efficient of all three modalities . ( 2 ) although clotrimazole ( group ii ) scored 100% on two indices , it came only a close second in overall efficiency . ( 3 ) conservative management ( group iii ) was the least efficient of all three treatments . the allopathic antifungal agents are effective in the treatment of oral fungal infection , but present several major negative effects that can not be overlooked . polyene antifungals have a broad spectrum of antifungal activities , but because of their poor absorption through the gut their use in the treatment of oral candidiasis is limited . fewer cases of polyene toxicity to animal cells have been reported ; however , at therapeutic doses , some ( e.g. , amphotericin b ) may bind to animal membrane cholesterol , thus increasing the risk of human toxicity . some recent case reports also suggest the toxicity of nystatin if it is used in the long run . as a polyene 's hydrophobic chain is shortened , its sterol binding activity is increased . therefore , further reduction of the hydrophobic chain may result in it binding to cholesterol , making it toxic to humans . among azole antifungals , however , several mechanisms of azole resistance have been reported including : ( 1 ) an alteration in the chemical structure of the demethylase enzyme , ( 2 ) removal of the azole from the cell by multidrug transporter pumps , and ( 3 ) compensation by other sterol synthesis enzymes in membrane biosynthesis . these resistance mechanisms of fungal microorganisms can increase the chances of failure of these antifungal drugs in the future . the clinical effectiveness of agents that can only be delivered topically , such as amphotericin or nystatin , is limited owing to problems in maintaining sufficient levels of these drugs at the site of infection . the taste of topical agents stimulates salivary secretion , which rapidly dilutes and removes the antifungal agent from the mouth . in view of this , their clinical use is limited ( k. hammer et al 2002 ) . fluconazole has a good safety profile when given systemically , with few contraindications or side effects . important interactions occur with coumarin anticoagulants and sulfonylurea antidiabetic agents though . apart from certain side effects such as liver damage or effect on estrogen levels hence , allopathic antifungals is beneficial but also has several major side effects . in vitro studies regarding tto the literature suggests that tto in vitro substantially alters the membrane permeability of c. albicans and candida glabrata . therefore , compared to clotramizole tto will be less misicible in saliva and will remain at the effected site for a longer length of time , thereby producing a better pharmacological effect . as discussed in detail , allopathic antifungals have numerous toxic effects if used in the long run , whereas tto , being purely a herbal plant , has no toxic effects if used for a long duration.17 , 18 although the authors have tried their best to include all possible parameters in the study , certain limitations / drawbacks remain ( as detailed below ) . research can be elaborated with an increase in the sample size with special emphasis on a specific candidal pathogen that is mainly the causative factor for denture - induced candidiasis . as candida is one of the predisposing factors for potentially premalignant and malignant lesions , specific candida species can be investigated . the efficacy of tto can be compared on the different grades of denture - induced candidiasis . the future of tea trees is interesting and challenging , and more aspects of its properties and potential therapeutic applications can be investigated . within the limitations of the study , it was apparent that group i patients showed improvement in relation to symptoms associated with lesion(s ) . tto , being a better alternative to clotrimazole , is justifiable because it has good membrane permeability and has no toxic effects , whereas allopathic antifungals have several side effects that can not be overlooked . from the study results , we conclude that efficiency of both medications are at a par , but tto , being nontoxic , economical , and compliable , remains a better alternative to allopathy in the treatment of oral fungal infections . the components of tto are known to have lipophilic properties that facilitate its diffusion through the epithelium . if tto is readily absorbed after its topical application or rinse into gingival tissues and has antifungal properties once it has entered connective tissues , it would be a unique nontoxic agent that would be a useful addition to the current range of chemotherapeutic antifungal options .

brain metastasis of uterine leiomyosarcoma ( lms ) is rare ( wroski et al . , 1994 aug , honeybul and ha , 2009 mar , yamada et al . several case reports suggest a resection of brain metastases of uterine lms could result in longer survival , for which controlling systemic disease is a prerequisite ( wroski et al . , 1994 aug , honeybul and ha , 2009 mar , yamada et al . , 2011 dec , gadducci et al . radiotherapy after brain surgery can lower recurrence rates , but long - term toxic side effects of radiation , such as cognitive decline , decrease quality of life , and radiation can not control systemic disease . pazopanib is approved for soft tissue sarcomas and penetrates the blood - brain barrier ( bbb ) ( iwamoto et al . , 2010 aug ) . several case reports in other malignancies suggest that pazopanib treatment could elicit survival benefits for patients with brain metastases in addition to controlling systemic disease ( jacobs et al . , 2013 we present the first case report of long - term disease stabilization with pazopanib treatment after a resection of solitary brain metastasis from uterine lms . a 48-year - old multiparous woman underwent a total abdominal hysterectomy for what was thought to be multiple uterine leiomyomas . subsequent positron emission tomography / computed tomography ( pet / ct ) showed no metastatic lesions , and she was followed up without further surgery or adjuvant therapy . forty - seven months after the initial diagnosis , a biannual follow - up computed tomography ( ct ) scan revealed multiple lung metastases , and video - assisted thoracic surgery ( vats ) was performed . shortly after the vats procedure , the patient developed pelvic pain , and pet / ct imaging suggested a pelvic bone metastasis . she was treated with six cycles of adjuvant chemotherapy with gemcitabine and docetaxel , and local radiation therapy was administered to control the pain . seventy - seven months after the initial diagnosis , she had a gradually worsening headache , and 2 weeks later , she developed left hemiparesis and aphasia with a karnofsky performance scale ( kps ) score of 40 . magnetic resonance imaging ( mri ) revealed a solitary 58 mm 45 mm lesion in the right frontal lobe with a midline shift ( fig . 1 ) . the patient underwent a craniotomy and complete resection of the lesion , after which she showed no neurological deficit , and her kps score improved to 90 . immunohistochemical findings of the resected metastatic brain tumor were positive for alpha - smooth muscle actin , vimentin , desmin , and epithelial membrane antigen staining , and lms metastasis to the brain was confirmed ( fig . one month after the craniotomy , she experienced lower abdominal pain that required opioids , and a ct scan revealed a pelvic mass that was suggestive of recurrence and was unresectable ( fig . one month after the brain surgery , she began oral pazopanib ( 800 mg per day ) . one month later , she was free of pain , and no opioid was necessary . she experienced mild diarrhea and mild hypertension , both of which were well - controlled . however , the pelvic lesions gradually enlarged , causing a severe pain . therefore , 14 months after starting pazopanib , it was discontinued . to date , she remains alive with disease , 18 months after the brain surgery . uterine lms is aggressive in nature , and prognosis of recurrent lms is very poor . the risk of recurrence after complete resection of uterus - limited lms , which was estimated to be 70% after 2 years in a retrospective study , is high ( major et al . , 1993 feb 15 ) . the most common sites of uterine lms metastasis are the lung , pelvis , and vagina ( rose et al . , 1989 , 1999 aug ) , but brain metastasis is rare . to the best of our knowledge , only case reports of uterine lms metastasis to the brain are available ( wroski et al . , 1994 aug , honeybul and ha , 2009 mar , yamada et al . , 2011 dec ) . yamada et al . reviewed in their case report that there had been 21 such cases ( yamada et al . , 2011 dec ) . brain metastasis incidence has been reported to be 1 in 19 ( rose et al . , 1989 mar 1 ) and 1 in 4 from autopsies of patients with uterine lms ( fleming et al . in a retrospective study of brain metastases from gynecologic malignancies , 4 cases out of 139 cases of gynecologic malignancies were uterine lms ( nasu et al . because of its rarity , there has been no established management for uterine lms metastasis to the brain . for recurrence of uterine lms , combination chemotherapy , such as gemcitabine and docetaxel , may be a treatment option ( hensley et al . , 2002 jun 15 ) , but it is meant to be palliative and is not curative . additionally , conventional cytotoxic agents that have proven to be effective for lms do not cross the bbb . only surgical resection can prolong survival . in a retrospective study by leitao et al . , pulmonary and extrathoracic metastasectomy of recurrent uterine lms resulted in a survival benefit when complete resection was achieved ( leitao et al . , 2002 dec ) . in cases of a solitary brain metastasis of uterine lms with well - controlled systemic disease , long - term survival , namely 12 years , after brain surgery can be expected , as presented and reviewed in multiple case reports ( wroski et al . , 1994 aug , honeybul and ha , 2009 mar , yamada et al . , 2011 dec , gadducci et al . , 1996 jul ) . on the other hand , in patients with progressive and uncontrolled systemic metastases , survival is very poor , even after an aggressive brain surgery ( honeybul & ha , 2009 mar ) . moreover , whole - brain radiotherapy ( wbrt ) after brain surgery can greatly lower brain metastasis recurrence rates ( patchell et al . , 1998 nov 4 ) , but long - term toxic effects of wbrt , such as cognitive decline , should be considered when systemic disease remains under control and long - term survival is expected . pointed out that in patients with uterine lms metastasis to the brain , postoperative radiation therapy might not offer survival benefits ( yamada et al . , 2011 dec ) . furthermore , when brain metastasis recurrence can be decreased , systemic disease should be controlled . adjuvant therapy that is effective for both systemic and brain - specific disease after brain surgery is needed . pazopanib is a multi - targeted tyrosine kinase inhibitor that impairs angiogenesis and is approved for soft tissue sarcoma treatment . it is small enough to penetrate the bbb and has been used in a phase ii study for patients with glioblastoma ( iwamoto et al . , 2010 aug ) . one case report showed prolonged survival after pazopanib therapy for patients with papillary renal cell carcinoma and brain metastases ( jacobs et al . another case report described a patient with renal cancer and brain metastasis who survived for 3 years after wbrt and subsequent pazopanib ( hingorani et al . , 2014 ) . pazopanib was used to treat the patient in our case report to control the pelvic lesions and prevent brain metastasis recurrence . it was well - tolerated , and to date , there has been no brain metastasis recurrence , and , although they have since progressed , the pelvic lesions remained under control for a year . we can study the role of pazopanib by relieved symptoms , tumor size reduction assessed by imaging studies including ultrasonography , ct and mri , and treatment response assessed by fluorodeoxyglucose pet / ct ( except for brain metastases ) . if resected specimens are obtained , the immnohistochemical expression of platelet - derived growth factor receptor ( pdgfr ) can be investigated as a predictive marker for response of pazopanib , although it has not been clinically applicable . as for brain metastasis , resectable uterine leiomyosarcoma metastatic to the brain is very rare , and to the best of our knowledge , immunohistochemical investigation has not proceeded . it is unknown whether pazopanib was actually effective for preventing recurrence of brain metastasis or whether complete resection of the brain metastasis contributed to the favorable prognosis in this patient . the patient enjoyed a prolonged symptom - free survival from the surgery for the brain metastasis . long - term disease stabilization was obtained with pazopanib treatment after resection of a uterine lms brain metastasis . pazopanib , with its ability to penetrate the bbb , could replace wbrt after resection of solitary brain metastasis and might lower brain metastasis recurrence rates and control systemic disease .

nearly 10% of the us population has diabetes mellitus , of whom 90% have type 2 diabetes ( t2 dm ) 1 . as the condition progresses , metabolic control often can not be maintained with just one agent , and multiple agents are required to treat the later stages of t2 dm . based on joint guidelines issued by the american diabetes association ( ada ) and the european association for the study of diabetes ( easd ) , metformin is considered the first - line therapy unless not tolerated or contraindicated 2 . some individuals , however , experience adverse gastrointestinal events , such as nausea , vomiting and diarrhoea , with metformin , and thus require a different treatment approach . sulphonylureas depend on the remaining pancreatic -cell mass and function , making them less than optimal for those with long - standing t2 dm . additionally this class of drugs is associated with hypoglycaemia and weight gain , both undesirable in the t2 dm population . thiazolidinediones , in comparison , can cause weight gain , induce fluid retention , worsen congestive heart failure and increase the incidence of bone fractures in women 3 . thus , there is a need for newer agents with acceptable safety profiles that can be used alone or with other agents at any stage of t2 dm . new treatment strategies for t2 dm have largely focused on the incretin system and , despite the important role of renal pathways in glucose homeostasis , have only recently targeted the kidney 4 . dipeptidyl peptidase ( dpp)-4 inhibitors enhance postprandial insulin secretion and suppress glucagon secretion by preventing the degradation of endogenously released incretins [ glucagon - like peptide ( glp)-1 and glucose - dependent insulinotropic polypeptide ( gip ) ] , two intestinal peptides whose concentration increases after food intake , thus playing a vital role in glucose homeostasis 5 . dpp-4 inhibitors stimulate insulin secretion in a glucose - dependent fashion and inhibit glucagon secretion , thus minimizing hypoglycaemia and improving hyperglycaemia 6 . in addition , dpp-4 inhibitors are weight - neutral and have been shown to improve -cell function in in vitro and animal studies 7,8 . these characteristics may provide benefits to those people with t2 dm who have impaired -cell function , excessive hepatic glucose production , postprandial hyperglycaemia and who are overweight or obese . plasma glucose is freely filtered in the kidney glomeruli and must be returned to the circulation to prevent urinary excretion . this process is accomplished with two types of carrier proteins : the active sodium - glucose co - transporters ( sglts ) and the passive glucose transporters 1113 . the sglt inhibitors are a family of membrane - bound transport proteins , of which sglt type 1 ( sglt1 ; a low - capacity , high - affinity transporter ) and sglt type 2 ( sglt2 ; a high - capacity , low - affinity transporter ) mediate glucose reabsorption from the glomerular filtrate independent of insulin and induce urinary glucose excretion ( i.e. glucosuria ) . an indication of what to expect from pharmacological sglt2 inhibition was derived from observations of individuals with familial renal glucosuria , a mutation of the sglt2 gene that serves as a model for sglt2 inhibition 8 . the impaired function of sglt2 in affected individuals can lead to daily urinary glucose excretion of up to 200 g , but most individuals are asymptomatic and do not seem to develop significant clinical problems over time 9.these findings suggest that pharmacologic sglt2 inhibition could be a safe option in the attempt to reduce hyperglycaemia in individuals with t2 dm . the lack of a common mechanistic pathway between sglt2 inhibitors and other agents suggests that they can be given in combination with any of the existing therapeutic classes of glucose - lowering agents , including dpp-4 inhibitors , glp-1 agonists and insulin . together , the dpp-4 and sglt2 inhibitors fulfil a need for agents with complementary mechanisms of action that can be used in combination with a low risk of adverse events , such as hypoglycaemia or weight gain . the present review provides an overview of the pharmacology , pharmacokinetics , efficacy and safety of these two classes of agents and a rationale for their use as dual - combination therapy for the treatment of t2 dm . it has been more than 8 years since the first dpp-4 inhibitor , sitagliptin , was approved for the treatment of t2 dm 14,15 . currently , eight dpp-4 inhibitors are available worldwide : sitagliptin , vildagliptin , saxagliptin , linagliptin , alogliptin , gemigliptin , anagliptin and teneligliptin . the latter three are only available in asia 14 . in individuals with t2 dm , the incretin effect is markedly impaired ; whereas exogenously administered glp-1 retains its effect and improves hyperglycaemia , the insulinoptropic effect of gip is lost in these individuals 6 . new therapeutic advances have focused on the development of glp-1 agonists that are resistant to dpp-4 inactivation as well as on inhibitors of dpp-4 , in an effort to prevent incretins from degradation . in addition to their glucose - dependent anti - hyperglycaemic actions that ultimately lead to reduced postprandial glucose , fasting plasma glucose ( fpg ) and glycated haemoglobin ( hba1c ) levels , the incretins also exert beneficial pleiotropic actions on the cardiovascular system 16 , including improvements in blood pressure and left ventricular function 17,18 , and improved endothelium - dependent vasodilation and increased levels of endothelial progenitor cells 19,20 . clinical trial data for dpp-4 inhibitor monotherapy have shown that these agents improve glycaemic control by reducing hba1c [ from 0.6 to 1.1% ( 6.6 to 12.0 mmol / mol ) ] 21 and fpg concentrations ( from 0.73 to 1.57 mmol / l ) 21 , with a similar incidence of hypoglycaemia and change in body weight as that found with placebo 2231 . dpp-4 inhibitors are approved for treatment of hyperglycaemia as mono- , dual and triple oral therapy as well as in combination with insulin . they are usually well tolerated , and the most frequent adverse event in clinical trials was nasopharyngitis . three sglt2 inhibitors are currently approved for the treatment of t2 dm in the usa and the european union : canagliflozin , dapagliflozin and empagliflozin . these agents cause reduced reabsorption of glucose from the glomerular filtrate and increased excretion of glucose into the urine , and cause urinary glucose excretion to occur at a lower plasma glucose concentration . sglt2 inhibition results in the loss of 6080 g of glucose in the urine per day 32 , which helps to reduce hyperglycaemia in individuals with t2 dm . in addition to improvements in glycaemic control , sglt2 inhibitors provide other effects that are desirable in a t2 dm agent , such as weight loss 33 , moderate reductions in systolic blood pressure and no increase in hypoglycaemia risk 34 . clinical trial data have shown that sglt2 inhibitors improve glycaemic control by reducing hba1c , postprandial glucose and fpg concentrations , and produce modest reductions in body weight and blood pressure 3539 . when used as monotherapy , these agents have been found to lead to reductions in hba1c [ from 0.34 to 1.03% ( 3.711.3 mmol / mol ) ] , body weight ( from 2.0 to 3.4 kg ) , and systolic and diastolic blood pressure ( from 1.7 to 6.4 mmhg and from 0.3 to 2.6 mmhg , respectively ) 40 . specific adverse reactions of sglt2 inhibitors are related to urinary glucose excretion , in that the continual presence of glucose in the urine may increase the risk of urinary tract infections and/or genital mycotic infections . the progressive deterioration of -cell function in t2 dm often necessitates the use of combination therapy in order for individuals to reach their glycaemic goals ; however , the use of anti - hyperglycaemic agents in combination may lead to an increase in the risk of adverse events , including weight gain and hypoglycaemia , which occur with sulphonylureas and thiazolidinediones . individuals need treatments that can be used in combination with other agents without adverse events limiting their use . as previously mentioned , the potential complementary mechanistic pathway for sglt2 inhibitors and dpp-4 inhibitors suggests they could be given in combination with each other without any obvious detrimental effects ; however , the use of the individual agents with an insulin secretagogue or insulin may increase the risk of hypoglycaemia . furthermore , a mechanism of action that is independent of pancreatic -cell function makes sglt2 inhibitors an appropriate option for those with advanced t2 dm , particularly if their glycaemic control is inadequate with existing oral glucose - lowering agents . a study assessed the effects of dapagliflozin on muscle insulin sensitivity in 18 men with t2 dm 41 and found that lowering fpg using an sglt2 inhibitor improves insulin - stimulated tissue glucose disposal . this study also showed that dapagliflozin treatment resulted in a substantial increase in endogenous glucose production , which was accompanied by an increase in fasting plasma glucagon concentrations . a study of empagliflozin that assessed the response to pharmacologically induced acute or chronic glycosuria in 66 participants with t2 dm showed that glycosuria induced by a single dose of empagliflozin lowered both fpg and postprandial glucose , despite a compensatory increase in endogenous glucose production 42 . this study also showed a glucagon increase that almost normalized after 4 weeks of empagliflozin treatment . it has been estimated that the increase in endogenous glucose production offsets approximately half of the glucose excreted as a result of sglt2 inhibition with dapagliflozin 41 . thus , the addition of a dpp-4 inhibitor which inhibits glucagon and stimulates insulin secretion may have the potential to block the increase in endogenous glucose production and enhance the glucose - lowering ability of sglt2 inhibitors . taken together , these findings suggest that the combination of an sglt2 inhibitor with a dpp-4 inhibitor would potentially provide additional help to individuals with t2 dm in reaching their glycaemic goal 41 . the combination of dpp-4 inhibitors and sglt2 inhibitors also has the potential to exert beneficial effects on the kidney . both classes have been reported to lower urinary albumin excretion , a risk factor for renal disease , although these findings need to be confirmed in larger studies 4345 . a recent study in individuals with t1 dm found that 8 weeks of empagliflozin treatment attenuated renal hyperfiltration , a surrogate marker of intraglomerular pressure 46 . furthermore , the canagliflizon and renal events in diabetes with established nephropathy clinical evaluation study ( credence study ; nct02065791 ) with canagliflozin was recently initiated and will assess renal outcomes with sglt2 inhibition . in addition , the carmelina study with linagliptin will explore the effect of dpp-4 inhibition on renal outcomes ( renal death , end - stage renal disease and a sustained estimated glomerular filtration rate decrease 50% ) . the potential of combining both classes to maximize renal opportunities is intriguing and may be of interest to the readers of the present review . the drug drug interactions of empagliflozin and linagliptin 47 or sitagliptin 48 were studied in healthy male volunteers . both studies showed that administration of either linagliptin or sitagliptin with empagliflozin had no clinically relevant effect on the pharmacokinetics of either agent ; therefore , empagliflozin can be co - administered with either linagliptin or sitagliptin without dose adjustments . a 24-week , randomized , double - blind , placebo - controlled study assessed dapagliflozin ( 10 mg ) as an add - on therapy to sitagliptin ( 100 mg , with and without metformin , 1500 mg / day ) in 447 participants 49 . after 24 weeks , the addition of dapagliflozin significantly reduced mean hba1c concentration compared with placebo , excluding data after rescue [ placebo - corrected difference 0.5% ; 95% confidence interval ( ci ) 0.6 to 0.3 ( 6 mmol / mol ; 95% ci 7 to 3 ) ; p < 0.0001 ] , even in a subset of participants with hba1c baseline levels 8% [ 64 mmol / mol ; placebo - corrected difference 0.8% ; 95% ci 1.0 to 0.6 ( 9 mmol / mol ; 95% ci 11 to 7 ) ; p < 0.0001 ] . similarly , < 0.0001 ) and body weight ( placebo - corrected difference 1.9 kg ; 95% ci 2.4 to 1.4 ; p < 0.0001 ) . these results suggest that add - on treatment with dapagliflozin in individuals inadequately controlled with sitagliptin with or without metformin can provide additional clinical benefits and is well tolerated . recently the combination of dapagliflozin with saxagliptin vs saxagliptin or dapagliflozin alone was assessed in a 24-week , randomized , active - controlled study in 534 participants with t2 dm receiving background metformin 50 . after 24 weeks , the reductions in hba1c from baseline [ 8.9 , 9.0 and 8.9% ( 74 , 75 and 74 mmol / mol ) , respectively ] , were larger in the group receiving dual combination treatment [ saxagliptin 5 mg + dapagliflozin 10 mg ; adjusted mean change from baseline 1.5% ( 16 mmol / mol ) ] compared with the groups receiving either agent alone [ saxagliptin 0.9% ( 10 mmol / mol ) ; difference 0.6% ( 7 mmol / mol ) ; p < 0.0001 ; dapagliflozin 1.2% ( 13 mmol / mol ) ; difference 0.3% ( 3 mmol / mol ) ; p < 0.02 ] . the adjusted proportion of participants achieving an hba1c concentration of < 7% ( < 53 mmol / mol ) was 41% with dual combination therapy vs 18 and 22% vs saxagliptin or dapagliflozin alone , respectively . this study shows that , when added to background metformin therapy , the combination of a dpp-4 inhibitor and an sglt2 inhibitor resulted in greater improvements in glucose control than each agent alone , and helped > 40% of individuals achieve their glycaemic goal . the canagliflozin cardiovascular assessment study ( canvas ) is an ongoing randomized , placebo - controlled study assessing the efficacy and safety of canagliflozin 100 and 300 mg added to a range of therapies compared with placebo in patients with t2 dm 51 . a post hoc analysis from canvas in individuals with t2 dm and a history or high risk of cardiovascular disease assessed the effects of canagliflozin 100 and 300 mg versus placebo in subsets of participants on dpp-4 inhibitors ( n = 316 ) or glp-1 agonists ( n = 95 ) , with or without other antihyperglycaemic agents 52 . in that study , canagliflozin 100 and 300 mg improved hba1c at 18 weeks compared with placebo , added to dpp-4 inhibitors [ placebo - corrected change from baseline in hba1c : canagliflozin 100 mg , 0.6% ( 7 mmol / mol ) ; 300 mg , 0.8% ( 9 mmol / mol ) ] or glp-1 agonists [ canagliflozin 100 mg , 1.0% ( 11 mmol / mol ) ; 300 mg , 1.1% ( 12 mmol / mol ) ] . additionally , canagliflozin reduced body weight in both subsets ( placebo - corrected change from baseline , dpp-4 inhibitors : canagliflozin 100 mg , 2.3 kg ; 300 mg , 3.0 kg or glp-1 agonists : canagliflozin 100 mg , 2.5 kg ; 300 mg , 3.2 kg ) . the canvas study continues in a blind fashion in order to collect additional safety data , including cardiovascular endpoints . two recent studies of the single - pill combination of empagliflozin and linagliptin assessed the efficacy and safety of empagliflozin / linagliptin in participants with t2 dm 53,54 . in both studies , the key secondary endpoints included changes from baseline in fpg and body weight , as well as the percentage of participants with hba1c 7.0% ( 53 mmol / mol ) at baseline who achieved an hba1c target of < 7% ( < 53 mmol / mol ) , all assessed at week 24 . one study randomized drug - nave individuals with t2 dm to receive empagliflozin / linagliptin ( 25 mg/5 mg , n = 137 or 10 mg/5 mg , n = 136 ) , empagliflozin alone ( 25 mg , n = 135 or 10 mg , n = 134 ) or linagliptin alone ( 5 mg , n = 135 ) 53 . both single - pill combinations significantly reduced hba1c from baseline [ from 7.99 to 8.05% ( 64 mmol / mol ) ] compared with linagliptin alone [ 25 mg/5 mg , difference 0.4% ( 4 mmol / mol ) ; p < 0.001 ; 10 mg/5 mg , difference 0.6% ( 7 mmol / mol ) ; p < 0.001 ] . hba1c reductions were greater with empagliflozin / linagliptin 10 mg/5 mg than with empagliflozin 10 mg [ difference 0.41% ( 4.5 mmol / mol ) ; p < 0.001 ] , but were not significantly different between empagliflozin / linagliptin 25 mg/5 mg and empagliflozin 25 mg [ difference 0.14% ( 1.5 mmol / mol ) ; p = non - significant ] . the single - pill combinations also caused greater reductions in fpg and body weight than linagliptin alone . in addition , the proportion of individuals who achieved their goal hba1c concentration [ < 7% ( < 53 mmol / mol ) ] at 24 weeks was significantly greater with the single - pill combination than with the individual components : 55.4 and 62.3% , respectively , for the empagliflozin / linagliptin 25 mg/5 mg and 10 mg/5 mg groups compared with 41.5 , 38.8 and 32.3% , respectively , for the empagliflozin 25 mg , empagliflozin 10 mg and linagliptin 5 mg groups . confirmed hypoglycaemic adverse events [ glucose 3.8 mmol / l ( 70 mg / dl ) and/or requiring assistance ] were reported in two subjects on empagliflozin 25 mg and one each on empagliflozin 10 mg and linagliptin 5 mg ; none required assistance . the second study assessed the single - pill combination of empagliflozin and linagliptin as an add - on to stable metformin in participants with t2 dm 54 . in 674 participants , the single - pill combinations significantly reduced hba1c compared with the respective monotherapies : empagliflozin / linagliptin 25 mg/5 mg vs empagliflozin 25 mg [ difference 0.58% ( 6.3 mmol / mol ) ; p < 0.001 ] and vs linagliptin 5 mg [ difference 0.50% ( 5.5 mmol / mol ) ; p < 0.001 ] ; empagliflozin / linagliptin 10 mg/5 mg vs empagliflozin 10 mg [ difference 0.42% ( 4.6 mmol / mol ) ; p < 0.001 ] and vs linagliptin 5 mg [ difference 0.39% ( 4.3 mmol / mol ) ; p < 0.001 ] . combination treatment also lowered fpg compared with the individual agents and body weight vs linagliptin . < 7% ( < 53 mmol / mol ) ] at 24 weeks was significantly greater with the single - pill combination than with the individual components : 61.8 and 57.8% , respectively , for the 25 mg/5 mg and the 10 mg/5 mg groups compared with 32.6 , 28.0 and 36.1% , respectively , for the empagliflozin 25 mg , empagliflozin 10 mg and linagliptin 5 mg groups . confirmed hypoglycaemic adverse events [ glucose 3.8 mmol / l ( 70 mg / dl ) and/or requiring assistance ] were reported in two subjects each on empagliflozin / linagliptin 25 mg/5 mg , empagliflozin / linagliptin 10 mg/5 mg and empagliflozin 10 mg and linagliptin 5 mg , and four subjects on empagliflozin 25 mg ; none required assistance . glycaemic control with empagliflozin / linagliptin was maintained at week 52 with statistically significant improvements from baseline in hba1c , and a higher proportion of individuals achieving hba1c < 7% [ 53 mmol / mol ] , in both single - pill combination groups vs the linagliptin 5 mg group , and the empagliflozin 10 mg / linagliptin 5 mg group vs empagliflozin 10 mg group 53 , and for the empagliflozin 25 mg / linagliptin 5 mg combination vs the empagliflozin 25 mg group in the metformin add - on study 54 . in addition , all therapies were well tolerated . a number of clinical trials assessing the combination of dpp-4 inhibitors with sglt2 inhibitors in t2 dm are underway . two studies are investigating the efficacy and safety of adding on empagliflozin to linagliptin vs linagliptin alone ( nct01734785 ) and adding on linagliptin to empagliflozin vs empagliflozin alone in participants with t2 dm ( nct01778049 ) , both studies are scheduled to be completed in 2015 . several other ongoing studies are assessing the combination of dapagliflozin and saxagliptin ; nct01662999 is looking at the drug interaction of both agents in healthy individuals . other phase iii studies that are currently recruiting include assessments on the efficacy and safety of saxagliptin ( nct01619059 ) or dapagliflozin ( nct01646320 ) in triple therapy in participants with t2 dm who have inadequate glycaemic control with dual therapy including metformin . the drug drug interactions of empagliflozin and linagliptin 47 or sitagliptin 48 were studied in healthy male volunteers . both studies showed that administration of either linagliptin or sitagliptin with empagliflozin had no clinically relevant effect on the pharmacokinetics of either agent ; therefore , empagliflozin can be co - administered with either linagliptin or sitagliptin without dose adjustments . a 24-week , randomized , double - blind , placebo - controlled study assessed dapagliflozin ( 10 mg ) as an add - on therapy to sitagliptin ( 100 mg , with and without metformin , 1500 mg / day ) in 447 participants 49 . after 24 weeks , the addition of dapagliflozin significantly reduced mean hba1c concentration compared with placebo , excluding data after rescue [ placebo - corrected difference 0.5% ; 95% confidence interval ( ci ) 0.6 to 0.3 ( 6 mmol / mol ; 95% ci 7 to 3 ) ; p < 0.0001 ] , even in a subset of participants with hba1c baseline levels 8% [ 64 mmol / mol ; placebo - corrected difference 0.8% ; 95% ci 1.0 to 0.6 ( 9 mmol / mol ; 95% ci 11 to 7 ) ; p < 0.0001 ] . similarly , mmol / l ; 95% ci 1.92 to 1.19 ; p < 0.0001 ) and body weight ( placebo - corrected difference 1.9 kg ; 95% ci 2.4 to 1.4 ; p < 0.0001 ) . these results suggest that add - on treatment with dapagliflozin in individuals inadequately controlled with sitagliptin with or without metformin can provide additional clinical benefits and is well tolerated . recently the combination of dapagliflozin with saxagliptin vs saxagliptin or dapagliflozin alone was assessed in a 24-week , randomized , active - controlled study in 534 participants with t2 dm receiving background metformin 50 . after 24 weeks , the reductions in hba1c from baseline [ 8.9 , 9.0 and 8.9% ( 74 , 75 and 74 mmol / mol ) , respectively ] , were larger in the group receiving dual combination treatment [ saxagliptin 5 mg + dapagliflozin 10 mg ; adjusted mean change from baseline 1.5% ( 16 mmol / mol ) ] compared with the groups receiving either agent alone [ saxagliptin 0.9% ( 10 mmol / mol ) ; difference 0.6% ( 7 mmol / mol ) ; p < 0.0001 ; dapagliflozin 1.2% ( 13 mmol / mol ) ; difference 0.3% ( 3 mmol / mol ) ; p < 0.02 ] . the adjusted proportion of participants achieving an hba1c concentration of < 7% ( < 53 mmol / mol ) was 41% with dual combination therapy vs 18 and 22% vs saxagliptin or dapagliflozin alone , respectively . this study shows that , when added to background metformin therapy , the combination of a dpp-4 inhibitor and an sglt2 inhibitor resulted in greater improvements in glucose control than each agent alone , and helped > 40% of individuals achieve their glycaemic goal . the canagliflozin cardiovascular assessment study ( canvas ) is an ongoing randomized , placebo - controlled study assessing the efficacy and safety of canagliflozin 100 and 300 mg added to a range of therapies compared with placebo in patients with t2 dm 51 . a post hoc analysis from canvas in individuals with t2 dm and a history or high risk of cardiovascular disease assessed the effects of canagliflozin 100 and 300 mg versus placebo in subsets of participants on dpp-4 inhibitors ( n = 316 ) or glp-1 agonists ( n = 95 ) , with or without other antihyperglycaemic agents 52 . in that study , canagliflozin 100 and 300 mg improved hba1c at 18 weeks compared with placebo , added to dpp-4 inhibitors [ placebo - corrected change from baseline in hba1c : canagliflozin 100 mg , 0.6% ( 7 mmol / mol ) ; 300 mg , 0.8% ( 9 mmol / mol ) ] or glp-1 agonists [ canagliflozin 100 mg , 1.0% ( 11 mmol / mol ) ; 300 mg , 1.1% ( 12 mmol / mol ) ] . additionally , canagliflozin reduced body weight in both subsets ( placebo - corrected change from baseline , dpp-4 inhibitors : canagliflozin 100 mg , 2.3 kg ; 300 mg , 3.0 kg or glp-1 agonists : canagliflozin 100 mg , 2.5 kg ; 300 mg , 3.2 kg ) . the canvas study continues in a blind fashion in order to collect additional safety data , including cardiovascular endpoints . two recent studies of the single - pill combination of empagliflozin and linagliptin assessed the efficacy and safety of empagliflozin / linagliptin in participants with t2 dm 53,54 . in both studies , the key secondary endpoints included changes from baseline in fpg and body weight , as well as the percentage of participants with hba1c 7.0% ( 53 mmol / mol ) at baseline who achieved an hba1c target of < 7% ( < 53 mmol / mol ) , all assessed at week 24 . one study randomized drug - nave individuals with t2 dm to receive empagliflozin / linagliptin ( 25 mg/5 mg , n = 137 or 10 mg/5 mg , n = 136 ) , empagliflozin alone ( 25 mg , n = 135 or 10 mg , n = 134 ) or linagliptin alone ( 5 mg , n = 135 ) 53 . both single - pill combinations significantly reduced hba1c from baseline [ from 7.99 to 8.05% ( 64 mmol / mol ) ] compared with linagliptin alone [ 25 mg/5 mg , difference 0.4% ( 4 mmol / mol ) ; p < 0.001 ; 10 mg/5 mg , difference 0.6% ( 7 mmol / mol ) ; p < 0.001 ] . hba1c reductions were greater with empagliflozin / linagliptin 10 mg/5 mg than with empagliflozin 10 mg [ difference 0.41% ( 4.5 mmol / mol ) ; p < 0.001 ] , but were not significantly different between empagliflozin / linagliptin 25 mg/5 mg and empagliflozin 25 mg [ difference 0.14% ( 1.5 mmol / mol ) ; the single - pill combinations also caused greater reductions in fpg and body weight than linagliptin alone . in addition , the proportion of individuals who achieved their goal hba1c concentration [ < 7% ( < 53 mmol / mol ) ] at 24 weeks was significantly greater with the single - pill combination than with the individual components : 55.4 and 62.3% , respectively , for the empagliflozin / linagliptin 25 mg/5 mg and 10 mg/5 mg groups compared with 41.5 , 38.8 and 32.3% , respectively , for the empagliflozin 25 mg , empagliflozin 10 mg and linagliptin 5 mg groups . confirmed hypoglycaemic adverse events [ glucose 3.8 mmol / l ( 70 mg / dl ) and/or requiring assistance ] were reported in two subjects on empagliflozin 25 mg and one each on empagliflozin 10 mg and linagliptin 5 mg ; none required assistance . the second study assessed the single - pill combination of empagliflozin and linagliptin as an add - on to stable metformin in participants with t2 dm 54 . in 674 participants , the single - pill combinations significantly reduced hba1c compared with the respective monotherapies : empagliflozin / linagliptin 25 mg/5 mg vs empagliflozin 25 mg [ difference 0.58% ( 6.3 mmol / mol ) ; p < 0.001 ] and vs linagliptin 5 mg [ difference 0.50% ( 5.5 mmol / mol ) ; p < 0.001 ] ; empagliflozin / linagliptin 10 mg/5 mg vs empagliflozin 10 mg [ difference 0.42% ( 4.6 mmol / mol ) ; p < 0.001 ] and vs linagliptin 5 mg [ difference 0.39% ( 4.3 mmol / mol ) ; combination treatment also lowered fpg compared with the individual agents and body weight vs linagliptin . in addition , the proportion of participants who were at goal [ hba1c < 7% ( < 53 mmol / mol ) ] at 24 weeks was significantly greater with the single - pill combination than with the individual components : 61.8 and 57.8% , respectively , for the 25 mg/5 mg and the 10 mg/5 mg groups compared with 32.6 , 28.0 and 36.1% , respectively , for the empagliflozin 25 mg , empagliflozin 10 mg and linagliptin 5 mg groups . confirmed hypoglycaemic adverse events [ glucose 3.8 mmol / l ( 70 mg / dl ) and/or requiring assistance ] were reported in two subjects each on empagliflozin / linagliptin 25 mg/5 mg , empagliflozin / linagliptin 10 mg/5 mg and empagliflozin 10 mg and linagliptin 5 mg , and four subjects on empagliflozin 25 mg ; none required assistance . glycaemic control with empagliflozin / linagliptin was maintained at week 52 with statistically significant improvements from baseline in hba1c , and a higher proportion of individuals achieving hba1c < 7% [ 53 mmol / mol ] , in both single - pill combination groups vs the linagliptin 5 mg group , and the empagliflozin 10 mg / linagliptin 5 mg group vs empagliflozin 10 mg group 53 , and for the empagliflozin 25 mg / linagliptin 5 mg combination vs the empagliflozin 25 mg group in the metformin add - on study 54 . a number of clinical trials assessing the combination of dpp-4 inhibitors with sglt2 inhibitors in t2 dm are underway . two studies are investigating the efficacy and safety of adding on empagliflozin to linagliptin vs linagliptin alone ( nct01734785 ) and adding on linagliptin to empagliflozin vs empagliflozin alone in participants with t2 dm ( nct01778049 ) , both studies are scheduled to be completed in 2015 . several other ongoing studies are assessing the combination of dapagliflozin and saxagliptin ; nct01662999 is looking at the drug interaction of both agents in healthy individuals . other phase iii studies that are currently recruiting include assessments on the efficacy and safety of saxagliptin ( nct01619059 ) or dapagliflozin ( nct01646320 ) in triple therapy in participants with t2 dm who have inadequate glycaemic control with dual therapy including metformin . combination therapy with dpp-4 and sglt2 inhibitors may prove to be a useful approach in a broad range of participants , such as those insufficiently controlled with metformin as dual or triple therapy or those with a contraindication or intolerance to metformin . combination therapy using agents with complementary mechanisms of action is recommended in the 2013 american association of clinical endocrinologists ' comprehensive diabetes management algorithm 55 as well as in the 2015 position statement of the ada / easd 2 . in addition , initial combination therapy is recommended for those with an hba1c 7.5% 55 or 9.0% 2 , either with metformin or other background treatment for those unable to use metformin . other populations who could benefit from this combined oral treatment approach include those desiring to lose weight or to offset any potential weight gain with other oral anti - hyperglycaemic agents or insulin , those with advanced t2 dm inadequately controlled on their antihyperglycaemic regimen , including participants who would otherwise need to progress to insulin therapy but are unwilling or unable to commence injections , and older individuals at high risk of hypoglycaemia . use of multiple oral agents can increase the efficacy of treatment but can also cause more adverse events and increase the pill burden ; therefore , choosing oral agents which can be combined safely is important . among the oral agents , sulphonylureas can cause weight gain , hypoglycaemia and are associated with progressive -cell failure in the long run and thiazolidinediones can cause fluid retention and weight gain . dpp-4 inhibitors and sglt2 inhibitors are well tolerated , weight - neutral and have a low propensity for hypoglycaemia ; hence , their combination can help improve glycaemic control while avoiding some of the trade - offs of the traditional oral glucose - lowering treatments . a single - pill combination of a dpp-4 inhibitor and a sglt2 inhibitor , when available , would offer several advantages over the free combination of individual pills , including a reduced pill burden , which could possibly translate into improved compliance . in summary , combining dpp-4 inhibitors with sglt2 inhibitors has the potential to exert benefits beyond lowering glucose , such as beneficial effects on cardiovascular and renal risk factors , including albuminuria , and lowering body weight and systolic blood pressure . boehringer ingelheim pharmaceuticals , inc . was given the opportunity to review the manuscript for medical and scientific accuracy as well as intellectual property considerations . the author has served on the speaker 's bureau for boehringer ingelheim pharmaceuticals , inc . and the author meets criteria for authorship as recommended by the international committee of medical journal editors ( icmje ) .

most cases of intrathoracic goiter are asymptomatic and are discovered at chest radiography by chance . it is difficult to gain a definite diagnosis for malignancy by magnetic resonance imaging ( mri ) and iodine-123-radioisotope ( i - ri ) . then fluorine-18-fluorodeoxyglucose positron emission tomography ( f - fdg - pet ) is expected to be useful in this issue but the specificity is not always clear . we experienced a case of intrathoracic benign goiter which showed high uptake of f - fdg - pet . a 55-year - old woman was referred to our hospital because of a suspicion of mediastinal tumor incidentally found through a medical - checkup plain x - ray photography ( x - p ) ( figure 1a ) . chest mri revealed a 3 cm diameter tumor which seemed to connect to the right lobe of thyroid and projected into the mediastinum ( figure 1b ) . by i - ri , no unusual accumulation was detected ( figure 1c ) . a fine needle aspiration biopsy , which is viewed as the gold standard for diagnosis in most cases , was tried but could not reach a conclusive diagnosis . thereby , f - fdg - pet was performed and a high accumulation was revealed with standardized uptake value ( suv ) of 3.8 ( figure 1d ) . a 3 cm diameter tumor which seemed to connect to the right lobe by mri ( panel b ) . no unusual accumulation by i - ri ( panel c ) . a high accumulation with suv of 3.8 by f - fdg - pet ( panel d ) . thus , as a possibility of malignancy could not be excluded , the right lobe excision procedure for thyroid gland was enforced . the surface was flat and smooth and the exfoliation from the circumference organization was easy ( figure 2a ) . microscopically , the encapsulated tumor consisted of atypical large - sized follicles without malignant characteristics , the background thyroid tissue showing no remarkable change ( figure 2b ) . flat and smooth surface of the tumor connected to the right thyroid lobe ( panel a ) . atypical large - sized follicles without malignant characteristics and the background thyroid tissue showing no remarkable change ( panel b ) . ethical approval was not thought to be necessary because all the clinical course of the case was completely within usual medical cares . informed consent was given from the case on each occasion of diagnostic examinations and therapeutic procedures . especially , intrathoracic goiter is a rare condition . in the early years of applying f - fdg - pet for the differentiation of thyroid nodule malignancy , high sensitivity , and selectivity however , recent reports did not confirm the high selectivity by suv ( 4060% ) . thus , follicular adenoma of thyroid could present negative i - ri uptake and positive f - fdg - pet accumulation .

collagen , the most abundant protein in mammal , plays a critical role in tissue development and regeneration by supporting proliferation and differentiation of cells . while fibrous collagens ( e.g. type i , ii ) , in connective tissues give mechanical strength to the tissues , the network - like collagen ( type iv ) form basic scaffold of the basement membrane where cells attach and form organized tissues . collagen remodeling involves both collagen degradation ( by proteases ) and synthesis ( promoted by growth factors ) . although collagen remodeling , for example in bone , is part of the normal tissue renewal process , excess remodeling activity , or its occurrence in abnormal locations , typically indicates wound healing response to an injury or chronic pathological conditions such as cancer , osteoporosis , arthritis , and fibrosis . the ability to directly image collagens undergoing remodeling in vivo could lead to understanding of the progression of these diseases , as well as new diagnostics and therapeutics . for example , live imaging can provide information about the severity and the location of the diseases , and can also be used to assess the efficacy of new therapeutic agents . multiphoton laser scanning microscopy and second harmonic generation have been applied to image fibrillar collagens for monitoring extracellular matrix remodeling in tumor in live mice . however , this technique requires animals to be mounted with transparent dorsal skinfold chambers , which is an invasive procedure . direct and noninvasive imaging of collagen remodeling will benefit from a probe that specifically targets collagen undergoing remodeling . such probe is difficult to prepare since it needs to distinguish the remodeling collagens from the intact and mature collagens , which are abundant in normal tissues . collagen is made up of extremely rare protein structure called triple helix , which is cleaved by proteases such as matrix metalloproteinases ( mmp ) during collagen remodeling . the cleaved collagen fragments lose their triple helical structure and become unfolded strands ( gelatin ) , which are further digested by nonspecific proteases . it was recently discovered that the collagen mimetic peptide ( cmp ) which has the propensity to fold into triple helical structure can specifically target collagen strands which are dissociated from its triple helical state by either heat denaturation or by enzymatic degradation . the binding is primarily driven by triple - helix hybridization between monomeric cmps and the denatured collagen strands . because cmps self - assemble into homotrimeric triple helices at room temperature with little driving force for collagen hybridization , a caged cmp [ ( gpo)4gpo(gpo)4 , designated as ( gpo)9 , o : hydroxyproline ] , was developed , which contains a photo - cleavable nitrobenzyl group ( nb ) attached to the central glycine of the peptide . the nb cage group sterically prevents the cmp from folding into triple helix ; yet , removal of the cage group by uv irradiation immediately triggers the triple helical folding and collagen hybridization . when monomeric cmps labeled with near infrared ( nir ) fluorophores are systemically delivered to model mice , they can specifically target and allow in vivo imaging of denatured collagens in tissues undergoing normal ( e.g. in bone and cartilage ) and pathological ( e.g. in tumors ) remodeling . fluorescently labeled cmps can also be used for imaging collagens in histological tissue sections . in histological study , harvested tissues are often preserved by fixation to keep the cellular components and overall tissue morphology from deterioration . the fixing procedures , which include heat , and treatment with organic solvents , and chemical cross - linking reagents ( e.g. paraformaldehyde ) , denature the triple helical structure of collagen . it has been shown that fluorescently labeled cmps can specifically bind to collagens in fixed tissue sections ( e.g. skin , cornea , and bone ) even more effectively than an anti - collagen antibody , which allowed facile identification of pathologic conditions in fibrotic liver tissues . the cmp targets denatured collagen strands containing amino acid sequence of triple helical motifs , which is common to all types of collagens . , we present detailed experimental procedures for i ) imaging denatured collagen strands in vivo and ii ) visualizing collagens in ex vivo tissue sections using fluorescently labeled caged cmps . a nir tag , ir680 , was conjugated to the caged cmp for live imaging , while carboxyfluorescein ( cf ) was used in tissue staining work for its compatibility with standard fluorescence microscopes . methods for cmp synthesis can be found in previous reports . in this video report , imaging skeletal tissues in normal mice and tissue sections of mouse cornea were chosen for demonstration purpose ; however the methods presented here can be readily applied to many pathologies and biological models involving collagen remodeling ( e.g. tumors , wound healing ) , as well as to almost any prefixed tissue sample that contains collagens . all animal studies were undertaken in compliance with the regulations of the johns hopkins animal care and use committee . photo - activation of the caged cmp and tail vein injection prepare 110 l of caged cmp solution for each mouse ( 20 - 30 g mouse weight ) : 100 l of solution for dosing , with an extra 10 l cushion for the possible loss during transfer and injection . mix 11 l of ir680-ahx-(gpo)9 stock solution ( 400 m ) with 11 l of 100 m cysteine in sterile pbs solution ; then bring the total volume to 110 l with 1x pbs buffer . the final peptide solution contains 4 nmol of ir680-ahx-(gpo)9 and 1 nmol of cysteine in 100 l of 1x pbs solution.slowly withdraw the peptide solution into a 0.5 ml insulin syringe with a transparent barrel and a small gauge ( 28 - 30 g ) needle , and carefully remove the air bubbles . turn on the uv lamp to allow the lamp to warm up for 2 - 5 min.place the peptide - containing syringe directly under the uv lamp ( 365 nm , > 25 mw / cm ) for 5 min to allow photo-activation.meanwhile , position the mouse in a restrainer under a heated lamp ; label the mouse with a permanent marker on the tail or other identification methods such as ear tag or toe tattoo , and disinfect the tail with 70% alcohol.immediately after the uv activation , inject 100 l of uv - activated ir680-ahx-(gpo)9 solution into the tail vein , preferentially at the posterior part of the tail . prepare 110 l of caged cmp solution for each mouse ( 20 - 30 g mouse weight ) : 100 l of solution for dosing , with an extra 10 l cushion for the possible loss during transfer and injection . mix 11 l of ir680-ahx-(gpo)9 stock solution ( 400 m ) with 11 l of 100 m cysteine in sterile pbs solution ; then bring the total volume to 110 l with 1x pbs buffer . the final peptide solution contains 4 nmol of ir680-ahx-(gpo)9 and 1 nmol of cysteine in 100 l of 1x pbs solution . slowly withdraw the peptide solution into a 0.5 ml insulin syringe with a transparent barrel and a small gauge ( 28 - 30 g ) needle , and carefully remove the air bubbles . turn on the uv lamp to allow the lamp to warm up for 2 - 5 min . place the peptide - containing syringe directly under the uv lamp ( 365 nm , > 25 mw / cm ) for 5 min to allow photo - activation . meanwhile , position the mouse in a restrainer under a heated lamp ; label the mouse with a permanent marker on the tail or other identification methods such as ear tag or toe tattoo , and disinfect the tail with 70% alcohol . immediately after the uv activation , inject 100 l of uv - activated ir680-ahx-(gpo)9 solution into the tail vein , preferentially at the posterior part of the tail . nir fluorescence imaging of collagen remodeling activity set the heater plate of the imaging bed of the impulse imager to 37 c using pearl impulse 2.0 software.put the nude or shaved mouse into the anesthesia induction chamber containing 2% isoflurane in oxygen . verify depth of anesthesia plane by lack of mouse movement during handling and by monitoring the frequency of respirations ( ~1/sec).after the mouse is anesthetized , open the pearl imager drawer , and place the animal on the imaging bed . quickly remove the nose cone plug , and slide the mouse 's muzzle inside the nose cone to keep the mouse under anesthesia ( by 2% isoflurane in oxygen delivered at 2 l / min through the nose cone ) during the imaging process . once the mouse is in place , close the drawer.when the instrument indicates " ready " on the image software panel , click the " 700 channel , " " white light " and " 85 micron " boxes followed by the " acquire image " button . nir ( excitation 685 nm , emission 720 nm ) and white light photographs will then be taken using automatic focus and exposure.when the recording is completed , open the drawer , turn the mouse , and acquire images from another angle . the mouse can be placed with its front ( ventral ) , back ( dorsal ) or sides facing the camera . narrow strips of tape can be used as restraints to stretch its limbs to get the best view of the region of interest ( e.g. rib cage , ankles , and wrists).a proper time to observe skeletal uptake of ir680-cmp is between 24 - 96 hr post injection ( h.p.i . ) , based on the dosage ( ~4 nmol / mouse ) . to acquire high resolution images , sacrifice the mouse by cervical dislocation while under deep anesthesia after 72 h.p.i , remove the skin ( and hair ) using surgical forceps and scissors , and image it by nir fluorescence as described above.analyze the acquired nir fluorescence images . set the heater plate of the imaging bed of the impulse imager to 37 c using pearl impulse 2.0 software . put the nude or shaved mouse into the anesthesia induction chamber containing 2% isoflurane in oxygen . verify depth of anesthesia plane by lack of mouse movement during handling and by monitoring the frequency of respirations ( ~1/sec ) . after the mouse is anesthetized , open the pearl imager drawer , and place the animal on the imaging bed . quickly remove the nose cone plug , and slide the mouse 's muzzle inside the nose cone to keep the mouse under anesthesia ( by 2% isoflurane in oxygen delivered at 2 l / min through the nose cone ) during the imaging process . when the instrument indicates " ready " on the image software panel , click the " 700 channel , " " white light " and " 85 micron " boxes followed by the " acquire image " button . nir ( excitation 685 nm , emission 720 nm ) and white light photographs will then be taken using automatic focus and exposure . when the recording is completed , open the drawer , turn the mouse , and acquire images from another angle . the mouse can be placed with its front ( ventral ) , back ( dorsal ) or sides facing the camera . narrow strips of tape can be used as restraints to stretch its limbs to get the best view of the region of interest ( e.g. rib cage , ankles , and wrists ) . a proper time to observe skeletal uptake of ir680-cmp is between 24 - 96 hr post injection ( h.p.i . ) , based on the dosage ( ~4 nmol / mouse ) . to acquire high resolution images , sacrifice the mouse by cervical dislocation while under deep anesthesia after 72 h.p.i , remove the skin ( and hair ) using surgical forceps and scissors , and image it by nir fluorescence as described above . analyze the acquired nir fluorescence images . material preparation prepare 1 ml of 10% ( w / v ) bsa solution in deionized water . cut a few pieces of parafilm in the size of approximately 2 cm x 5 cm.prepare 10 ml of blocking buffer by mixing 500 l of goat serum , 1 ml of 10x pbs and 8.5 ml of deionized water . prepare 5 ml of cmp dilution buffer by diluting 50 l of 10% ( w / v ) bsa with 4.45 ml of deionized water and 500 l 10x pbs.dilute stock solutions of carboxyfluorescein - labeled caged cmp , cf(gpo)9 ( 480 m ) , to 5 m using the cmp dilution buffer . optimal cmp concentration varies from 2.5 - 30 m , depending on the type of tissues and the nature of tissue samples . store the formulated cmp solutions in dark.let the mouse cornea tissue slides equilibrate to room temperature . the mouse cornea tissues used in this demonstration have been prefixed with 4% paraformaldehyde in pbs solution for 1 hr , cryopreserved in tissue - tek o.c.t . medium , cryosectioned to 8 m thickness , and mounted on charged glass slides . prepare 1 ml of 10% ( w / v ) bsa solution in deionized water . cut a few pieces of parafilm in the size of approximately 2 cm x 5 cm . prepare 10 ml of blocking buffer by mixing 500 l of goat serum , 1 ml of 10x pbs and 8.5 ml of deionized water . prepare 5 ml of cmp dilution buffer by diluting 50 l of 10% ( w / v ) bsa with 4.45 ml of deionized water and 500 l 10x pbs . dilute stock solutions of carboxyfluorescein - labeled caged cmp , cf(gpo)9 ( 480 m ) , to 5 m using the cmp dilution buffer . optimal cmp concentration varies from 2.5 - 30 m , depending on the type of tissues and the nature of tissue samples . the mouse cornea tissues used in this demonstration have been prefixed with 4% paraformaldehyde in pbs solution for 1 hr , cryopreserved in tissue - tek o.c.t . medium , cryosectioned to 8 m thickness , and mounted on charged glass slides . to each slide , apply 0.5 ml of blocking solution and incubate the slides for 30 min at room temperature . remove the blocking solution by blotting the slides on a paper towel.apply approximately 100 l of cf(gpo)9 solutions to cover the tissue sections of each slide . incubate the slides for 2 min to allow the cmp solution to permeate the tissues.turn on the uv lamp . when the lamp is warmed up , expose the cmp - covered tissue sections to the uv light for 6 min ( 365 nm , ~8 mw / cm ) to deprotect the caged cmps . after uv exposure , cover the tissue sections of each slide with a piece of parafilm to prevent drying . place the slides in the humidified chamber and incubate them at 4 c for 2 hr.prepare a 1:3,000 dilution of dapi solution in 1x pbs . after cmp staining , gently remove the parafilm with forceps , and blot the slides on a paper towel to remove excess cmp solution . apply approximately 100 l of diluted dapi solutions to each tissue slide and incubate for 1 min at room temperature.after staining , immerse the slides in 1x pbs buffer in a staining jar for 5 min . repeat this 3x in fresh 1x pbs to wash off unbound staining agents.add a drop of mounting medium on the tissue section and cover it with a glass cover slip while avoiding trapping air bubbles . put the slides in a cardboard slide tray to protect them from light.image the collagen strands ( fitc channel ) and cell nuclei ( dapi channel ) in the tissue slides using a fluorescence microscope . place the slides in a humidified chamber . to each slide , apply 0.5 ml of blocking solution and incubate the slides for 30 min at room temperature . apply approximately 100 l of cf(gpo)9 solutions to cover the tissue sections of each slide . incubate the slides for 2 min to allow the cmp solution to permeate the tissues . when the lamp is warmed up , expose the cmp - covered tissue sections to the uv light for 6 min ( 365 nm , ~8 mw / cm ) to deprotect the caged cmps . after uv exposure , cover the tissue sections of each slide with a piece of parafilm to prevent drying . place the slides in the humidified chamber and incubate them at 4 c for 2 hr . prepare a 1:3,000 dilution of dapi solution in 1x pbs . after cmp staining , gently remove the parafilm with forceps , and blot the slides on a paper towel to remove excess cmp solution . apply approximately 100 l of diluted dapi solutions to each tissue slide and incubate for 1 min at room temperature . after staining , immerse the slides in 1x pbs buffer in a staining jar for 5 min . repeat this 3x in fresh 1x pbs to wash off unbound staining agents . add a drop of mounting medium on the tissue section and cover it with a glass cover slip while avoiding trapping air bubbles . image the collagen strands ( fitc channel ) and cell nuclei ( dapi channel ) in the tissue slides using a fluorescence microscope . photo - activation of the caged cmp and tail vein injection prepare 110 l of caged cmp solution for each mouse ( 20 - 30 g mouse weight ) : 100 l of solution for dosing , with an extra 10 l cushion for the possible loss during transfer and injection . mix 11 l of ir680-ahx-(gpo)9 stock solution ( 400 m ) with 11 l of 100 m cysteine in sterile pbs solution ; then bring the total volume to 110 l with 1x pbs buffer . the final peptide solution contains 4 nmol of ir680-ahx-(gpo)9 and 1 nmol of cysteine in 100 l of 1x pbs solution.slowly withdraw the peptide solution into a 0.5 ml insulin syringe with a transparent barrel and a small gauge ( 28 - 30 g ) needle , and carefully remove the air bubbles . turn on the uv lamp to allow the lamp to warm up for 2 - 5 min.place the peptide - containing syringe directly under the uv lamp ( 365 nm , > 25 mw / cm ) for 5 min to allow photo-activation.meanwhile , position the mouse in a restrainer under a heated lamp ; label the mouse with a permanent marker on the tail or other identification methods such as ear tag or toe tattoo , and disinfect the tail with 70% alcohol.immediately after the uv activation , inject 100 l of uv - activated ir680-ahx-(gpo)9 solution into the tail vein , preferentially at the posterior part of the tail . prepare 110 l of caged cmp solution for each mouse ( 20 - 30 g mouse weight ) : 100 l of solution for dosing , with an extra 10 l cushion for the possible loss during transfer and injection . mix 11 l of ir680-ahx-(gpo)9 stock solution ( 400 m ) with 11 l of 100 m cysteine in sterile pbs solution ; then bring the total volume to 110 l with 1x pbs buffer . the final peptide solution contains 4 nmol of ir680-ahx-(gpo)9 and 1 nmol of cysteine in 100 l of 1x pbs solution . slowly withdraw the peptide solution into a 0.5 ml insulin syringe with a transparent barrel and a small gauge ( 28 - 30 g ) needle , and carefully remove the air bubbles . turn on the uv lamp to allow the lamp to warm up for 2 - 5 min . place the peptide - containing syringe directly under the uv lamp ( 365 nm , > 25 mw / cm ) for 5 min to allow photo - activation . meanwhile , position the mouse in a restrainer under a heated lamp ; label the mouse with a permanent marker on the tail or other identification methods such as ear tag or toe tattoo , and disinfect the tail with 70% alcohol . immediately after the uv activation , inject 100 l of uv - activated ir680-ahx-(gpo)9 solution into the tail vein , preferentially at the posterior part of the tail . nir fluorescence imaging of collagen remodeling activity set the heater plate of the imaging bed of the impulse imager to 37 c using pearl impulse 2.0 software.put the nude or shaved mouse into the anesthesia induction chamber containing 2% isoflurane in oxygen . verify depth of anesthesia plane by lack of mouse movement during handling and by monitoring the frequency of respirations ( ~1/sec).after the mouse is anesthetized , open the pearl imager drawer , and place the animal on the imaging bed . quickly remove the nose cone plug , and slide the mouse 's muzzle inside the nose cone to keep the mouse under anesthesia ( by 2% isoflurane in oxygen delivered at 2 l / min through the nose cone ) during the imaging process . once the mouse is in place , close the drawer.when the instrument indicates " ready " on the image software panel , click the " 700 channel , " " white light " and " 85 micron " boxes followed by the " acquire image " button . nir ( excitation 685 nm , emission 720 nm ) and white light photographs will then be taken using automatic focus and exposure.when the recording is completed , open the drawer , turn the mouse , and acquire images from another angle . the mouse can be placed with its front ( ventral ) , back ( dorsal ) or sides facing the camera . narrow strips of tape can be used as restraints to stretch its limbs to get the best view of the region of interest ( e.g. rib cage , ankles , and wrists).a proper time to observe skeletal uptake of ir680-cmp is between 24 - 96 hr post injection ( h.p.i . ) , based on the dosage ( ~4 nmol / mouse ) . to acquire high resolution images , sacrifice the mouse by cervical dislocation while under deep anesthesia after 72 h.p.i , remove the skin ( and hair ) using surgical forceps and scissors , and image it by nir fluorescence as described above.analyze the acquired nir fluorescence images . set the heater plate of the imaging bed of the impulse imager to 37 c using pearl impulse 2.0 software . put the nude or shaved mouse into the anesthesia induction chamber containing 2% isoflurane in oxygen . verify depth of anesthesia plane by lack of mouse movement during handling and by monitoring the frequency of respirations ( ~1/sec ) . after the mouse is anesthetized , open the pearl imager drawer , and place the animal on the imaging bed . quickly remove the nose cone plug , and slide the mouse 's muzzle inside the nose cone to keep the mouse under anesthesia ( by 2% isoflurane in oxygen delivered at 2 l / min through the nose cone ) during the imaging process . when the instrument indicates " ready " on the image software panel , click the " 700 channel , " " white light " and " 85 micron " boxes followed by the " acquire image " button . nir ( excitation 685 nm , emission 720 nm ) and white light photographs will then be taken using automatic focus and exposure . when the recording is completed , open the drawer , turn the mouse , and acquire images from another angle . the mouse can be placed with its front ( ventral ) , back ( dorsal ) or sides facing the camera . narrow strips of tape can be used as restraints to stretch its limbs to get the best view of the region of interest ( e.g. rib cage , ankles , and wrists ) . a proper time to observe skeletal uptake of ir680-cmp is between 24 - 96 hr post injection ( h.p.i . ) , based on the dosage ( ~4 nmol / mouse ) . to acquire high resolution images , sacrifice the mouse by cervical dislocation while under deep anesthesia after 72 h.p.i , remove the skin ( and hair ) using surgical forceps and scissors , and image it by nir fluorescence as described above . analyze the acquired nir fluorescence images . material preparation prepare 1 ml of 10% ( w / v ) bsa solution in deionized water . cut a few pieces of parafilm in the size of approximately 2 cm x 5 cm.prepare 10 ml of blocking buffer by mixing 500 l of goat serum , 1 ml of 10x pbs and 8.5 ml of deionized water . prepare 5 ml of cmp dilution buffer by diluting 50 l of 10% ( w / v ) bsa with 4.45 ml of deionized water and 500 l 10x pbs.dilute stock solutions of carboxyfluorescein - labeled caged cmp , cf(gpo)9 ( 480 m ) , to 5 m using the cmp dilution buffer . optimal cmp concentration varies from 2.5 - 30 m , depending on the type of tissues and the nature of tissue samples . store the formulated cmp solutions in dark.let the mouse cornea tissue slides equilibrate to room temperature . the mouse cornea tissues used in this demonstration have been prefixed with 4% paraformaldehyde in pbs solution for 1 hr , cryopreserved in tissue - tek o.c.t . medium , cryosectioned to 8 m thickness , and mounted on charged glass slides . prepare 1 ml of 10% ( w / v ) bsa solution in deionized water . thaw 1 ml of goat serum in a water bath at room temperature . cut a few pieces of parafilm in the size of approximately 2 cm x 5 cm . prepare 10 ml of blocking buffer by mixing 500 l of goat serum , 1 ml of 10x pbs and 8.5 ml of deionized water . prepare 5 ml of cmp dilution buffer by diluting 50 l of 10% ( w / v ) bsa with 4.45 ml of deionized water and 500 l 10x pbs . dilute stock solutions of carboxyfluorescein - labeled caged cmp , cf(gpo)9 ( 480 m ) , to 5 m using the cmp dilution buffer . optimal cmp concentration varies from 2.5 - 30 m , depending on the type of tissues and the nature of tissue samples . the mouse cornea tissues used in this demonstration have been prefixed with 4% paraformaldehyde in pbs solution for 1 hr , cryopreserved in tissue - tek o.c.t . medium , cryosectioned to 8 m thickness , and mounted on charged glass slides . tissue staining and imaging place the slides in a humidified chamber . to each slide , apply 0.5 ml of blocking solution and incubate the slides for 30 min at room temperature . remove the blocking solution by blotting the slides on a paper towel.apply approximately 100 l of cf(gpo)9 solutions to cover the tissue sections of each slide . incubate the slides for 2 min to allow the cmp solution to permeate the tissues.turn on the uv lamp . when the lamp is warmed up , expose the cmp - covered tissue sections to the uv light for 6 min ( 365 nm , ~8 mw / cm ) to deprotect the caged cmps . after uv exposure , cover the tissue sections of each slide with a piece of parafilm to prevent drying . place the slides in the humidified chamber and incubate them at 4 c for 2 hr.prepare a 1:3,000 dilution of dapi solution in 1x pbs . after cmp staining , gently remove the parafilm with forceps , and blot the slides on a paper towel to remove excess cmp solution . apply approximately 100 l of diluted dapi solutions to each tissue slide and incubate for 1 min at room temperature.after staining , immerse the slides in 1x pbs buffer in a staining jar for 5 min . repeat this 3x in fresh 1x pbs to wash off unbound staining agents.add a drop of mounting medium on the tissue section and cover it with a glass cover slip while avoiding trapping air bubbles . put the slides in a cardboard slide tray to protect them from light.image the collagen strands ( fitc channel ) and cell nuclei ( dapi channel ) in the tissue slides using a fluorescence microscope . place the slides in a humidified chamber . to each slide , apply 0.5 ml of blocking solution and incubate the slides for 30 min at room temperature . apply approximately 100 l of cf(gpo)9 solutions to cover the tissue sections of each slide . incubate the slides for 2 min to allow the cmp solution to permeate the tissues . expose the cmp - covered tissue sections to the uv light for 6 min ( 365 nm , ~8 mw / cm ) to deprotect the caged cmps . after uv exposure , cover the tissue sections of each slide with a piece of parafilm to prevent drying . place the slides in the humidified chamber and incubate them at 4 c for 2 hr . prepare a 1:3,000 dilution of dapi solution in 1x pbs . after cmp staining , gently remove the parafilm with forceps , and blot the slides on a paper towel to remove excess cmp solution . apply approximately 100 l of diluted dapi solutions to each tissue slide and incubate for 1 min at room temperature . after staining , immerse the slides in 1x pbs buffer in a staining jar for 5 min . add a drop of mounting medium on the tissue section and cover it with a glass cover slip while avoiding trapping air bubbles . image the collagen strands ( fitc channel ) and cell nuclei ( dapi channel ) in the tissue slides using a fluorescence microscope . figure 1 shows a typical result of how photo - triggered ir680-ahx-(gpo)9 distributes in a healthy female skh1 nude mouse after 24 - 96 hr post injection . the process of cmp hybridizing with the remodeling collagen strands seems relatively slow , especially in contrast to other peptide imaging probes ( e.g. rgd ) . this most likely is because of the slow folding rate of the collagen triple helix . however , once hybridized , the cmp strands are strongly bound to the collagenous tissues , as only slight signal reduction is seen after 24 h.p.i . ( figure 1 ) . at 96 h.p.i . , the nir fluorescence image of the mouse after skin removal clearly demonstrates the skeletal uptake of ir680-ahx-(gpo)9 in spine and ribs , as well as within the knees , ankles , wrists , and lower mandibles ( figure 2a ) . in ex vivo tissue staining , photo - triggered fluorescently - labeled caged cmps specifically hybridize to denatured collagen strands in tissue sections . in figure 3 , cmp staining clearly reveals the fine parallel collagen fibrils in the corneal stroma , which demonstrates its use as a collagen specific staining agent . serial nir fluorescence images of a nude mouse administered intravenously with photo - decaged ir680-ahx-(gpo)9 showing skeletal uptake over four days . nir fluorescence images of mice administered with photo - decaged ir680-ahx-(gpo)9 ( a ) , caged ir680-ahx-(gpo)9 without uv exposure ( b ) , prefolded triple - helical [ ir680-ahx-(gpo)9]3 ( c ) , or heat - dissociated single - strand ir680-ahx-(gpo)9 ( d ) at 96 h.p.i . . skeletal targeting by the cmp can only be observed in a and d. nir fluorescence signals are shown in rainbow scale . fluorescence micrographs of a mouse corneal tissue section prefixed in paraformaldehyde , and stained with dapi and photo - triggered cf(gpo)9 using protocol 2 , displaying dense collagenous stroma ( stained by cmp , in green ) as well as cellular epithelium and endothelium ( stained by dapi , in blue ) ( scale bar : 100 m ) . as can be seen in this protocol , the delivery of cmp is straightforward since the uv - activation of the caged cmps is the only additional step to the common tail vein injection protocols . the key is to inject the peptide probes in an uncaged and metastable single - strand state . the cage group prevents cmp from self - assembly and binding to collagens ( figure 2b ) until it is removed by uv light at which point the cmp starts to fold into triple helix . the injection formulation contains cysteine , which can quickly react with the cleaved cage group during uv irradiation to minimize toxicity . numerous mice with different strains ( e.g. skh-1 and dr-1 ) have been tested using this formulation , and we did not detect any behavioral or other health defects in these mice up to six months . if the injection does not follow immediately after uv exposure , the cmps will fold into homotrimeric triple helices , which have no hybridization capacity . figure 2c shows an extreme case where prior to injection the cmps were fully reassembled into triple helices by long delay time ( > 2 days ) . to circumvent this problem , cmp solution should be prepared in relatively low concentration ( e.g. ~40 m ) , and injected into the mice immediately after uv - decaging . because of the slow triple helix folding rate of the cmps in dilute conditions ( half time of refolding : > 50 min ) and the short uv exposure and injection time ( 5~7 min total ) , most of the cmps enter the bloodstream in single - strand form when this protocol is followed . once injected , cmps are expected to remain as single - strands , as the concentration is reduced by a factor of 20 in the blood pool , leading to dramatic reduction in the reassembling rate . if injection is delayed ( e.g. due to animal handling ) and homotrimeric refolding is suspected , the partially assembled peptides can be reactivated by heating to above 70 c to dissociate the cmps to single - strands , followed by prompt cooling and injection . although less convenient , such heat - dissociated cmps also show expected results of in vivo targeting ( figure 2d ) . in this demonstration , nude mice were used for nir fluorescence imaging because up to 50% of the nir signal can be blocked by hair . when working with haired mouse models ( especially the ones with black hairs ) , we recommend shaving the mouse in the region of interest prior to imaging using an electric shaver or hair remover . in figure 1 , there are false positive signals from the animal 's abdominal region , which is caused by auto - fluorescence of chlorophylls in the animal diet . such background signals can be significantly lowered by switching the mouse to purified diets approximately 4 days prior to imaging . as seen in figures 1 and 2a , therefore , to avoid artifacts resulting from imperfect tail vein injections , we recommend injecting at the posterior part of the tail so that the injection site is not captured in the fluorescence image . the labeled cmp enables targeting and imaging of specific locations of collagen remodeling in vivo that are difficult to detect using other methods . for instance , the elisa - based blood and urine assays are sensitive for cleaved collagen telopeptide fragments , but the method can not localize the source of the collagen turnover , because it targets soluble antigens . the main limitations of the in vivo imaging technique using nir labeled cmps are depth - of - penetration attenuation and quenching by proximal pooled red cells as hemoglobin is an endogenous quencher of 680/710 nm nir dyes . future applications of cmp in vivo imaging include spect or pet imaging using cmps labeled with direct gamma - emitting radionuclides or with positron emitters , for diagnostic of disease states involving collagen remodeling ( e.g. osteoporosis , arthritis , atherosclerosis , and fibrosis ) . these radio - labeled cmp analogs will eliminate the limitations of signal loss due to tissue depth and presence of pooled red cells , and will allow quantitative measurements of diseased tissues . in addition , the relatively late imaging time ( e.g. 48 - 96 h.p.i . ) resulting from the slow binding and clearance of cmps could make it difficult to follow rapid changes in collagen remodeling . such limitation could be overcome by further engineering of the binding kinetics and affinity of cmp imaging agents . since cmp binds to denatured collagens which have little structure , the cmp - based collagen imaging is complementary to second harmonic generation microscopy which can only image collagen fibers . when staining tissue sections with fluorescently labeled cmps , we found it beneficial to incubate the samples in the uv - activated peptide solutions at 4 c , because the triple helical hybridization is facilitated at lower temperature . it was also found that immersing the slides in fresh pbs buffer in a staining jar is the most effective way to wash off unbound cmps , which works much better than merely pipetting washing buffers over the samples . the cmp - collagen strand hybridization is very specific and robust ; therefore the simple staining protocol presented in this video can be readily modified for costaining additional biomarkers , and for identifying degraded collagens in unfixed tissue sections . this is an effective and convenient alternative to using anti - collagen antibodies for detecting fibrous collagens in various histological samples .

regional anaesthesia has been coming and going since 1885 for surgeries on parts of the body where general anaesthesia is not mandatory and/or perilous to the patient . with the application of ultrasound to regional blocks , there is resurgence of regional anaesthesia which can now be administered more precisely with lesser doses of local anaesthetics , more successfully and safely . ultrasound guidance is emerging as the gold standard for regional anaesthesia . due to the cost factor and other constraints hence , sound knowledge of anatomy including approximate depth of nerve would be beneficial to avoid unnecessary complications , especially for those who are still performing regional blocks using the conventional landmark or peripheral nerve stimulator ( pns)-guided techniques . the brachial plexus block is the most popular regional block for surgeries of the upper limb by various approaches . supraclavicular approach is the most favoured , fulfilling all surgical requirements with the potential disadvantages of accidental pneumothorax , inadvertent vascular puncture , inter - scalene block and neurological complications . the incidence of pneumothorax without usg monitoring is 6.1% , whereas with usg guidance , it comes down to 0.06% . we hypothesised that to avoid this complication , the variation in depth of brachial plexus could be estimated to guide the needle advancement during the procedure . recently , usg predictors of corner pocket depth have been studied for usg - guided supraclavicular block in indian population . however , to the best of our knowledge , there was no study where the variability of depth of the neural elements of brachial plexus in supraclavicular area had been assessed with ultrasound in indian population . hence , this study was conceived to give some approximation of the depth of the brachial plexus from the skin . this cross - sectional study was conducted from april 2015 to june 2015 after approval from our institutional ethical committee . the patients of either sex in the age group of 2050 years with american society of anesthesiologists physical status i and ii were included in our study . patients not willing to participate in the study , patients with any pathology , deformity or any history of the previous surgical intervention of the supraclavicular area were excluded from the study . the sample size was calculated based on a pilot study on ten patients with the formula for cross - sectional study with the quantitative variable . considering 5% type-1 error ( p < 0.05 ) , 0.1 cm as the absolute error or precision and 0.405 cm as standard deviation of variable ( longest distance [ ld ] ) from the pilot study , minimum 63 subjects were required . considering the potential drop - outs , after thorough pre - anaesthetic check - up , patients satisfying the inclusion criteria were selected . written informed consent was taken from each of the ninety enrolled patients for participation in the study . in the operation theatre , the patient was positioned supine with pillow between the shoulder blades and head was turned to contralateral side and arm adducted by the side of the body . brachial plexus was scanned with a high - frequency linear probe ( 813 mhz ) of the usg machine ( m - turbo , micromaxx , fujifilm sonosite inc . the footprint of the probe was placed lateral to the clavicular head of the sternocleidomastoid muscle in a coronal oblique plane with 60 angle with the horizontal plane [ figure 1 ] . position of ultrasonography probe on right supraclavicular fossa ( r ) once an optimal image was obtained , the brachial plexus ( usually appearing as bundle of hypoechoic round nodules or bunch of grapes ) was kept in the middle of the screen and the image was frozen . the following two distances were measured : sd , distance from skin to the most superficial hypoechoic nodule / neural element [ figure 2a ] and ld , distance from skin to the deepest hypoechoic nodule / neural element [ figure 2b ] . ( a ) shortest distance = distance from skin to the most superficial hypoechoic nodule / neural element . ( b ) longest distance = distance from skin to the deepest hypoechoic nodule / neural element the demographic parameters and the distances ( in centimetres ) were expressed as mean standard deviation . pearson correlation was used to calculate the strength and significance of the relation between sd and ld from skin to the brachial plexus with the demographic parameters such as height , weight and body mass index ( bmi ) . p value of < 0.05 was considered as statistically significant and < 0.001 was considered as statistically highly significant . sonographic assessment and analysis were done in 87 patients , out of which 69 patients were male and 18 patients were female . hence , they were excluded from the final analysis . the mean age , weight , height and bmi of the study population demographic variables and its correlations with the depths of brachial plexus the mean sd was 0.60 0.262 cm ( minimum 0.21 cm , maximum 1.0 cm ) and mean ld was 1.34 0.385 cm ( minimum 0.72 cm , maximum 2.14 cm ) . the demographic profile parameters and depth measurements according to gender are presented in table 2 . analysis of data with regards to gender the distribution of population according to the range of depths is displayed in table 3 . distribution of patients according to shortest distance and longest distance the strength of correlation between these two measured distances and different demographic variables was calculated . we found significant correlation between weight , bmi and sd ( p < 0.00001 ) [ table 1 ] . similarly , we also found significant correlation between ld and weight as well as bmi ( p < 0.00001 ) [ table 1 ] . we did not find any statistically significant correlation between age , height and the two distances . distribution of shortest distance with weight distribution of shortest distance with body mass index distribution of longest distance with weight distribution of longest distance with body mass index in our study , we observed that the difference between mean ld and mean sd was 0.74 cm . in majority of the patients , there were no statistically significant correlations between age , height and depth of brachial plexus ( p > 0.05 ) . significant correlation was observed between the depths ( sd as well as ld ) and weight and bmi ( p < 0.0001 ) . an anatomic study on cadavers was performed by apan et al . and the results were correlated with a surface landmark - based technique later , using ultrasound and magnetic resonance imaging on healthy volunteers . the mean distances between skin and superficial lying part of the brachial plexus were found to be 16.5 0.7 mm in male and 14.5 0.5 mm in female volunteers , which are longer than the sd we have observed in our study . this dissimilarity might be due to the difference in the surface landmark , the ethnicity of the study population and the demographic profile between the two studies . in a study on 15 healthy volunteers , the high - resolution ultrasound probe was used to scan the supraclavicular region in coronal oblique plane . the mean skin - to - nerve distance was found to be 0.9 0.3 cm . in another study on 20 healthy volunteers , sonographic assessment revealed that the brachial plexus is relatively superficial in supraclavicular region with a depth of 12 cm . however , the distance between the skin and the superficial and deep neural element was not measured separately . moreover , all these studies were done in western population and the findings may not be applicable to the indian population . the deposition of drug at the corner pocket is practised by some sonography users for ultrasound - guided supraclavicular brachial plexus block . significant correlations have been observed between weight , bmi and the depth of corner pocket in a study on indian population . we also found significant correlation between the weight , bmi and sd and ld between skin and the neural element . supraclavicular brachial plexus block is effective but may sometimes be complicated by pneumothorax because of needle advancement beyond the plexus and injury to pleura . pre - procedural scan and measurement of the depth would be beneficial for selection of the needle size as well as advancement of needle during ultrasound - guided brachial plexus block . we suggest that use of needle with 3 cm shaft would be sufficient to reach the sheath of the brachial plexus and proximal to the neural elements during performance of the block in patients with weight and bmi ranging from 55 to 79 kg and 17.75 to 28.54 kg / m , respectively . our study population did not have older ( > 50 years ) or obese ( bmi > 30 we studied the variation in depth on 87 patients only . a study on larger number is needed to derive a formula that can predict the depth of neural element based on weight and/or bmi . additionally , measurement of neck circumference would probably be helpful in predicting the depth of brachial plexus and needle size to be used . the difference between the most superficial and deep neural elements of brachial plexus was < 1 cm . if brachial plexus is not encountered within 1 cm from skin during conventional or pns - guided technique , it is advisable to be cautious before further advancement of the needle tip .

although most mature cystic teratoma ( mct ) occurs in the ovary , some cases of cystic teratoma of extra gonadal origin have been reported . it is usually found in neonates , and the ovary is most often implanted in the omentum.123 ectopic ovary , used synonymously with accessory ovary , supernumerary ovary , or ovarian implant syndrome , is a rare gynecologic anomaly . ovarian auto amputation , especially occurring in ovaries with dermoid cysts , is a complication of ovarian torsion that may lead to formation of an ectopic ovary . here , we present a rare case of an autoamputated ovary with mct , and it is the first case of successful spontaneous pregnancy within seven months of resection . a 34-year - old woman , gravida 0 , para 0 was referred to our clinic for presumed left ovarian tumor . her past medical history was not significant ; the patient had a history of chronic abdominal pain for two years . on pelvic examination , tumor markers were as follows , cancer antigen ( ca ) 125 ; 10.4 u / ml , ca 19 - 9 ; 2 u / ml . an ultrasonography examination was suggested the presence of 5.0 2.7 cm sized echogenic cyst in left ovary . a computed tomography ( ct ) scan was carried out and demonstrated a cystic mass measuring 5.7 3.1 cm at left ovary ( fig . 2 ) . there was no ligamentous or direct connection with the pelvic organs , including the uterus , and there was no apparent blood supply to the tumor . histological examination revealed a typical mct with adipose tissue and hair root sheaths . the cyst wall consisted of collagen fibers with marked infiltration of lymphocytes and histiocytes with calcification , which indicated ischemic and inflammatory changes ( fig . mct is one of the most common types of ovarian tumors , with incidence ranging from 5% to 25% of all ovarian neoplasia.4 it is a germ cell neoplasm composed of various tissues , including tissues not normally found in the organ from which it arises . embryologically , ovaries arise from the primordial germ cells that migrate from the wall of the yolk sac , along the dorsal mesentery , to the gonadal ridges.5 theses totipotential cells may give rise to a variety of tissues originating from the three primitive germ cell layers . dermoid cysts occur most commonly in the ovary , other favorite sites are the mediastinum , sacral region , and retroperitoneum . there are three proposed theories on the cause of these extragonadal sites : ( 1 ) primary dermoids originating from displace germ cells ; ( 2 ) dermoids developing in a supernumerary ovary ; and ( 3 ) autoamputation of an ovarian dermoid and implantation into an extra gonadal site.456 autoamputation could result from the torsion of the pedicle , as torsion is reported to be the most frequent complication of ovarian teratomas , occurring in 16.1% of case.4 torsion interferes with the blood supply , causing venous congestion and aseptic inflammation of the tumor wall . in acute torsion , the tumor undergoes necrosis and subsequent atrophy as a result of ischemia . in subacute or chronic torsion , the tumor may become adherent to adjacent structures , with a new collateral circulation formed . infrequently , the tumor completely detaches from its pedicle , thus resulting in a parasitic dermoid cyst.7 this parasitic dermoid cyst may reimplant in adjacent structures and form a new blood supply . thus torsion of ovary and its cystic contents may lead to development of a new ectopic ovary . the reason for the predilection for the omentum is because of its defensive role in intraabdominal inflammation , and adhesion formation , allowing the secondary implantation of the autoamputated ovary.8 autoamputation is the most plausible mechanism for our patient whose history of chronic abdominal pain for two years . in contrast to other cases , it is suspected that the blood supply is cut off not long after autoamputation . in our case , the ultrasonography and ct suggested that the tumor might be mct , but failed to demonstrate the exact localization of the tumor . however , it is suggested that the color flow doppler may play an important role in the tumor localization . in summary , our case report presents a patient with autoamputation of an ovary with mct that was treated by laparoscopic surgery . one of the possible differential diagnosis is lipoleiomyoma of uterus , which contain lipid portion within mass that may lead to misdiagnosis of ovarian dermoid cyst.9 some cases of gynecologic emergency may arise from postmenopausal women.10 physician should keep in mind of this rare case when encountered postmenopausal women complaining of abrupt abdominal and pelvic pain . furthermore , most of ovarian dermoid cyst can be treated by laparoscopic surgery with preservation of ovary , this kind of case only have consequence of oophorectomy which may lead to premature ovarian failure in patient with history of previous unilateral salpingo - oophorectomy .

regional lymph node metastasis ( lnm ) is a well - known prognostic indicator in many types of solid cancer , including head and neck squamous cell carcinomas ( hnscc ) . lnm negatively influences the overall survival and increases the likelihood of distant metastases . in hnscc , the correlation between lnm and distant metastases is quite strong , especially with the presence of lnm in the neck being one of the strongest prognostic factors . moreover , the presence of lnm , number and size of involved lymph nodes , and nodal characteristics , including extra - capsular spread , significantly influence regional recurrence , distant metastases , and even overall survival . the accepted treatment protocols for patients with hnscc with clinical lnm are surgical ablation or chemoradiation therapy . however , even in a clinically negative neck , surgical data from prophylactic neck dissection have demonstrated high rates of occult lnm for up to 34% of the patients in the majority of hnscc . recently , several clinical and pathological features of primary tumors have been claimed to be predictive for occult metastases . as the primary determinant of t classification , the maximum tumor diameter and invasion depth of primary tumors have been considered an important risk factor for regional lnm . these data reflect the concept that regional lnm can be induced by time - dependent tumor burden or chronological age of primary tumors . however , lnm from undetected or small primary lesions definitely exist in some cases , indicating that lnm is not always dependent upon the physical dimension of the primary tumors . furthermore , a series of data presenting the significant correlation between lymphovascular invasion ( lvi ) and perineural invasion ( pni ) with concomitant lnm suggest that the tumors with aggressive biological characteristics have higher risks of regional lnm . a recent report has confirmed that tumor dimension and biology have a similar impact on the presence of lnm in breast cancer . despite this body of knowledge thus , this study was designed to identify the relative impact of tumor dimension and tumor biology on regional lnm in patients with hnscc , particularly focusing on the subsites of the head and neck . medical information was extracted from a prospectively collected data in accordance to the standardized protocols in our institute for hnscc , which included tumor size , thickness , and pathology details . participants provided written informed consents . among various subgroups of hnscc , oral tongue , oropharynx ( tonsil or base of tongue ) , and hypopharynx ( patients who had undergone surgery as the initial treatment between 2003 and 2012 were included . all pathology diagnoses were squamous cell carcinomas . among them , the subjects were excluded from the analyses because of incomplete pathological information ( n=48 ) and follow - up duration of less than 2 years in case of no events ( n=55 ) ; the final number of study subjects was 295 . of 295 hnscc , oral tongue cancer was predominated ( n=174 ) , followed by oropharynx and hypopharynx cancer . lnm was more prevalent in oropharynx and hypopharynx cancer compared to tongue cancer ( p < 0.0001 and p=0.0028 , respectively ) . there was a significant correlation between t classification and lnm ( p < 0.0001 ) . in patients with lnm ( n=153 ) , distribution of n stage was n1 ( n=43 ) , n2 ( n=104 ) , and n3 ( n=6 ) , respectively . the five major variables were selected as determinants of tumor dimension and biology . as tumor dimensional variables the estimated tumor volume was calculated by the equation : 1/2maximal diameterminimal diametertumor thickness . as biological variables , we evaluated the presence of lvi , pni , and differentiation status of primary tumors ( = tumor grade ) . patients with all available data for the five major variables were only included in the analyses . to investigate the impact of tumor dimension and biology on disease recurrence/ systemic metastasis and survival , the survival analyses were conducted . follow - up durations were defined as time gap between the completion of initial treatment , including adjuvant therapy , and the occurrence of events of interest . the mean follow - up duration was 32.5 months ( range , 0 to 154 months ) . the events of interest within 1 month after completion of treatment were defined as a follow - up duration of 0 . the baseline characteristics were analyzed for association of lnm , using a chi - square test for categorical variables and wilcoxon rank sum tests for continuous variables . post - hoc comparison of the association with primary tumor subsites and lnm was assessed by fisher exact test using permutation method for multiple testing . the association between the selected variables and the presence of lnm with adjustment of primary tumor subsite was determined by a logistic regression analysis . to evaluate the predictive ability for five variables of tumor dimension and biology on the presence of lnm , the area under the curves ( auc ) the combined measures for tumor dimension and tumor biology were defined by a logistic regression model for two and three variables . in order to assess the significance for the added predictive ability of the combined measure from the single variable , the integrated discrimination improvement ( idi ) method was used . subgroups analyses of lnm predictability , in accordance to the primary tumor subsites , were used . univariate survival analyses of the possible clinical factors , tumor dimensional , and biological variables were conducted , using log - rank tests , and then cox proportional hazards model was used to assess the association of the selected variables on disease - free and overall survival . if needed , bonferroni s correction was used for multiple testing , such as subgroup analysis . medical information was extracted from a prospectively collected data in accordance to the standardized protocols in our institute for hnscc , which included tumor size , thickness , and pathology details . participants provided written informed consents . among various subgroups of hnscc , oral tongue , oropharynx ( tonsil or base of tongue ) , and hypopharynx ( patients who had undergone surgery as the initial treatment between 2003 and 2012 were included . all pathology diagnoses were squamous cell carcinomas . among them , the subjects were excluded from the analyses because of incomplete pathological information ( n=48 ) and follow - up duration of less than 2 years in case of no events ( n=55 ) ; the final number of study subjects was 295 . of 295 hnscc , oral tongue cancer was predominated ( n=174 ) , followed by oropharynx and hypopharynx cancer . lnm was more prevalent in oropharynx and hypopharynx cancer compared to tongue cancer ( p < 0.0001 and p=0.0028 , respectively ) . there was a significant correlation between t classification and lnm ( p < 0.0001 ) . in patients with lnm ( n=153 ) , distribution of n stage was n1 ( n=43 ) , n2 ( n=104 ) , and n3 ( n=6 ) , respectively . the five major variables were selected as determinants of tumor dimension and biology . as tumor dimensional variables , we evaluated the tumor thickness and estimated tumor volume calculated from pathology specimen . the estimated tumor volume was calculated by the equation : 1/2maximal diameterminimal diametertumor thickness . as biological variables , we evaluated the presence of lvi , pni , and differentiation status of primary tumors ( = tumor grade ) . patients with all available data for the five major variables were only included in the analyses . to investigate the impact of tumor dimension and biology on disease recurrence/ systemic metastasis and survival , the survival analyses were conducted . follow - up durations were defined as time gap between the completion of initial treatment , including adjuvant therapy , and the occurrence of events of interest . the mean follow - up duration was 32.5 months ( range , 0 to 154 months ) . the events of interest within 1 month after completion of treatment were defined as a follow - up duration of 0 . the baseline characteristics were analyzed for association of lnm , using a chi - square test for categorical variables and wilcoxon rank sum tests for continuous variables . post - hoc comparison of the association with primary tumor subsites and lnm was assessed by fisher exact test using permutation method for multiple testing . the association between the selected variables and the presence of lnm with adjustment of primary tumor subsite was determined by a logistic regression analysis . to evaluate the predictive ability for five variables of tumor dimension and biology on the presence of lnm , the area under the curves ( auc ) the combined measures for tumor dimension and tumor biology were defined by a logistic regression model for two and three variables . in order to assess the significance for the added predictive ability of the combined measure from the single variable , the integrated discrimination improvement ( idi ) method was used . subgroups analyses of lnm predictability , in accordance to the primary tumor subsites , were used . univariate survival analyses of the possible clinical factors , tumor dimensional , and biological variables were conducted , using log - rank tests , and then cox proportional hazards model was used to assess the association of the selected variables on disease - free and overall survival . if needed , bonferroni s correction was used for multiple testing , such as subgroup analysis . univariate analyses of five major variables regarding tumor dimension and biology revealed significant association with the presence of lnm , except pni ( table 1 ) . because tumor subsite was significantly associated with the presence of lnm , we performed multivariate analyses for the variables of tumor dimension and biology adjusting to tumor subsite , which also showed a significant correlation of all these five variables with the occurrence of lnm ( table 2 ) . next , to evaluate the predictability of lnm by variables of tumor dimension and biology , we performed roc curve analysis . both tumor volume and thickness could predict the presence of lnm with an auc of 0.7546 and 0.7570 , respectively ( fig . variables of lvi , pni , and tumor grade could also predict lnm with auc of 0.6666 , 0.5342 and 0.6518 , respectively ( fig . the combined measure of tumor biology increased auc of 0.7717 , and significantly increased the sensitivity . combined measure of all five variables representing tumor dimension and biology strikingly increased the predictability of lnm with auc of 0.8315 ( fig . . comparisons of predictive sensitivity between combined measures of tumor dimension and biology indicated no significant difference ( idi , 0.0223 ; 95% confidence interval , 0.0607 to 0.0954 ; p > 0.999 ) , suggesting that each combined measure of tumor dimension and biology had a similarly equivalent impact on the occurrence of lnm in hnscc . these results led us to investigate the roles of tumor dimension and biology on each subsite of hnscc . interestingly , in oral tongue cancers , the predictive ability of tumor dimensional factors ( auc , 0.8403 ) was higher than biological factors ( auc , 0.7515 ) ( fig . . a combined measure of all variables did not significantly increase the predictive sensitivity for lnm compared to tumor dimension ( p=0.076 ) , but was statistically significant in comparison with tumor biology ( p < 0.001 ) , indicating that the predictive sensitivity of lnm in oral tongue cancer is mainly dependent on tumor dimensional factors . on the other hand , in oropharynx cancers , the predictive ability for lnm was rather low in tumor dimensional factors ( auc , 0.7125 ) in comparison to tumor biological factors ( auc , 0.8070 ) ( fig . a combined measure of all variables significantly increased the predictive sensitivity for lnm compared to tumor dimension ( p=0.006 ) , but had no statistical significance with respect to tumor biology ( p=0.789 ) , suggesting that the predictive sensitivity of lnm in oropharyngeal cancer is highly dependent on tumor biological factors . similarly , in hypopharynx cancers , tumor dimensional factors had almost no predictive ability for lnm ( auc , 0.4354 ) and only biological factors had predictive ability for lnm ( auc , 0.7094 ) ( fig . 2c ) . a combined measure of all variables significantly increased the predictive sensitivity for lnm compared to tumor dimension ( idi=0.1391 , p=0.040 ) , but had no statistical significance in comparison to tumor biology ( idi=0.0040 , p > 0.999 ) . thus , the impact of tumor dimension and biology was context - dependent in terms of tumor subsites in hnscc ; particularly in oro- and hypo - pharyngeal cancers , tumor progression to regional lymph nodes was influenced more by biological factors of primary tumor , rather than tumor dimension . previous studies have indicated a strong correlation between lnm in hnscc and distant metastasis . to evaluate whether the dimension or biology of the primary tumor affects the occurrence of distant metastasis or recurrence of loco - regional sites , 281 study patients were analyzed for disease - free survival . we excluded 14 patients from survival analyses due to incomplete medical and follow - up data among 295 initial subjects . estimated disease - free survival rate of the total subjects was 74.9% at 2 years and 70.4% at 5 years . first , we divided the study subjects into three subsite groups to identify the difference of relative impact of tumor dimensional or biological factors . univariate analyses included age , sex , nodal status ( n ) , treatment modalities , and tumor dimensional or biological factors . t classification was significantly correlated with tumor dimensional variables ; thus t classification was excluded from the subsequent analyses . using potential factors significant in univariate analyses ( p < 0.2 ) , we conducted multivariate analyses , via a stepwise - selection method ( table 3 ) . in concordance to the previous findings , analyses using cox proportional hazards model also confirmed the subsite differences of relative tumor dimensional or biological impacts . in oral cavity cancer , one of the tumor dimensional variables , tumor thickness , remained significant in a multivariate analysis ( hazard ratio [ hr ] , 4.518 ; p=0.016 ) ; meanwhile , one of the biological variables , pni , had a significant impact on disease - free survival in hypopharyngeal cancer ( hr , 20.314 ; p=0.007 ) ( table 3 ) . however , the results indicated that each variable of tumor dimension and tumor biology had no significant association with overall survival in our study patients ( data not shown ) . univariate analyses of five major variables regarding tumor dimension and biology revealed significant association with the presence of lnm , except pni ( table 1 ) . because tumor subsite was significantly associated with the presence of lnm , we performed multivariate analyses for the variables of tumor dimension and biology adjusting to tumor subsite , which also showed a significant correlation of all these five variables with the occurrence of lnm ( table 2 ) . next , to evaluate the predictability of lnm by variables of tumor dimension and biology , we performed roc curve analysis . both tumor volume and thickness could predict the presence of lnm with an auc of 0.7546 and 0.7570 , respectively ( fig . variables of lvi , pni , and tumor grade could also predict lnm with auc of 0.6666 , 0.5342 and 0.6518 , respectively ( fig . the combined measure of tumor biology increased auc of 0.7717 , and significantly increased the sensitivity . combined measure of all five variables representing tumor dimension and biology strikingly increased the predictability of lnm with auc of 0.8315 ( fig . . comparisons of predictive sensitivity between combined measures of tumor dimension and biology indicated no significant difference ( idi , 0.0223 ; 95% confidence interval , 0.0607 to 0.0954 ; p > 0.999 ) , suggesting that each combined measure of tumor dimension and biology had a similarly equivalent impact on the occurrence of lnm in hnscc . these results led us to investigate the roles of tumor dimension and biology on each subsite of hnscc . interestingly , in oral tongue cancers , the predictive ability of tumor dimensional factors ( auc , 0.8403 ) was higher than biological factors ( auc , 0.7515 ) ( fig . a combined measure of all variables did not significantly increase the predictive sensitivity for lnm compared to tumor dimension ( p=0.076 ) , but was statistically significant in comparison with tumor biology ( p < 0.001 ) , indicating that the predictive sensitivity of lnm in oral tongue cancer is mainly dependent on tumor dimensional factors . on the other hand , in oropharynx cancers , the predictive ability for lnm was rather low in tumor dimensional factors ( auc , 0.7125 ) in comparison to tumor biological factors ( auc , 0.8070 ) ( fig . a combined measure of all variables significantly increased the predictive sensitivity for lnm compared to tumor dimension ( p=0.006 ) , but had no statistical significance with respect to tumor biology ( p=0.789 ) , suggesting that the predictive sensitivity of lnm in oropharyngeal cancer is highly dependent on tumor biological factors . similarly , in hypopharynx cancers , tumor dimensional factors had almost no predictive ability for lnm ( auc , 0.4354 ) and only biological factors had predictive ability for lnm ( auc , 0.7094 ) ( fig . 2c ) . a combined measure of all variables significantly increased the predictive sensitivity for lnm compared to tumor dimension ( idi=0.1391 , p=0.040 ) , but had no statistical significance in comparison to tumor biology ( idi=0.0040 , p > 0.999 ) . thus , the impact of tumor dimension and biology was context - dependent in terms of tumor subsites in hnscc ; particularly in oro- and hypo - pharyngeal cancers , tumor progression to regional lymph nodes was influenced more by biological factors of primary tumor , rather than tumor dimension . previous studies have indicated a strong correlation between lnm in hnscc and distant metastasis . to evaluate whether the dimension or biology of the primary tumor affects the occurrence of distant metastasis or recurrence of loco - regional sites , 281 study patients were analyzed for disease - free survival . we excluded 14 patients from survival analyses due to incomplete medical and follow - up data among 295 initial subjects . estimated disease - free survival rate of the total subjects was 74.9% at 2 years and 70.4% at 5 years . first , we divided the study subjects into three subsite groups to identify the difference of relative impact of tumor dimensional or biological factors . univariate analyses included age , sex , nodal status ( n ) , treatment modalities , and tumor dimensional or biological factors . t classification was significantly correlated with tumor dimensional variables ; thus t classification was excluded from the subsequent analyses . using potential factors significant in univariate analyses ( p < 0.2 ) , we conducted multivariate analyses , via a stepwise - selection method ( table 3 ) . in concordance to the previous findings , analyses using cox proportional hazards model also confirmed the subsite differences of relative tumor dimensional or biological impacts . in oral cavity cancer , one of the tumor dimensional variables , tumor thickness , remained significant in a multivariate analysis ( hazard ratio [ hr ] , 4.518 ; p=0.016 ) ; meanwhile , one of the biological variables , pni , had a significant impact on disease - free survival in hypopharyngeal cancer ( hr , 20.314 ; p=0.007 ) ( table 3 ) . however , the results indicated that each variable of tumor dimension and tumor biology had no significant association with overall survival in our study patients ( data not shown ) . lnm acts as a very strong prognostic indicator in many solid cancers , as well as hnscc , which led to investigations of the determinants of nodal metastatic cascade . in this study , a prediction of lnm in hnscc based on primary tumor characteristics was analyzed using variables that represent tumor dimension and biology . the overall impact of tumor dimension and biology on lnm was similarly significant , with auc of 0.7682 and 0.7717 , respectively . interestingly , the combined measure of the variables strikingly increased the predictive value for lnm with auc of 0.8315 . these results are comparable with recent data performed in breast cancer , which indicated the impact of tumor dimension and biology on lnm , with an auc 0.6700 and 0.6852 , respectively . however , the overall predictive value was significantly high in this study in a comparison between hnscc and those in breast cancer ( auc , 0.8315 vs. 0.7462 ) . these results reflect important characteristics of hnscc , in which there exist a strong association between primary tumor and metastatic lymph node . generally , the dimension of a tumor has been considered to be a product of tumor size . however , the maximal tumor dimension , which is currently used in tnm staging , represents just one - dimensional information . recently , the total tumor volume as a three - dimensional parameter has been proposed as an important prognostic factor in hnscc . therefore , we adopted an estimated tumor volume based on the pathological reports for three - dimensional analyses . moreover , tumor thickness is a particularly strong independent predictive factor for lnm in the majority of hnscc cases . hence , we performed a combined measure of estimated tumor volume and tumor thickness as the dimensional variables , which showed a significant and context - dependent impact on lnm in hnscc . in this study , the impact of primary tumor biological factors on lnm was determined by a combined measure of lvi , pni , and tumor grade , which showed a significant correlation with lnm in hnscc . as expected , lvi , pni , and tumor grade had significant and synergistic roles on the prediction of lnm with auc of 0.6666 , 0.5342 , and 0.6518 , respectively . in addition , a recent investigation also suggested that several molecular markers , including epidermal growth factor receptor ( egfr ) expression , tp53 mutation , and human papillomavirus ( hpv ) infection status , are emerging prognostic factors and therapeutic targets in hnscc . hpv - positive tumors have a relatively favorable prognosis , while tumors with tp53 mutations or egfr over - expression have a relatively poor prognosis . thus , further analyses for these markers as biological factors might give rise to an increase of the predictive value for lnm . one may argue that our statistical models oversimplified the primary tumor characteristics , just divided into two arbitrary categories : tumor dimension and biology . indeed , the tumor dimension , such as tumor volume and thickness , are possibly affected by tumor infiltrative activity or tumor - doubling time as biological phenomena . thus , we cautiously selected and divided the indicated variables into a more probable side of the categories based on a previous report . despite the concerns , our simplified model has benefits , providing an acceptable understanding of nodal metastatic cascade and translating easily into the clinics for a more accurate prediction of lnm in different subsites of hnscc . one of the intriguing aspects of this study is the tumor subsite - specific analyses of hnscc , which showed subsitedependent different predictive value of the indicated variables . in fact , each primary tumor site of hnscc , such as oral tongue cancer and hypopharynx cancer , have been known to have different clinical characteristics , treatment protocols , and prognosis . concomitantly , we tried to analyze the impact of tumor dimension and biology on lnm in each subsite of hnscc and showed the context - dependent impact of these variables by different subsites . in oral tongue cancers , a surprisingly high predictability of tumor dimension on lnm with auc of 0.8403 indicated why local control , through either surgery or radiation therapy , is important . meanwhile , in oropharynx and hypopharynx cancers , a more dominant impact of tumor biological factors and relatively low predictability of tumor dimensional factors on lnm suggest that understanding and therapeutic control of the tumor biology is necessary . considering lnm in hnscc as a strong prognostic factor that influences distant metastasis and survival , we tried to confirm our findings through the survival analyses . when we analyzed disease - free ( recurrence or distant metastasis ) survivals , we could observe a similar trend in survival analyses ; tumor dimension ( thickness ) had a significant prognostic impact on disease - free survival in oral tongue cancer , as biological factor ( pni ) did in hypopharynx cancer . however , it is interesting that the variables of primary tumor dimension or biology did not significantly impact the overall survival , despite the impact on lnm in our series . we cautiously interpreted these results to indicate that many host factors , which were not included in the analysis or biological factors in metastatic sites , may have a more direct impact on overall survival . recently , it has been suggested that the biology and phenotype of primary tumors differ from those of metastatic lymph nodes , and tumor cells in different microenvironments respond differently to therapy . possibly , metastatic nodal characteristics can be independent prognostic factors for distant metastasis and survival in hnscc . both dimension and biology of primary tumors have a significant , tumor subsite - dependent impact on the occurrence of lnm in hnscc . particularly in oro- and hypo - pharyngeal cancers , tumor biological properties , which can be estimated through a biopsy or surgical pathology of primary tumors , could guide adjuvant treatment for regional lymph nodes . meanwhile , the primary tumor dimension should be considered in decision making of neck management in oral tongue cancer ; as such , an accurate evaluation of tumor dimension is important .

in middle - aged to elderly patients , a coronary anomaly is often an incidental finding . the clinical presentation varies from asymptomatic up to presentation as an acute myocardial infarction or even sudden cardiac death ( scd ) . we present a case of an aberrant right coronary artery ( arca ) and reviewed the literature concerning the best diagnostic and therapeutic strategy . a 40-year - old male farmer attended the outpatient clinic complaining of chest pain during work . when he tilted heavy straw bales he felt a piercing and oppressive left - sided thoracic pain , which ceased at rest . cardiovascular risk factors consisted of a familial predisposition for coronary artery disease ( father had coronary bypass at age of 65 ) and hypercholesterolemia . resting electrocardiogram ( ecg ) showed a regular sinus rhythm at 65 bpm , without further abnormalities . during his first visit , a treadmill exercise test and echocardiography were performed . during the maximal treadmill test ( fig . 1 ) up to 200 w , he mentioned the chest pain , he felt when working and tilting heavy weights . this was associated with st depression up to 1.5 mm in leads v5 and v6 and some solitary monomorphic premature ventricular complexes . 2 ) revealed a normal left and right ventricular function with normal function of the heart valves . treadmill test showing 1.5 mm st - segment depression in leads v5 to v6 and a solitary ventricular extrasystole . transthoracic echocardiography apical 4-chamber view showing normal left ventricle function , and moderate right ventricle hypertrabeculation . a further diagnostic work - up was initiated with stress echocardiography and cardiac magnetic resonance ( cmr ) . supine bicycle stress echocardiography once again provoked recognizable chest pain , electrocardiographic st depression in leads v5 to v6 , and revealed severe hypokinesia in the inferoposterior region . angiography revealed nonsignificant atherosclerosis in the 3 main coronary arteries in a right dominant coronary system . however , an anomalous take off of the right coronary artery ( rca ) in the left sinus of valsalva was diagnosed ( fig . 3 ) . coronary angiography showing the anomalous origin of the right coronary artery from the left sinus of valsalva . cmr confirmed some hypertrabeculation of the right ventricle , without signs of arrhythmogenic right ventricular dysplasia or noncompaction syndrome . the left and right ventricular function was preserved and no delayed enhancement or myocardial fibrosis was visualized . this imaging technique confirmed the aberrant origin of the rca , originating from the left coronary cusp and with a course of the proximal rca between the pulmonary artery and aorta . in conclusion , our patient presented with recurrent stable angina and only an arca could explain the symptoms . this was clearly confirmed by the abnormal myocardial ischemia tests ( treadmill exercise ecg and stress echocardiography ) in the territory of the rca . the case was debated in the heart team , and the patient was referred for a surgical correction . medication at the time of discharge included low - dose aspirin , a statin , and a beta - blocker . after 4 weeks the patient was already slowly resuming daily life , including his work as a farmer . four months after the operation , the patient consulted because of exertional shortness of breath ( new york heart association class ii ) . several forms of anatomical variants in coronary arteries exist . some are believed to be potentially dangerous , others are benign . potential malignant coronary artery anomalies are an ectopic coronary origin from the pulmonary artery , an ectopic coronary origin from the opposite sinus of valsalva , a single coronary artery , and large coronary fistulae or muscular bridging . the proximal course of an arca may vary : preaortic , interarterial ( between aorta and pulmonary artery , as in our patient ) , retrocardiac , retroaortic , intraseptal , and precardiac ( prepulmonary ) . the hemodynamic important variant ( i.e. , interarterial course ) runs between the aorta and pulmonary artery , either intramural ( within the aortic wall ) or separated from the aortic wall ( like 2 distinct arteries ) . it is supposed that only this interarterial course can cause symptoms or is potentially dangerous . with newer imaging techniques , newer anatomical variants of the proximal course of an arca the coronary takeoff can be high ( above the level of the pulmonary valve with a course between the aorta and pulmonary artery ) or low ( below the level of the pulmonary valve with a course between the aorta and right ventricular outflow tract [ rvot ] ) . this high takeoff variant is believed to be more hostile than an arca with low takeoff . post hoc analysis of the cmr images of our case , revealed a low takeoff . during ca studies , an arca has a higher incidence than an anomalous left coronary artery ( alca ) ( 0.020.15% ) and is presumed to be the most common type of hemodynamically significant coronary anomalies . in mdct coronary studies , the incidence of an arca is comparable ( 0.54% ) . among 18,950 autopsy cases in a los angeles hospital , 54 cases of coronary anomalies were detected of whom 39 with anomalies of the coronary ostia , totaling to an incidence of 0.206% . in young athletes , 12% of scds is caused by an anomalous coronary artery from the opposite aortic sinus . in young athletes , however , with increasing age of athletes , coronary anomaly becomes a less frequent cause of scd . mortality data derived from autopsy studies ( risk on scd in arca 057% , in alca 30100% ) are far too high when converted to the general population . these numbers can obviously not be extrapolated from autopsy studies to the general population and are probably severely overestimated as mortality rates are biased as we are not aware of the exact prevalence of an arca in asymptomatic patients . notwithstanding , it is assumed that there is a 3- to 6-fold increased risk for scd in people with an arca doing physical activity . there are several hypotheses : a slit - like orifice caused by an acute angle of takeoff , which could cause reduced coronary flow during exercise ; compression of the interarterial segment caused by systolic compression between the aorta and pulmonary artery , aggravated during increased flow , for example , with exercise ; ventricular arrhythmias caused by ischemia ; acute or repetitive ischemia provoking myocardial fibrosis or reperfusion ; intramural proximal intussusception of the anomalous artery at the aortic - root wall , as proposed by angelini et al during intravascular ultrasound ( ivus ) studies . , studies showed that a coronary anomaly is not associated with an increased risk for development of coronary atherosclerosis . it would be interesting to identify specific anatomical or clinical risk factors for scd in order to predict the possible hemodynamic significance . logically , the dominance of an anomalous coronary artery is an important risk factor . in a large pathology study of 30 cases of anomalous arteries , it was not possible to identify a certain anatomical feature related to increased mortality . in an mdct study , clinical symptoms were not related to the relative luminal narrowing nor angle of takeoff . however , a significant difference in the prevalence of major adverse cardiac events ( mace ) and typical angina was observed in a retrospective review of 22,925 consecutive mdct scans , in which 124 cases with an interarterial anomalous coronary artery were found . they differentiated the anatomical takeoff in a high and a low takeoff ( above or below the level of pulmonary valve ) . the group with high takeoff ( coursing between aorta and pulmonary artery ) proved to have a significant higher prevalence of typical angina ( 43% vs 6% , p = 0.001 ) and mace ( 28% vs 6% , p = 0.012 ) compared to those with a low takeoff ( coursing between aorta and rvot ) . age is an important clinical parameter that is related with the risk of scd . under the age of 30 it has been hypothesized that the aortic wall stiffens with age , which reduces compression . as mentioned above , exercise is a risk factor for scd in people with an arca . in a necropsy study of 242 patients who died suddenly , 62% of the patients were asymptomatic until the event . in a japanese review of 56 patients with coronary anomalies of whom 44 with an arca and with a mean age of 55.9 years old of 22 patients with an arca who had undergone a treadmill test , 10 proved abnormal . three out of 4 had a positive myocardial perfusion single photon emission computed tomography ( spect ) exercise test . these latter results show a much higher rate of positive stress testing than seen in other studies . this is why a negative stress test does not exclude a potentially dangerous coronary anomaly . when a stress test proves negative but symptoms are suspicious ( e.g. , exertional syncope or chest pain ) anatomical examinations ( i.e. , mdct scan ) should be considered . in young patients , or echogenic patients one study showed significant 2d strain impairment in 25 patients with a coronary anomaly ( of whom 15 with arca ) , suggesting subtle left ventricular contractility disorder in these patients . one study evaluated the accuracy of coronary artery calcium scanning for detecting coronary anomalies , which was found out to have a great diagnostic accuracy . however , nowadays , mdct ca is accepted as the gold standard for the evaluation of coronary anomalies . magnetic resonance angiography ( mra ) is similar successful , but the identification of the distal coronary course can be more difficult . some disadvantages of mra are also to be considered : availability is less , mra is not useful in patients with pacemakers or claustrophobia , and total study time takes much longer . one large benefit of mra is that it can assess and locate scarred tissue and viability in the course of the affected artery , which could have an important prognostic value . it is not always possible to define the exact proximal anatomical course of the coronary anomaly . in a ca study , only in 53% of the cases the exact proximal course could be defined . ivus showed in some studies intussusception or lateral compression during systole , aggravated by saline , atropine , or dobutamine infusion . fractional flow reserve ( ffr ) was tested by angelini et al , and showed results within normal limits during adenosine provocation ( ffr > 0.9 , cutoff 0.8 ) , indicating that ffr is not a good parameter to diagnose hemodynamic significance in this setting . stress - rest myocardial perfusion spect can be used to detect reversible perfusion defects , although results are often negative , as it is for stress ecg . long - term holter monitoring can be helpful in screening for arrhythmia , although being an aspecific tool . in summary , mdct ca seems to be the best examination for anatomical diagnosis and ischemic testing proves often negative . the 2008 american heart association guidelines for the management of adults with congenital disease , recommend that surgical coronary revascularization should be performed when there is evidence of ischemia in an anomalous rca coursing between aorta and pulmonary artery ( level of evidence class i , b ) . indication to treat should be individualized , and depends on age , symptoms , the anatomical variant , and ischemic testing . in literature , patients are separated by age ( younger and older than 35 to 40 years old ) . new anatomical understandings separate high and low coronary takeoff of the arca ( above or under level of pulmonary valve ) as a possible prognostic finding . lee et al suggest to operate all patients , younger than 40 years old , with high takeoff , regardless of symptoms . symptomatic patients older than 40 years old and with high takeoff should be operated if symptomatic , following their ideas . in patients with a low takeoff , close observation is suggested , unless when related symptoms are present surgery might be considered . in a japanese retrospective study of 56 patients ( mean age 55.9 years old ) , with anomalous origin of coronary artery ( 78% arca ) and without coexisting atherosclerosis , a conservative treatment was applied ( nitrates , calcium channel blockers , beta - adrenergic antagonists , or antiarrhythmic drugs ) . during follow - up ( 2 months to 14.5 years ) , there were no cardiac - related deaths . the authors concluded that the prognosis of middle - aged to elderly patients with an anomalous origin of the coronary artery is relatively good . a few other studies proved comparable results , suggesting a conservative approach ( beta - blockers and physical restriction ) may be safe . this somehow conflicting evidence illustrates that further research is warranted , matching anatomical with clinical and functional test data . one case series of 14 patients with proximal coronary artery stenting showed a normalization of stress test results and angiographical patency 6 months after the percutaneous coronary intervention was demonstrated . there are also different surgical strategies : reimplantation of the rca ( as in our case ) , unroofing of the intramural course with creation of a neo - orifice , the modified unroofing technique , patch augmentation , and classical bypass grafting . unroofing of the anomalous artery can be applied when the proximal course runs intramural , and when there is no involvement of aortic valve commissures , which otherwise could create aortic insufficiency . this technique relieves the ostial stenosis , creates a large neo - orifice , and removes the intramural segment . in the modified unroofing technique , the anomalous orifice is closed , a neo - orificium is created in the appropriate sinus , without extensive unroofing of the proximal intramural part of the anomalous coronary artery . the proximal course of an arca may vary : preaortic , interarterial ( between aorta and pulmonary artery , as in our patient ) , retrocardiac , retroaortic , intraseptal , and precardiac ( prepulmonary ) . the hemodynamic important variant ( i.e. , interarterial course ) runs between the aorta and pulmonary artery , either intramural ( within the aortic wall ) or separated from the aortic wall ( like 2 distinct arteries ) . it is supposed that only this interarterial course can cause symptoms or is potentially dangerous . with newer imaging techniques , newer anatomical variants of the proximal course of an arca the coronary takeoff can be high ( above the level of the pulmonary valve with a course between the aorta and pulmonary artery ) or low ( below the level of the pulmonary valve with a course between the aorta and right ventricular outflow tract [ rvot ] ) . this high takeoff variant is believed to be more hostile than an arca with low takeoff . post hoc analysis of the cmr images of our case , revealed a low takeoff . an arca has a higher incidence than an anomalous left coronary artery ( alca ) ( 0.020.15% ) and is presumed to be the most common type of hemodynamically significant coronary anomalies . in mdct coronary studies , the incidence of an arca is comparable ( 0.54% ) . among 18,950 autopsy cases in a los angeles hospital , 54 cases of coronary anomalies were detected of whom 39 with anomalies of the coronary ostia , totaling to an incidence of 0.206% . in young athletes , 12% of scds is caused by an anomalous coronary artery from the opposite aortic sinus . in young athletes , however , with increasing age of athletes , coronary anomaly becomes a less frequent cause of scd . mortality data derived from autopsy studies ( risk on scd in arca 057% , in alca 30100% ) are far too high when converted to the general population . these numbers can obviously not be extrapolated from autopsy studies to the general population and are probably severely overestimated as mortality rates are biased as we are not aware of the exact prevalence of an arca in asymptomatic patients . notwithstanding , it is assumed that there is a 3- to 6-fold increased risk for scd in people with an arca doing physical activity . there are several hypotheses : a slit - like orifice caused by an acute angle of takeoff , which could cause reduced coronary flow during exercise ; compression of the interarterial segment caused by systolic compression between the aorta and pulmonary artery , aggravated during increased flow , for example , with exercise ; ventricular arrhythmias caused by ischemia ; acute or repetitive ischemia provoking myocardial fibrosis or reperfusion ; intramural proximal intussusception of the anomalous artery at the aortic - root wall , as proposed by angelini et al during intravascular ultrasound ( ivus ) studies . , studies showed that a coronary anomaly is not associated with an increased risk for development of coronary atherosclerosis . it would be interesting to identify specific anatomical or clinical risk factors for scd in order to predict the possible hemodynamic significance . logically , the dominance of an anomalous coronary artery is an important risk factor . in a large pathology study of 30 cases of anomalous arteries , it was not possible to identify a certain anatomical feature related to increased mortality . in an mdct study , clinical symptoms were not related to the relative luminal narrowing nor angle of takeoff . however , a significant difference in the prevalence of major adverse cardiac events ( mace ) and typical angina was observed in a retrospective review of 22,925 consecutive mdct scans , in which 124 cases with an interarterial anomalous coronary artery were found . they differentiated the anatomical takeoff in a high and a low takeoff ( above or below the level of pulmonary valve ) . the group with high takeoff ( coursing between aorta and pulmonary artery ) proved to have a significant higher prevalence of typical angina ( 43% vs 6% , p = 0.001 ) and mace ( 28% vs 6% , p = 0.012 ) compared to those with a low takeoff ( coursing between aorta and rvot ) . age is an important clinical parameter that is related with the risk of scd . under the age of 30 it has been hypothesized that the aortic wall stiffens with age , which reduces compression . as mentioned above , in a necropsy study of 242 patients who died suddenly , 62% of the patients were asymptomatic until the event . in a japanese review of 56 patients with coronary anomalies of whom 44 with an arca and with a mean age of 55.9 years old of 22 patients with an arca who had undergone a treadmill test , 10 proved abnormal . three out of 4 had a positive myocardial perfusion single photon emission computed tomography ( spect ) exercise test . these latter results show a much higher rate of positive stress testing than seen in other studies . this is why a negative stress test does not exclude a potentially dangerous coronary anomaly . when a stress test proves negative but symptoms are suspicious ( e.g. , exertional syncope or chest pain ) anatomical examinations ( i.e. , mdct scan ) should be considered . in young patients , or echogenic patients one study showed significant 2d strain impairment in 25 patients with a coronary anomaly ( of whom 15 with arca ) , suggesting subtle left ventricular contractility disorder in these patients . one study evaluated the accuracy of coronary artery calcium scanning for detecting coronary anomalies , which was found out to have a great diagnostic accuracy . however , nowadays , mdct ca is accepted as the gold standard for the evaluation of coronary anomalies . magnetic resonance angiography ( mra ) is similar successful , but the identification of the distal coronary course can be more difficult . some disadvantages of mra are also to be considered : availability is less , mra is not useful in patients with pacemakers or claustrophobia , and total study time takes much longer . one large benefit of mra is that it can assess and locate scarred tissue and viability in the course of the affected artery , which could have an important prognostic value . ca it is not always possible to define the exact proximal anatomical course of the coronary anomaly . in a ca study , only in 53% of the cases the exact proximal course could be defined . ivus showed in some studies intussusception or lateral compression during systole , aggravated by saline , atropine , or dobutamine infusion . fractional flow reserve ( ffr ) was tested by angelini et al , and showed results within normal limits during adenosine provocation ( ffr > 0.9 , cutoff 0.8 ) , indicating that ffr is not a good parameter to diagnose hemodynamic significance in this setting . stress - rest myocardial perfusion spect can be used to detect reversible perfusion defects , although results are often negative , as it is for stress ecg . long - term holter monitoring can be helpful in screening for arrhythmia , although being an aspecific tool . in summary , mdct ca seems to be the best examination for anatomical diagnosis and ischemic testing proves often negative . the 2008 american heart association guidelines for the management of adults with congenital disease , recommend that surgical coronary revascularization should be performed when there is evidence of ischemia in an anomalous rca coursing between aorta and pulmonary artery ( level of evidence class i , b ) . indication to treat should be individualized , and depends on age , symptoms , the anatomical variant , and ischemic testing . in literature , patients are separated by age ( younger and older than 35 to 40 years old ) . new anatomical understandings separate high and low coronary takeoff of the arca ( above or under level of pulmonary valve ) as a possible prognostic finding . lee et al suggest to operate all patients , younger than 40 years old , with high takeoff , regardless of symptoms . symptomatic patients older than 40 years old and with high takeoff should be operated if symptomatic , following their ideas . in patients with a low takeoff , close observation is suggested , unless when related symptoms are present surgery might be considered . in a japanese retrospective study of 56 patients ( mean age 55.9 years old ) , with anomalous origin of coronary artery ( 78% arca ) and without coexisting atherosclerosis , a conservative treatment was applied ( nitrates , calcium channel blockers , beta - adrenergic antagonists , or antiarrhythmic drugs ) . during follow - up ( 2 months to 14.5 years ) , there were no cardiac - related deaths . the authors concluded that the prognosis of middle - aged to elderly patients with an anomalous origin of the coronary artery is relatively good . a few other studies proved comparable results , suggesting a conservative approach ( beta - blockers and physical restriction ) may be safe . this somehow conflicting evidence illustrates that further research is warranted , matching anatomical with clinical and functional test data . one case series of 14 patients with proximal coronary artery stenting showed a normalization of stress test results and angiographical patency 6 months after the percutaneous coronary intervention was demonstrated . there are also different surgical strategies : reimplantation of the rca ( as in our case ) , unroofing of the intramural course with creation of a neo - orifice , the modified unroofing technique , patch augmentation , and classical bypass grafting . with patch augmentation , unroofing of the anomalous artery can be applied when the proximal course runs intramural , and when there is no involvement of aortic valve commissures , which otherwise could create aortic insufficiency . this technique relieves the ostial stenosis , creates a large neo - orifice , and removes the intramural segment . in the modified unroofing technique , the anomalous orifice is closed , a neo - orificium is created in the appropriate sinus , without extensive unroofing of the proximal intramural part of the anomalous coronary artery . the interarterial form of an anomalous rca ( coursing between the aorta and pulmonary artery ) can lead to symptoms or even scd . coronary anomaly is the second most common cause of cardiac sudden death in young athletes , which demonstrates that young age and vigorous exercise are risk factors for sudden death in patients with an arca . further ischemic testing can help to guide the therapy strategy , although functional tests often prove negative . in middle - aged patients , this anomaly is often an incidental finding . notwithstanding the lack of randomized trials , we consider it advisable to operate when an arca is seen in young patients ( < 40 years old ) or , in older patients with proven related symptoms or positive ischemic testing . reimplantation of the rca or unroofing the proximal course of the anomalous artery seems to be the best surgical strategies . when a conservative approach is proposed , avoidance of vigorous exercise and prescribing beta - blockers are advised .

a 44-year - old male patient visited the outpatient department with complaints of a chronic epiphora , and discharge from the right eye . a palpable mass had been noted in the right medial canthal area 10 days prior to the visit . on ophthalmological examination , the visual acuity was 1.0 in both eyes without correction and there was no relative afferent pupillary defect . dacryocystography showed evidence of a soft tissue mass as an uneven mottled density of the contrast media in the right lacrimal sac ( fig . 1 ) . magnetic resonance imaging ( mri ) of the orbit revealed a well - defined heterogeneous enhancing mass lesion , approximately 189 mm , filling the lacrimal sac and extending to the upper nld . the mass extended anteriorly to the right facial soft tissues with no any evidence of bony structure destruction ( fig . when the lacrimal sac was removed , the mass was found within lacrimal sac , extending into the nld . adhesions were adjacent to the sac and obstruction of the nld occurred at the junction with the bony canal due to tumor extension to the excision margins . we removed as much of the tumor as possible , but could not excise a portion that was located in the bony canal duct . surgical biopsy demonstrated an exophytic schneiderian papilloma with focal moderate to severe dysplasia on the histopathology . three months after the operation , the orbital mri showed a recurrent mass in the right lacrimal sac invading the nasal cavity via the inferior meatus ( fig . , the patient underwent endoscopic biopsy of the nasal cavity lateral wall through a nasal approach . an identical tumor was confirmed , and endoscopic medial maxillectomy and dacryocystorhinostomy has been planned for its removal . histological examination revealed a fungiform mass with projecting finger - like proliferation of the epithelium . the tumor revealed an exophytic growth pattern with branching fibrovascular stalks covered by an epithelial cell layer ( fig . 4 ) . according to the cytological features , the lesion was a transitional cell papilloma . occasionally , a single layer of columnar ciliated respiratory epithelial cells lined the surface of the hyperplastic transitional cells ( fig . pathologic examination confirmed the diagnosis of an exophytic schneiderian papilloma with moderate to severe dysplasia . a 44-year - old male patient visited the outpatient department with complaints of a chronic epiphora , and discharge from the right eye . a palpable mass had been noted in the right medial canthal area 10 days prior to the visit . on ophthalmological examination , the visual acuity was 1.0 in both eyes without correction and there was no relative afferent pupillary defect . dacryocystography showed evidence of a soft tissue mass as an uneven mottled density of the contrast media in the right lacrimal sac ( fig . 1 ) . magnetic resonance imaging ( mri ) of the orbit revealed a well - defined heterogeneous enhancing mass lesion , approximately 189 mm , filling the lacrimal sac and extending to the upper nld . the mass extended anteriorly to the right facial soft tissues with no any evidence of bony structure destruction ( fig . when the lacrimal sac was removed , the mass was found within lacrimal sac , extending into the nld . adhesions were adjacent to the sac and obstruction of the nld occurred at the junction with the bony canal due to tumor extension to the excision margins . we removed as much of the tumor as possible , but could not excise a portion that was located in the bony canal duct . surgical biopsy demonstrated an exophytic schneiderian papilloma with focal moderate to severe dysplasia on the histopathology . three months after the operation , the orbital mri showed a recurrent mass in the right lacrimal sac invading the nasal cavity via the inferior meatus ( fig . , the patient underwent endoscopic biopsy of the nasal cavity lateral wall through a nasal approach . an identical tumor was confirmed , and endoscopic medial maxillectomy and dacryocystorhinostomy has been planned for its removal . histological examination revealed a fungiform mass with projecting finger - like proliferation of the epithelium . the tumor revealed an exophytic growth pattern with branching fibrovascular stalks covered by an epithelial cell layer ( fig . 4 ) . according to the cytological features , the lesion was a transitional cell papilloma . occasionally , a single layer of columnar ciliated respiratory epithelial cells lined the surface of the hyperplastic transitional cells ( fig . pathologic examination confirmed the diagnosis of an exophytic schneiderian papilloma with moderate to severe dysplasia . the mucosal epithelium that lines the nasal cavity , paranasal sinuses , and lacrimal apparatus is unique in its embryogenesis , in that it is ectodermal in origin.1,2 a common synonym for this ectodermally derived mucosa and papilloma is the " schneiderian mucosa " or " schneiderian papilloma".4 the schneiderian papilloma is an uncommon neoplasm . the lateral nasal wall is the most common site of origin.2 the lacrimal system is one of the most unusual primary locations,3 having been reported in korea in only two cases reported.5,6 this case is a recurrent schneiderian papilloma of the lacrimal sac that eventually invaded the nasal cavity through the nasolacrimal duct after mass excision of the lacrimal sac tumor . there is no prior report of recurrence and invasion of the nasolacrimal duct by an exophytic papilloma originating in the lacrimal sac . valenzuela et al.7 reported the clinical presentation of lacrimal drainage apparatus tumors in 37 patients . symptoms included epiphora , a palpable mass , dacryocysitis , discharge , a visible lesion , bloody discharge or tears , epistaxis , and non - axial globe displacement . this patient had chronic epiphora and a small palpable mass at the nasal canthal area . bloodstained discharge from the puncta should always be regarded as a significant finding whether spontaneous , secondary to pressure on the sac , or after irrigation.8 when found , surgical intervention is usually recommended , because it is suggestive of a lacrimal sac tumor . a characteristic dacryocystogram , as described by veirs,9 shows a distended sac shadow , uneven or mottled density of the contrast media , and patency with residual media present 30 minutes after injection . orbit and sinus ct scan , and mri provide the most useful information about the extent of tumors and their relationship with surrounding bony structures and soft tissues.10 in this case , dacryocystography showed uneven contrast media at the right lacrimal sac , and evidence of a soft tissue mass . mri of the orbit revealed a heterogeneous enhancing mass , approximately 189 mm , in the right lacrimal sac and upper nasolacrimal duct . the mass lesion extended anteriorly to the facial soft tissue , but no bony destruction was observed . histological examination of excised tissue is essential to confirm diagnosis . as presented by ryan and font,3 lacrimal papillomas can be subdivided by growth pattern into exophytic , inverted , or mixed in architecture . another classification , reported by ashton et al.11 defines transitional cell papillomas , intermediate types , and transitional cell carcinomas . in this case , the schneiderian papilloma had an exophytic growth pattern . the lesion was a transitional cell papilloma , with stratified columnar epithelium and scattered goblet cells , with partial severe dysplasia . the possibility of malignant change must always be considered . in the cases studied by ryan and font,3 7 out of the 12 inverted or mixed - type papillomas presented either as carcinomatous lesions or progressed to invasive carcinoma ; none of six exophytic papillomas developed into carcinomas , however . this patient had an exophytic schneiderian papilloma with moderate to severe dysplasia , which may precede the development of a malignancy . hyams,12 reported on papillomas of the sinonasal tract , and found a recurrence rate of 40 - 60% for all papillomas , regardless of type . all recurrences occurred at essentially the same anatomical site as the previous surgically removed tumor . we concluded that this case was a recurrent schneiderian papilloma caused by incomplete removal of the lacrimal sac papilloma . sham et al.13 recommended that guidelines for the surgical treatment of schneiderian papillomas include the following : 1 ) the papilloma should not be managed as a completely benign lesion ; 2 ) the best opportunity for successful control of the papilloma is the first surgical procedure and ; 3 ) the more open the approach , the better the accessibility , the more complete the resection , and the lower the chance of recurrence . in conclusion , a schneiderian papilloma of the lacrimal sac is a locally aggressive benign epithelial tumor with malignant potential . therefore , a schneiderian papilloma of the lacrimal sac must be accurately diagnosed by histopathological examination , and completely removed by surgical excision .

noncarious cervical lesions ( nccls ) are becoming an increasingly important factor when considering the long - term health of the dentition . in fact , the occurrence of this condition is steadily increasing [ 14 ] . according to the present literature available , it is not possible to determine a unique etiological factor , but there is a concern that it is a multifactorial condition [ 58 ] . these lesions can affect tooth sensitivity , plaque retention , caries incidence , structural integrity , and pulp vitality , and they present unique challenges for successful restoration [ 59 ] . these challenges involve each step of the restoration process , including isolation , adhesion , insertion technique , and finishing and polishing . a successful diagnosis and treatment plan requires keen observation , a thorough patient history , and careful evaluation . this work aims to provide some knowledge of the nccls ' characteristics and etiologic covariables as well as improve assessment of prognosis by aiding in proper case selection for treatment and in the selection of appropriate treatment protocols . this could be reached with a complete patient anamnesis accompanied by a careful clinical examination . some studies suggest that treatment provided for nccls may not be based on the correct diagnosis [ 3 , 4 ] . it is important to diagnose the tooth wear process in children and adults as early as possible . diagnosing early forms of erosion is difficult , as erosion is accompanied by few signs and fewer , if any , symptoms . therefore , clinical appearance is the most important feature for dental professionals when diagnosing this condition . commonly , when the nccl is painless and does not affect esthetics , there is no complaint by the patient . sometimes , it is not completely painless , but the dentin is partially ( or completely ) covered by dental plaque , tartar , or gum . a simple removal ( or displacement ) of this coverage followed by the application of some stimulus ( like a delicate air blast ) can initiate a pain process . when pain is present , the location of the lesion becomes easier to detect . pain is one of the factors that will directly influence the decision for restorative therapy as well as the technique employed . as soon as the dental caries is eliminated as primary cause , the possible factors involved have to be identified . these noncarious processes may include abrasion , corrosion , and ( possibly ) abfraction , acting alone or in combination . there are factors associated directly with the genesis of nccl , such as occlusion , saliva , age , sex , diet , and parafunctional habits [ 11 , 12 ] . if teeth are worn on their occlusal surfaces , incisal surfaces , or both by friction from the food bolus , this wear is termed masticatory abrasion . masticatory abrasion can also occur on the facial and lingual aspects of teeth , as coarse food is forced against these surfaces by the tongue , lips , and cheeks during mastication . we should not underestimate the relevance of some current diet habits , which are considered but potentially destructive to the teeth ( granolas , nuts , all bran cereal , and acid juices ) . abrasion can also occur as a result of overzealous tooth brushing , improper use of dental floss and toothpicks , or detrimental oral habits . frequently , they appear as painless cavities with polished surfaces , but pain is not an uncommon occurrence . typically , when improper tooth brushing is one of the causes of the nccls , the enamel resists differently than the dentin which erodes following the path made by the toothbrush [ 39 ] . in dentistry , the term erosion is used to define the loss of dental hard tissues by chemical action not involving bacteria . erosion , as defined by the american society for testing and materials committee on standards , is the progressive loss of a material from a solid surface due to mechanical interaction between that surface and a fluid , a multi component fluid , impinging liquid or solid particles . corrosion is the more appropriate term and represents tooth surface loss caused by chemical or electrochemical action . there are both endogenous and exogenous sources of corrosion . in cases of endogenous sources of corrosion , such as bulimia or gastro esophageal reflux disease ( gerd ) , the enamel appears thin and translucent , enamel is lost on the posterior occlusal and anterior palatal surfaces , and depressions occur at the cervical areas of upper anterior teeth . cupped , or invaginated , areas develop where dentin has been exposed on the occlusal surfaces of posterior teeth because of wear . in the exogenous sources of corrosion , the aspect is similar , but the tissue loss location modifies following the areas related to the passage of the corrosive element . it has been reported that any food substance with a critical ph value of less than 5.5 can become a corrodent and demineralize teeth . this may occur as a result of consuming highly acidic foods and beverages such as citrus fruits , carbonated soft drinks , and sucking on sour candies . acidulated carbonated soft drinks have become a major component of many diets , particularly among adolescents and young children . it is evident that this condition does not exclusively affect the cervical areas , but , in association with other factors , it will act synergistically . abfraction is thought to take place when excessive cyclic , nonaxial tooth loading leads to cusp flexure and stress concentration in the vulnerable cervical region of teeth . such stress is then believed to directly or indirectly contribute to the loss of cervical tooth substance [ 5 , 7 , 8 , 1623 ] . although there is theoretical evidence in support of abfraction , predominantly from finite element analysis studies , caution is advised when interpreting results of these studies due to their limitations [ 9 , 2426 ] . frequently , more than two mechanisms may be involved in the etiology of tooth surface lesions , featuring a multifactorial phenomenon . when to these two mechanisms are added the effect of stress ( abfraction ) resulting from bruxism or occlusal interference , these lesions then become corrosive - abrasive abfractive in nature . the interplay of chemical , biological , and behavioral factors is crucial and helps to explain why some individuals exhibit more erosion than others [ 5 , 7 ] . therefore , awareness of a multifactorial etiology in noncarious cervical lesions may help the clinician to formulate an appropriate treatment plan for the patient . abrasion is the most cited etiological factor for development of nccl . in clinical surveys , 94% of respondent dentists classified the lesion as abrasion , and 66% rated tooth brushing as the most likely cause . cervical toothbrush abrasions are generally thought to be a consequence of toothbrush factors such as frequent or forceful tooth brushing , faulty or vigorous techniques , filament stiffness or design , dominant hand dexterity , or abrasive dentifrices . however , investigations can not conclusively establish one factor as the primary etiology because of conflicting results . therefore , an array of aspects related to toothbrush factors may operate in conjunction with dental erosion and occlusal loading . the clarification of patients , their orientation about brushing techniques , and the change of some of the above factors can bring tangible results and must be performed . another etiology that can be effectively modified is the chemical corrosion ( also called dental erosion ) and should be correctly diagnosed . when derived from eating disorders ( bulimia ) or and gerd , the treatment may require the participation of a physician . the extrinsic etiology is more easily treatable ; removing or altering the harmful habit , as in the abrasion etiology , provides consistent results . when the abfraction etiology is diagnosed , no consensus on treatment strategies exists . it is important that oral health professionals understand that abfraction is still a theoretical concept , as it is not proved . as a result of the reported associations between occlusal interferences and abfraction lesions , and between loading direction ( influenced by cusp inclines ) and unfavorable tensile stresses , occlusal adjustment has been advocated to prevent their initiation and progression and to minimize failure of cervical restorations . occlusal adjustments may involve altering cusp inclines , reducing heavy contacts , and removing premature contacts . in fact , inappropriate occlusal adjustments may increase the risk of certain conditions such as caries , occlusal tooth wear , and dentine hypersensitivity . the science of occlusion is complex , and the treatment requires understanding , care , and experience . although it is desirable to reduce lateral forces on teeth with stress - induced cervical lesions , extensive restorative procedures , such as the reestablishment of anterior guidance or orthodontic movement , require cost - and - benefit justification . occlusal adjustment should be undertaken only in cases where the interferences are well established and diagnosed . the professional must be enabled to do the adjustments and be aware that this procedure must be performed only when strictly indicated . the adjustment must be carried out in order to remove only the interferences , preserving the original points of centric occlusion . it is a conservative procedure , since it involves only the application of a composite resin , but it is important to carefully observe the possibility of excessive stress concentrated on this tooth . in fact , it is recommended that destructive , irreversible treatments aimed at treating so - called abfraction lesions , such as occlusal adjustment , must be avoided or implemented only in exceptional cases . occlusal splints , aimed at reducing the amount of nocturnal bruxism and nonaxial tooth forces , have been recommended to prevent the initiation and progression of abfraction lesions . however , it should be noted that the use of occlusal splints to reduce bruxism is still a controversial topic . although they provide a conservative treatment option for managing suspected abfraction lesions , according to some authors , there is no evidence base to support their use [ 9 , 24 ] . in the presence of evidence of the relevance of the abfraction mechanism in the development of lesions , the occlusal splint should be considered as a good treatment strategy due to its conservative nature . it should be noted that when restoring nccls , clinicians are not treating the etiology but are merely replacing what has been lost . others recommend early intervention [ 6 , 16 , 24 , 26 , 27 ] . there are no generally accepted , specific guidelines in the literature stating that all lesions should be restored . logic and good clinical judgment would suggest that they should be restored when clinical consequences ( e.g. , dentine hypersensitivity ) have developed or are likely to develop in the near future . cervical restorations may contribute to increased plaque accumulation potentially leading to caries and periodontal disease [ 11 , 24 , 25 ] . there are different reasons for the need for restorative treatment : the structural integrity of the tooth is threatened , the exposed dentin is hypersensitive , the defect is esthetically unacceptable to the patient , or pulp exposure is likely to occur . when the dentist is against nonsensitive shallow cavities that do not provide additional plaque retention the possible causes of the nccls should be identified and eliminated ( or treated ) . if the abfraction etiology is considered , the occlusion should be marked with red and blue articulating paper to check whether there has been any change , and photographic records from an occlusal view should be taken . if a progression of the nccls is diagnosed , changes in the therapy should be considered , providing restorative treatment if necessary [ 6 , 19 ] . once the restorative treatment is indicated , the dentist has to know the different causes and aspects of each situation and choose the best strategy to employ . unfortunately , although nccl restorations are a very common occurrence in clinics , they also represent one of the less durable types of restorations and have a high index of loss of retention , marginal excess , and secondary caries . despite these restorations being a continuing problem in restorative dentistry , failure of cervical adhesive restorations is often attributed to inadequate moisture control , adhesion to different opposite substrates ( enamel and dentin ) , differences in dentin composition , and also cusp movement during occlusion . in order to help adopt the best restorative strategy , problems with restoring nccls include difficulty in obtaining moisture control and gaining access to subgingival margins [ 10 , 2830 ] . rubber dam clamps , gingival retraction cord , and periodontal surgery are methods that can be used to retract and control the gingival tissues , and thus facilitate access and also control moisture . the exudation of gingival fluid is possibly one of the challenges to adhesion in cervical region , which is already impaired by other factors ( such as the absence of enamel in the gingival wall of the cavity and the characteristics of the dentin in nccls ) . intrinsic anatomical and morphological characteristics of the cervical region create limitations in the placement of the rubber dam and clamp . proper isolation , is very difficult , sometimes impossible , when lesions extend proximally or under the gingiva . sometimes part of the structure can not be isolated and the dam promotes restorative material accumulation . when adequate rubber dam isolation is not possible another isolation method has to be employed . another option is a proposed association of mylar matrix with wood wedges and a photocured gingival barrier . in any case , a proper isolation is the first step for the success in restoring nccls but , despite being the basis for the other subsequent steps , is probably the most underestimated one . even with advanced destruction , minimally invasive restorative intervention , such as sealing or covering with composite material it is evident from the recent literature that there is no place for metallic materials such as amalgam and gold in the modern day restoration of nccls . glass ionomer cements ( gics ) , resin - modified gics ( rmgics ) , a gic / rmgic liner base laminated with a resin composite , and resin composite in combination with a dentine bonding agent are all restorative options [ 24 , 3135 ] . some authors recommend that rmgic should be the first preference for restoration of nccls or , in aesthetically demanding cases , a gic / rmgic liner base with resin composite [ 32 , 33 ] . indeed gic presents several characteristics that make them a good choice : biocompatibility , adhesion to calcified substrates ( especially in cases of dentin sclerosis where traditional adhesion may underperform ) , and elastic modulus similar to the dentin . however , some other characteristics make its use infrequent : technical difficulties related to the material 's stickiness , poor esthetics , solubility particularly in acidic oral environments , and retention failure occurrences . some authors claim that under the action of parafunctional loadings , fracture - induced failure of cervical gic restorations occurs at the cervical margin . it is further shown that prior to fracture , the restorative material undergoes strain softening , which in turn introduces damage and weakens the materials involved . the softening of the material occurs in the cervical region of the restoration area which has been linked to the location of most of the clinical observed failures . the author does not indicate gic or rmgic frequently , but it is a good indication in deep nccls , where a laminate technique ( sandwich technique with composite resins ) can be used . the best materials for restoration of nccls are the composite resins . within this group of materials , some authors recommend that nccls suspected of being caused primarily by abfraction should be restored with a microfilled resin composite or a flowable resin that has a low modulus of elasticity , as it will thus flex with the tooth and not compromise retention [ 34 , 3638 ] . however , no definitive conclusion can be found in the literature addressing the difference between failures rates of resin composites of different stiffness used to restore nccls . nevertheless , in must situation , the authors recommend low modulus composites or associations of composites with different modulus . after the isolation another important , and commonly neglected , step should be performed : the prophylaxis of the cavity . due to their nature , nccls are lined with a contaminated layer that resists adhesion . the gingival proximity ( sometimes partially or totally covering the cavity ) makes this procedure a more complex step . in some cases , rotary prophylactic brushes can not be used in order to avoid mechanical aggression and bleeding . in nonsensitive cavities , the authors recommend rubbing the cavity and its periphery with a cotton pellet soaked with an anionic detergent , followed by rinsing with water , drying , and conventional total acid etching ( 37% phosphoric acid10 seconds on dentins and 20 seconds on enamel ) with the aim of removing the sticky layer . even when the roughening procedure is performed , the same sequence is recommended . in the presence of sensitivity , rubbing with detergent is still indicated but the phosphoric acid should be applied only on enamel . when a conventional gic is chosen , the previous conditioning with polyacrylic acid is indicated in order to provide a good surface wetting . if an rmgic is chosen , pretreatment of dentin with self - etch adhesive systems , before filling , seems to be a good alternative to the conventional dentin conditioner provided by the manufacturer . some recent studies demonstrate important histological differences between prepared dentin and the affected dentin from nccls . one work based on raman analysis showed that the distinct compositional and structural alterations in mineral and matrix components of nccls affected dentin . a heterogeneous hypermineralized layer , with characteristic features such as high phosphate / low carbonate content , high degree of crystallinity , and partially denatured collagen , was revealed in the affected dentin substrate of nccls [ 39 , 40 ] . in another study focusing on adhesion to sclerotic dentin , the authors observed that most dentinal tubules were obliterated by rod - like sclerotic casts and could not be dissolved by acid etching . both the hybrid zone and the resin tags were observed in sclerotic dentin after restoration . although resin tags were fewer , and in lack of communications , the length of resin tags and the thickness of the hybrid zone were almost similar to those of the sound dentin . they concluded that bonding to sclerotic dentin is different from bonding to sound dentin and may be compromised by fewer resin tags and communications . transmission electron microscopy revealed that in addition to occlusion of the tubules by mineral crystals , many parts of wedge - shaped cervical lesions contain a hyper mineralized surface that resists the etching action of both self - etching primers and phosphoric acid . examination of both sides of the failed bonds revealed a wide variation in fracture patterns that involved all of these structures . microtensile bond strengths to the occlusal , gingival , and deepest portions of these wedge - shaped lesions were significantly lower than similar areas artificially prepared in normal teeth . further studies are required to understand the role that these alterations play in response to acid etching and bonding to these clinically relevant substrates . further , some authors agree that restorations placed in teeth whose dentin / enamel had been prepared , or roughened , showed a statistically significant higher retention rate than those placed in teeth with unprepared dentin [ 10 , 43 ] . considering these studies and the author 's clinical experience , a mild roughening of the superficial dentin with a diamond point is indicated when restoring polished nonsensitive nccls . this procedure does not create additional sensitivity and aims to get a more reliable adhesion in this specific situation . if the cavity is deep and provides sufficient thickness , a sandwich technique may be performed , taking advantage of the gic 's good adhesion to calcium . it is important to note that adhesives with direct interaction with calcium have been recently developed and present a promising option in these cases . , it is logical to conclude , based on hydrodynamic theory , that the dentin tubules are not obliterated ; on the contrary , they are probably opened . thus , the etching should be gentle in order to provide a good substrate to adhesion without enhancing sensitivity . based on this , and considering the available adhesives , the self - conditioning ( se ) adhesives should be the first choice . although several articles doubt their efficiency in aspects such as bond strength and marginal discoloration , others demonstrate acceptable clinical performance [ 4549 ] . a previous acid etching of the surrounding enamel is indicated because , as known , the microretentions created by the se adhesives are not enough to give adhesive strength similar to that achieved by conventional acid etching . within this group , the self - etching primers ( two steps ) present better results than the self - etching adhesives ( one step ) [ 5052 ] . one must always remember that an active application of these adhesives should be employed , rubbing the surface with a soaked microbrush for 15-seconds , waiting other 15 second period to allow volatilization of solvents . this is important because the cervical wall of the cavity tends to retain excess of adhesive which leads to future discoloration and gap formation . despite the apparent easy access and insertion , nccl presents some particularities that should be emphasized . this may justify the high documented failure rate [ 30 , 33 , 5355 ] and the number of published articles about this theme [ 10 , 34 , 36 , 5667 ] . the first point that creates difficulties is that the cavity limits are not well defined , especially the proximal limits location . . every effort should be made to delimit the future restoration , because the excess removal and the finishing and polishing present other difficulties . the simple fact of working with cavities on opposite walls from dissimilar tissues like dentin and enamel already creates intrinsic problems . several restorative techniques have been proposed to minimize shrinkage due to polymerization and also to achieve better marginal adaptation in class v cavities . because bond strength to enamel is usually greater than to dentin , it was suggested that cavities could be restored in multiple layers , starting with incremental placement in the occlusal wall of the preparation . it has also been suggested that the contraction gap at the gingival margin caused by polymerization shrinkage could be prevented by the incremental placement of a composite material starting in the dentin portion of the preparation . regarding the possibility of bulk placement , it has been stated that this often results in open dentin margins , thus increasing microleakage . since enamel adhesion is stronger , more stable , and more predictable , the insertion of material should begin from the gingival wall , without surrounding enamel . avoiding concomitant insertion on opposite walls and leaving a free surface , the adhesion to the cervical wall can be achieved without antagonistic forces . whenever possible , the cavity should be restored with three , or at least two , increments . employing a careful technique is possible to achieve a restoration with minimum or no finishing and polishing procedures needed . considering esthetics , the color of the cervical area is easy to obtain , usually with a higher saturation and smaller translucency compared to the color of the other two thirds of the tooth . any excess or roughness plaque retention , gingival inflammation , and occurrence of caries lesions represent not only a failure of the restoration but also a creation of new problems to the patient . poorly performed finishing and polishing procedures can lead to damage to the soft and hard tissues . techniques with minimum need of finishing and polishing are ideal , but properly contoured restorations are seldom achieved without the need to remove excess material [ 10 , 6872 ] . when they are needed , a good option is the use of delicate diamond finishing points followed by application of a surface sealant or a liquid polisher [ 10 , 72 , 73 ] . as emphasized before , treatment of nccls is not easy , and sometimes , new procedures or different approaches are needed . semiannual appointments should be performed in order to observe the evolution of the lesions , the conditions of the restorations , and other concerns of the patient . also , the maintenance of the surface polish can be performed with a new surface sealant application . treating nccls necessarily involves these steps : problem identification , diagnosis , etiological factor removal , or treatment , and , if necessary , restoration . due to the multifactorial character , it is not a simple procedure . a successful diagnosis and treatment plan requires a thorough patient history and careful observations and evaluations .

most signaling pathways include protein kinases and their substrates that serve as means to amplify signals from extracellular signals and other stimuli . however , the precise connectivity between protein kinases and their downstream substrates has not been fully elucidated for most protein kinases . c - src is a classic nonreceptor tyrosine kinase that has been implicated in regulation of cytoskeletal rearrangement and cell adhesion networks that control cell migration , cell proliferation and cell survival.(1 ) one vital step in understanding the role of c - src kinase in cellular transformation and signaling is systematic identification of all of its potential cellular substrates involved in these processes . recent studies based on advances in mass spectrometry - based proteomics have provided large - scale catalogs of phosphorylation sites . however , determination of kinases responsible for these phosphorylation events is not an easy task owing to the transient interaction between kinases and their substrates . such studies include use of an atp analogue that is a specific substrate for an analogue - specific allele of v - src,(6 ) screening of cdna expression libraries with anti - phosphotyrosine antibodies(7 ) and use of mutant inducible forms of c - src.(8 ) to date , several c - src substrates as well as interactors have been reported . human protein reference database ( hprd)(9 ) provides a list of 132 c - src mediated phosphorylation sites in 64 known substrates along with 204 proteins that interact with c - src . we have used the stable isotope labeling with amino acids in cell culture ( silac ) approach which enables identification of tyrosine kinase substrates based on a unique signature in mass spectrometry experiments . the main objective of this work was to identify novel c - src substrates by overexpression of a constitutively active form of c - src followed by enrichment of tyrosine - phosphorylated proteins . we have identified 26 novel c - src tyrosine kinase substrates in addition to 10 others , which were either known src family kinase substrates or proteins known to associate with src family kinases . we have experimentally confirmed 4 novel substrates , nice-4 , rna binding motif 10 , fuse - binding protein 1 and trk - fused gene , to be direct substrates of c - src using in vitro kinase assays . we were also able to implicate ews1 , rna binding motif 10 and bcl-2 associated transcription factor in pdgf signaling using a chemical inhibitor of c - src . our peptide microarray approach led to identification of a number of peptides that are phosphorylated by c - src . to our knowledge , this is the first reported integrated proteomics strategy that couples cell culture , mass spectrometry and peptide microarrays to identify tyrosine kinase substrates . stable isotope containing amino acids , c6-arginine , c6-arginine and c6-n4-arginine , were purchased from cambridge isotope labs ( andover , ma ) . complete protease inhibitor cocktail tablets were purchased from roche ( indianapolis , in ) , sodium orthovanadate and anti - flag m2 monoclonal antibody from sigma - aldrich co. ( st . ( san diego , ca ) , anti - phosphotyrosine antibodies ( 4g10 ) agarose - conjugate and streptavidin - agarose beads from upstate biotechnology ( lake placid , ny ) , antiphosphotyrosine - rc20 biotin conjugate from bd biosciences ( san jose , ca ) and pdgf - bb from invitrogen ( carlsbad , ca ) . ( chicago , il ) , p130cas and ews1 from santa cruz biotechnology , inc . phospho - src ( tyr416 ) antibody and phosphoscan kit ( p - tyr-100 ) were purchased from cell signaling technology ( boston , ma ) . human embryonic kidney 293 t cells were grown in dulbecco s modified eagle s medium ( dmem ) containing the 293 t cells were adapted to growing in isotope rich - medium supplemented with dialyzed serum prior to initiating these experiments . in each experiment , 20 10-cm dishes were used per condition and the cells were transfected with 15 g of dna using the standard calcium phosphate method ( invitrogen , carlsbad , ca ) . six hours after transfection , the cells were serum - starved for 10 or 20 h. after starvation , the cells were lysed in modified ripa buffer ( 50 mm tris - hcl , ph 7.4 , 150 mm nacl , 1 mm edta , 1% nonidet p-40 , 0.25% sodium deoxycholate , and 1 mm sodium orthovanadate in the presence of protease inhibitors ) . upon cell lysis , proteins lysates were either subjected to affinity purification of tyrosine phosphorylated proteins(13 ) or peptides containing phosphotyrosine were enriched directly from trypsin - digested cell lysates(14 ) using specific antibodies against phosphotyrosine and identified by tandem mass spectrometry . light , medium and heavy cell lysates were precleared with protein a - agarose , mixed , and incubated with 400 g of 4g10 monoclonal antibodies coupled with agarose beads , 75 g of biotin - conjugated rc20 antibody , and streptavidin - agarose beads overnight at 4 c . western blotting experiments were performed using anti - phosphotyrosine antibody ( 4g10 ) and reprobing was carried out using anti - flag antibody . nice-4 protein ( np_055662 ) , rna binding motif protein 10 isoform 1 ( np_05667 ) , far upstream element - binding protein ( np_003893 ) , and trk - fused gene ( np_006061 ) were subcloned into a flag epitope - tagged mammalian expression vector , pcmvtag4a . one dish transfected with wild - type c - src and pcmvtag4a vector as control ; one was cotransfected with wild - type c - src and flag - tagged cdnas . the expressed proteins were immunoprecipitated using anti - flag antibody , followed by sds - page and western blotting . the blots were probed with anti - phosphotyrosine antibody followed by stripping and reprobing with anti - flag antibodies . fusion proteins were made using tnt - coupled rabbit reticulocyte lysate system ( promega , madison , wi ) with the cdnas cloned in gst expression vector , pgex4t1 . the in vitro translated gst - tagged proteins were purified with 10 g of gst beads for 12 h at 4 c . after incubation , the beads were washed two times in lysis buffer and two times in kinase buffer ( 20 mm hepes , ph 7.4 , 5 mm mgcl2 , 2 mm mncl2 , 50 m sodium vanadate , 50 m dtt ) . immune complexes were incubated for 30 min at 30 c in 5 l of atp mixture ( 10 m cold atp and 10 ci of [ -p ] atp ) and c - src kinase . protein samples were then eluted by boiling in sample buffer and resolved by sds - page . the gel was dried and exposed to x - ray film to visualize the p - labeled protein bands . nih3t3 cells were grown in dmem containing 10% fbs supplemented with antibiotics . for all pdgf stimulation experiments , cells were stimulated with 100 ng / ml pdgf - bb for 5 min . for inhibition of c - src kinase , cells were treated with 2 m c - src kinase inhibitor , su6656 , for 1 h prior to stimulation of cells with pdgf - bb for 5 min . the silver - stained protein bands were excised and in - gel trypsin digestion was performed as described previously.(15 ) briefly , the gel slices were excised and incubated with trypsin overnight at 37 c to allow digestion of proteins after a reduction and alkylation step . after in - gel digestion , the tryptic peptides were extracted . the supernatants from the in - gel and in - solution trypsin digestion containing the peptide mixture were partially dried down in a vacufuge to approximately 10 l . for in - solution digestion and enrichment of phosphopeptides , the extracted peptide mixture was centrifuged for 2 min at 12 000 g and 4 c and resolved by reversed - phase liquid chromatography on agilent 1100 series lc system ( agilent technologies , palo alto , ca ) equipped with a well plate sampler , a vacuum degasser , and a capillary pump . each fraction from the digested peptide mixture was analyzed by automated nanoflow lc - ms / ms . an agilent technologies 1100 series system was used to deliver a flow of 1.5 l / min during desalting of the sample and 250 nl / min during elution of the peptides into the mass spectrometer as described before.(10 ) each sample was loaded onto an online analytical fused silica needle column ( proxion biosystems , odense , denmark ) packed with 5-m vydac c18 resin . washing and desalting was done with 95% mobile phase a ( h2o with 0.4% acetic acid and 0.005% heptafluorobutyric ( v / v ) ) and 5% mobile phase b ( 90% acetonitrile , 0.4% acetic acid , 0.005% heptafluorobutyric acid in water ) . samples were eluted from the analytical column by a linear gradient of 90% mobile phase a to 60% mobile phase a. a 34-min gradient was used for elution . the spectra were acquired on a quadrupole time - of - flight mass spectrometer ( q - tof us - api , micromass , manchester , u.k . ) equipped with an ion source sample introduction system designed by proxeon biosystems ( odense , denmark ) . ms to ms / ms switch was set to a threshold of 10 counts / s , and ms / ms to ms was set to an intensity below a threshold of 2 counts / s . scan time was set to 0.9 s , and interscan time was set to 0.1 s. the number of components ( i.e. , number of ms / ms per ms scan ) was set to three resulting in a total cycle time ( one ms and three ms / ms spectra ) of 10 s. the acquisition of data was performed using masslynx ( version 4.0 ) . the parameters used for generating peak lists from the raw data were the following : smooth window , 4.00 ; number of smooth , 2 ; smooth mode , savitzky golay ; and percentage of peak height to calculate centroid spectra , 80% with no baseline subtraction . the generated peak lists ( pkl - file ) were searched against the refseq human protein database ( build 33 , 29 572 sequences ) ( www.ncbi.nlm.nih.gov./refseq/ ) using mascot version 2.0 , with a mass accuracy of 1.1 da for the parent ion ( ms ) and 0.2 for the fragment ions ( ms / ms ) , allowing a maximum of two missed cleavages . carbamidomethylation of cysteines was considered as fixed modification , and oxidations of methionine residues , medium arginine ( + 6 da ) , heavy arginine ( + 10 da ) , and phosphorylation of tyrosine residue were considered as variable modifications . only proteins containing at least one unique peptide ( if the sequence has not been assigned to a different protein ) with a mascot score over 30 were considered in the data set . quantitation was performed on three to four peptides ( wherever available ) by comparing the extracted ion chromatogram of the corresponding light and heavy peptides using ms - quant.(16 ) reproducibility of measurements was performed by using two analysis of variance models as described earlier.(17 ) the tandem mass spectra were manually verified to assign the sequence and phosphorylation sites for all peptides identified in this study ( supplementary table 1 ) . the phosphorylation sites were manually verified and assigned after confirmation of a mass difference of 243 da corresponding to phosphotyrosine residue . the wt ( peptides containing a tyrosine residue in the center ) and mut ( peptides where the central tyrosine residue was replaced by phenylalanine ) peptides ( supplementary table 2 ) were each spotted as triplicates on glass slides ( pepscan systems , lelystad , netherlands ) as described earlier.(18 ) c - src kinase assays were carried out using these custom peptide microarrays by incubating 50 ng of recombinant c - src kinase ( invitrogen , carlsbad , ca ) in kinase reaction buffer and 300 ci / ml p - labeled atp ( ah9968 ; ge healthcare biosciences corp . , piscataway , nj ) at 25 c for 1 h in a 120 l reaction volume supplemented with 200 m atp . the reaction was stopped and the following washing steps were performed : 2 washes in 2 m sodium chloride containing 1% triton x-100 followed by 3 washes in phosphate buffered saline containing triton x-100 and 1 wash in distilled water . the glass slides were then air - dried and exposed to the phosphorimager screen for 12 h and scanned using biorad molecular imager fx ( bio - rad laboratories , inc . , hercules , ca ) . the image was processed using genepix pro 6.0 software ( molecular devices corporation , sunnyvale , ca ) . effects on intensity due to the position of the spot on the slide were estimated by performing a local regression analysis ( loess ) with respect to chip coordinates , and subtracted out.(19 ) normalized log 2 intensities for triplicate spots were averaged , and the mean log 2 mut intensity was subtracted from the corresponding mean log 2 wt intensity for each peptide . the resulting background adjusted values were averaged over replicate arrays . the mean background adjusted log intensity for each peptide on the y - axis , and the average of wt , and mut log intensities on the x - axis were plotted to estimate the distribution of the intensity values arising from the phosphorylated peptides . a key assumption for selection of positive ( phosphorylated ) peptides is that wt peptide intensity values are greater than mut peptide intensities . we note that , consistent with this assumption , the wt intensity is consistently higher than mut intensity for the high intensity peptides on the right side of the plot . likewise , there is greater symmetry on the left side quadrant of the mva plot , where we expect nonphosphorylated peptides to have wt intensities that are as likely to be lower than mut values as higher . the classical false positive rate ( expected error rate for a set of points ) is derived by evaluating symmetry in the y - axis . for each value of a , the classical fpr blue curve gives : ( number of points below the x - axis to the right of a)/(number of points above the x - axis to the right of a ) , and describes the expected number of false positives in the upper right quadrant of the plot . sometimes it is desirable to estimate the probability that a single given point is a false positive , allowing us to move the threshold to the left until the price of adding one more peptide is too high . the local fpr curve gives the probability of a positive peptide located at a being a false positive . we selected all peptides where the local false positive rate was lower than 0.15 and wt intensities were 2-fold greater compared to mut . silac involves metabolic labeling of cellular proteomes by growing the cells in media containing amino acids labeled with stable isotopes . silac enables identification of peptides labeled in vivo and relative quantitation of abundance of the peptides arising out of a mixture of labeled and unlabeled protein samples.(15 ) this method also allows one to distinguish contaminating proteins in immunoprecipitates that arise due to nonspecific binding . because of the complexity of cell lysates , and because kinase substrates generally exhibit low stoichiometry of tyrosine phosphorylation , specific identification of tyrosine kinase substrates can be facilitated by prior enrichment of tyrosine - phosphorylated proteins with anti - phosphotyrosine antibodies . we applied silac for the identification of the c - src kinase substrates in human embryonic kidney cells by overexpression of a constitutively active form of c - src followed by affinity purification of tyrosine - phosphorylated proteins . we transiently overexpressed either a kinase inactive c - src ( k298 m ) as a control or a constitutively active c - src ( y527f ) kinase in 293 t cells . phosphorylation of the c - terminal tyrosine by c - terminal src kinase ( csk ) allows inactivation of c - src . hence , mutation of this tyrosine residue to phenylalanine allows c - src kinase to be constitutively active by preventing its folding and by allowing the kinase domain access to its substrates . inhibition of c - src activity is often achieved by coexpression of the c - src - inactivating c - terminal src kinase ( csk ) , the kinase - inactive src mutant src k298 m,(25 ) or by treatment of the cells with c - src inhibitors . three populations of human embryonic kidney cells were grown in dmem containing c6-arginine ( light ) , c6-arginine ( medium ) and c6-n4-arginine ( heavy ) , respectively ( figure 1 ) . the cells grown in light medium were transfected with a kinase inactive c - src as a negative control.(26 ) cells grown in medium and heavy isotope containing media were transfected with a constitutively active form of c - src ( y527f ) and harvested at 12 or 24 h post - transfection , respectively . we found an increased tyrosine phosphorylation upon transfection of constitutively active form of c - src for 24 h ( figure 2 ) . we have also observed that the y416 in c - src gets phosphorylated more in this state which points to the increased c - src tyrosine kinase activity ( figure 2 ) . in addition , the trend of increasing tyrosine phosphorylation could also serve as one more surrogate signature of substrates as one would expect the phosphorylation level to increase during this time course . ( a ) schematic for the integrated proteomic approach for the identification of c - src kinase substrates . human embryonic kidney ( hek ) 293 t cells growing in arg 0 containing medium were transiently transfected with a kinase - dead src ( k298 m ) and 293 t cells growing in arg 6 and arg 10 were transiently transfected with constitutively active src kinase ( y527f ) . 10 refers to c6-n4-arginine , isotopic labeled forms of arginine used to differentially label 293 t cells for identification of src substrates . tyrosine phosphorylation profile of proteins on transfection with inactive and active forms of c - src . 293 t cells were transfected with inactive and active forms of c - src , cells were lysed , and tyrosine - phosphorylated proteins were immunoprecipitated from the cell lysates as described in . cell lysates and immunoprecipitates were then run on a 10% sds - page and transferred to nitrocellulose membranes . the membranes were probed with anti - phosphotyrosine antibodies and reprobed with phospho ( y416)-src antibody . mixing of light , medium and heavy isotope labeled cell lysates allowed us to compare the profile of proteins in a single ms experiment . in ms / ms spectra , fragmentation patterns generated by light , medium and heavy peptide pairs are identical except for the expected mass shift of the fragment ions . the ratio of the intensity of the heavier versus the light peptides provides information about the degree of phosphorylation of a protein and hence its enrichment upon expression of an active c - src kinase . thus , the greater the extent of phosphorylation of a protein by c - src kinase , the higher should be its abundance in anti - phosphotyrosine antibody immunoprecipitates . peptide sets with a little or no increase in intensity indicate that the protein is not different in abundance in the different states being compared . such proteins were not investigated further as they are likely nonspecifically bound proteins . an increase in heavy / light intensity ratio , indicating an increase in total phosphotyrosine content upon src kinase expression and activity , was found in peptides derived from 36 proteins ( tables 1 and 2 ) . of these , 10 proteins were either known src family kinase substrates or proteins known to interact with src family kinases ( table 1 ) , whereas the remaining 26 proteins have not previously been described as substrates of c - src or src family members ( table 2 ) in higher eukaryotes . the known substrates identified in this screen included ews1 ( ewing sarcoma breakpoint region 1),(27 ) cortactin,(28 ) calponin-3,(29 ) hnrnp - k ( heterogeneous nuclear ribonucleoprotein k ) , g3bp ( rasgap sh3-domain binding protein ) and c - src itself.(35 ) the protein with maximum increase in tyrosine phosphorylation upon c - src overexpression was c - src itself . other known and novel src family substrates displayed > 2-fold increase in intensity of phosphorylation . apart from signaling and cytoskeletal proteins , we also identified dna and rna binding proteins in our analysis . some of the reasons it is not possible in a kinasesubstrate identification screen of this type to possibly identify every known substrate are ( i ) previously described substrates might not be expressed in the cell line that we have used ; ( ii ) although the substrates might be expressed , they might not be abundant enough to be enriched and detected in our experiments ; ( iii ) the phosphorylation might not occur or occur at a lower level in the cells that we have used ; and ( iv ) the time course and kinetics of phosphorylation in the system that we have employed might be different from the systems previously used in the literature to describe substrates . thus , although it is not possible to identify all of the known substrates of src , identification of 6 known substrates of src along with validation of some novel ones is indicative of the success of this type of phosphoproteomic screen . heavy refers to arg 10 and light refers to arg 0 containing peptides . heavy refers to arg 10 and light refers to arg 0 containing peptides . we performed relative quantitation of tyrosine phosphorylation for each protein as a measure of increase in intensity ratios from light isotope to heavier isotope containing peptides ( supplementary table 1 ) . figure 3 shows representative ms spectra for six of the proteins identified from our screen . in addition to identification of proteins with increased phosphorylation and quantitation , we also mapped 6 phosphorylation sites ; y334 in cortactin(10 ) ( figure 4a ) and y72 in hnrnp - k(30 ) ( figure 4b ) identified in this study were reported earlier . we also identified 4 novel tyrosine phosphorylation sites on each on ews1 ( figure 4c ) , fuse - binding protein 1 ( figure 4d ) , calponin-3 ( figure 4e ) and fip1-like1 ( figure 4f ) . we note that , although ews1 was a known c - src substrate , no tyrosine phosphorylation sites were previously localized . the 3 spectral peaks in each figure represent the mass shift of the same peptide . the relative increase in intensity ratios between light to heavy ( a ) a doubly charged peptide from cortactin ( 1:7 ) ; ( b ) a triply charged peptide from nice-4 ( 1:7 ) ; ( c ) a doubly charged peptide from bcl2-associated transcription factor ( 1:7.5 ) ; ( d ) a doubly charged peptide from fuse - binding protein 1 ( 1:8 ) ; ( e ) a doubly charged peptide from fuse - binding protein 2 ( 1:4 ) ; ( f ) a doubly charged peptide from rna binding motif 10 ( 1:7.5 ) . ms / ms spectra of novel phosphorylation sites identified in this study . ( a ) phosphopeptide nastfedvtqvssapyqk derived from cortactin ; ( b ) phosphopeptide tdpynasvsvpdssgper derived from hnrnpk ; ( c ) phosphopeptide qdhpssmgvpygqesggfsgpgenr derived from ewing sarcoma breakpoint region 1 ; ( d ) phosphopeptide iggdagtslnsndpygyggqk derived from fuse - binding protein 1 ; ( e ) phosphopeptide gpspyglsaevk derived from calponin-3 ; ( f ) phosphopeptide tgapqpygsygtapvnlnik derived from fip1-like 1 . one of the drawbacks of our experiments is that lysine was not available as 3 different isotopic forms at the time we initiated our experiments . by using 3 isotopes of lysine along with arginine , we would have obtained a better peptide coverage for each protein and likely identified additional proteins as substrates of c - src . nevertheless , we performed a lysine and arginine labeled silac experiment in a similar manner but followed by enrichment of phosphopeptides using antibodies against phosphotyrosine to see if we could identify phosphorylated peptides , but unfortunately , we could only identify 8 tyrosine phosphorylation sites ( data not shown ) . however , a protein ip serves our purpose of identifying the proteins that are substrates of src even though the site is still not identified . this is the reason we coupled our approach with peptide microarrays to aid in identification of phosphopeptides . further validation of the proteins identified by silac to prove that they are bona fide substrates usually involves the use of antibodies against these proteins . the validation of all of the protein candidates is not always possible , especially for novel proteins , as it depends on the availability of good antibodies . as commercial antibodies were not available for many of the proteins identified , we chose to investigate a subset of proteins , if they were direct substrates of c - src using in vitro kinase assays . we selected nice-4 , rbm10 , fbp1 and trk - fused gene for this purpose , as their cdnas were readily available . these proteins were purified and incubated along with c - src kinase to investigate if it could phosphorylate these proteins . after incubation with c - src kinase , the proteins were resolved by sds - page and autoradiographs were obtained . we found that all of the tested proteins were tyrosine phosphorylated upon incubation with c - src kinase ( figure 5a ) . trk - fused gene from xenopus laevis has been shown to interact with sh3 domains of various proteins including v - src but did not bind to neuronal specific src in vitro.(36 ) experimental validation of tyrosine phosphorylation of proteins obtained from c - src kinase overexpression in 293 t cells . ( a ) in vitro kinase assays using gst tagged proteins and c - src using a rabbit reticulocyte in vitro transcription and translation system . ( b ) 293 t cells were cotransfected with genes of interest along with either empty vector pcmvtag4a or with c - src . culture media was changed 12 h after transfection and cells were serum - starved for 12 h and lysed 48 h after transfection . proteins were immunoprecipitated using anti - flag antibodies and western blotting was performed using phosphotyrosine antibodies and reprobed . nih3t3 cells have been grown to confluence and serum - starved for 12 h followed by stimulation with pdgf - bb ( 100 ng / ml for 5 min ) and pdgf stimulation after treatment with su6656 ( 2 m for 1 h prior to lysis or stimulation ) , and cell lysates were subjected to immunoprecipitation using anti - phosphotyrosine antibodies , probed with respective antibodies , and reprobed in whole cell lysates . we verified if the above c - src substrates were also substrates of c - src in vivo by cotransfecting these proteins with wild - type c - src in 293 t cells . we also subcloned these cdnas into a flag epitope - tagged vector , pcmvtag4a , and cotransfected 293 t cells with wild - type c - src kinase or with an empty vector . the proteins were immunoprecipitated using anti - flag antibodies , resolved by sds - page , and immunoblotted with anti - phosphotyrosine antibodies . upon cotransfection with c - src kinase , we again observed increased tyrosine phosphorylation of all the tested proteins suggesting that these proteins were also in vivo substrates of c - src ( figure 5b ) . we have previously identified nice-4 as a tyrosine - phosphorylated protein in a global phosphoproteomic study of hela cells.(10 ) rbm10 , fbp1 and trk - fused gene are novel tyrosine - phosphorylated proteins and further investigations need to be carried out to determine how they transduce signals downstream of c - src kinase and if tyrosine phosphorylation regulates this process . since kinase activity of c - src is required for modulating cellular responses to pdgf receptor stimulation,(37 ) we chose to study the role of a subset of novel substrates in pdgf signaling . the role of c - src has been well - studied in pdgf signaling.(38 ) we investigated the involvement of ews1 , btf and rbm10 in pdgf receptor signaling . cortactin and p130cas were used as positive controls . for this experiment , nih3t3 cells , which express endogenous pdgf receptors , were treated with pdgf - bb in the presence or absence of a c - src kinase inhibitor , su6656 ( 2-oxo-3-(4,5,6,7-tetrahydro-1 h - indol-2-ylmethylene)-2,3-dihydro-1h - indole-5-sulfonic acid dimethylamide).(38 ) by activating pdgf signaling in nih3t3 cells , we investigated the ability of these proteins to get tyrosine - phosphorylated upon ligand - induced stimulation of the pdgf receptor . we found that all of the novel proteins were tyrosine - phosphorylated upon stimulation of pdgf receptor ( figure 5c ) , as was the case with the two known substrates . using su6656 , a potent inhibitor of c - src kinase , we have shown the involvement of three novel proteins rbm10 , ews1 and btf as c - src substrates in pdgf signaling ( figure 5c ) . c - src has been shown to have been involved in the regulation of nuclear proteins and transcription factors downstream of pdgf signaling and plays an important role in controlling dna synthesis.(39 ) we have also examined the involvement of rasgap sh3-domain binding protein and thyroid hormone receptor associated protein 3 in pdgf signaling using su6656 but could not detect any tyrosine phosphorylation of these proteins in any state ( data not shown ) . further experiments need to be done in order to examine the precise role of these proteins and the importance of their phosphorylation in pdgf signaling . one important caveat of these experiments using su6656 for inhibition of src kinase is that it could bind and inhibit other related kinases . when better inhibitors become available , this would be a great approach to identify the kinasesubstrate relationships . ewing sarcoma breakpoint region 1 ( ews1 ) is an rna binding protein and has been shown to be involved in gene translocations and often appear as fusion of ews with ets family transcription factor genes and known to cause ewing sarcoma tumors.(40 ) one of the fusion proteins ews - fli1 has been implicated in insulin - like growth factor 1 ( igf-1)(41 ) as well as pdgf - bb(42 ) induced proliferation of ewing sarcoma cells . the implication of ews1 as a downstream substrate of c - src in pdgf - signaling might shed light into the mechanism of induction of proliferation by these genes in signaling and tumors . bcl-2-associated transcription factor 1 ( btf ) is an apoptotic transcriptional repressor(43 ) localized to the nucleus , whose function is still under investigation . rna binding motif 10 ( rbm10 ) is an rna binding protein with a zinc finger domain , associated with the expression of bax family members in breast cancers and vegf.(44 ) this was the first time btf and rbm10 were identified as a tyrosine - phosphorylated proteins and the finding that they are components of pdgf signaling downstream of c - src might help to understand the role of btf and rbm10 in growth factor receptor signaling . although we have observed tyrosine phosphorylation of a subset of proteins in pdgf signaling , it is not possible to validate all candidates in any proteomics experiment because of the following reasons : ( i ) availability of good immunoprecipitating antibodies against these proteins is limited , and hence , we have tagged a subset of these proteins to show that these are indeed substrates ; and ( ii ) to validate these proteins in a specific signaling pathway , it is not easy to predict in which signaling pathway(s ) these proteins are involved downstream of c - src . however , studies are currently in progress on a subset of proteins to show their physiological relevance and also to identify tyrosine - phosphorylated proteins in growth factor signaling pathways downstream of src kinase . although we have established above that a number of novel proteins are potential substrates of c - src , the exact residues that undergo phosphorylation have not been identified in most of these instances . hence , we developed a custom peptide microarray as a platform to rapidly identify the phosphopeptides on these proteins which are phosphorylated by c - src . we systematically designed 312 peptides encompassing all tyrosines from 14 selected proteins ( table 3 ) . these were synthesized in such a fashion that they contained the tyrosine residue being tested in the center . in parallel , an equal number of peptides that have the centric tyrosine residues mutated to phenylalanine were designed . in all , 624 wt or mutant ( 312 wt and 312 mut ) peptides from 14 proteins were spotted with each sequence being represented in triplicate , on to the glass slides as described earlier.(18 ) the design of these peptide microarrays is analogous to dna microarrays manufactured by affymetrix for mrna expression studies . c - src kinase assays were performed on the peptide microarrays and the arrays subsequently exposed to phosphorimager screen ( figure 6a ) . we normalized the intensity values and compared the log 2 intensities of the wt peptides against their corresponding mut peptides . the intensity values from 3 different experiments were averaged individually for wt and mut peptides and plotted ( figure 5b ) . the spots in the upper right quadrant were taken as true positives ( figure 6b ) . the false positive rates ( fpr ) were calculated by assuming that those peptides for which mut intensity exceeded wt intensity were not phosphorylated , and that the variation of wt - mut intensities for those points was representative of nonphosphorylated peptides ( figure 6c ) . on the basis of the calculated fprs , a line was drawn which separates the true positives from the remainder of the peptides . ( a ) peptide microarrays : in vitro kinase assays performed on peptide microarrays , where all tyrosine containing peptides and their corresponding y f mutant counterparts were spotted on glass slides . a representative section of the peptide microarray is magnified to show the signal corresponding to a peptide and its y f mutant . ( b ) a classical mva plot displaying data pertaining phosphorylation intensities on peptide microarrays . m on the y - axis represents differential of ( log 2 wt log 2 mut intensity values for each peptide and a on the x - axis represents average intensities ( [ log 2 wt + log 2 mut]/2 ) . ( c ) a plot displaying classical and local false positive rates ( fpr ) . the red line represents local false positive rate curve and the blue line represents the classical false positive rate . the vertical dotted line shows where the local fpr = 0.15 . all peptides to the right of the vertical dotted line and above 2-fold ( horizontal line ) were selected as true positives . from this analysis , we have identified phosphorylation sites on 12 out of 14 proteins that were spotted on peptide microarrays . peptides containing multiple tyrosines were mutated systematically and all the tyrosine containing peptides were looked at along with their corresponding mutated counterparts to deduce the correct phosphorylated peptide . a total of 34 peptides from 12 proteins ( table 3 ) out of 312 peptides from 14 proteins spotted ( supplementary table 2 ) were phosphorylated by c - src in our analysis . this included 6 phosphopeptides from bcl2-associated transcription factor , 5 from thyroid hormone receptor associated protein 3 , and 4 phosphopeptides from ars2 . we did not detect phosphorylation on any of the peptides derived from two proteins , zinc finger , cchc domain containing 8 and splicing factor proline / glutamine - rich . thus , peptide arrays allowed assignment of phosphorylation sites in a high - throughput fashion and also served as an additional validation step for potential substrates identified from our silac experiments . because of the tremendous potential and promise that peptide microarrays hold for the identification kinasesubstrate identification , studies are ongoing to spot the peptides from other proteins identified in this and other mass spectrometry based studies to identify the kinase - specific phosphorylation sites and build phosphorylation motifs . this study describes identification of substrates of a nonreceptor tyrosine kinase using a combination of proteomic approaches . use of silac methodology allowed us to identify a number of known and novel substrates of c - src in human embryonic kidney 293 t cells . we identified 4 new phosphorylation sites and also validated a subset of the novel substrates as direct c - src substrates using in vitro kinase assays . we also implicated three of the novel c - src substrates as tyrosine - phosphorylated proteins in pdgf receptor signaling . since identification of phosphopeptides is still a challenge in proteomics , we designed a custom peptide microarray platform for high - throughput identification of peptides phosphorylated by specific kinase , c - src , in this case . using peptide microarrays , we identified 34 phosphopeptides phosphorylated by c - src that are derived from 12 novel candidate substrate proteins that were identified by silac as potential c - src substrates . identification of the phosphopeptides from these 12 new substrates also provides validation of this approach . it is worth noting here that most tyrosine containing peptides were not phosphorylated by c - src . although it can help identify bona fide substrate peptides in many instances , it is possible that some sequences that are phosphorylated in vivo do not get phosphorylated because of lack of secondary and tertiary structure of the immobilized peptides . further , although the peptide microarray analysis has shed light on the phosphopeptides preferentially phosphorylated by c - src , these sites still remain to be investigated in vivo using other methodologies . the present study offers many encouraging leads , such as the identification of tyrosine phosphorylation of a subset of new c - src substrates that are components in the pdgf signaling downstream of c - src . clearly , further characterization of these novel sites and proteins will result in a significant expansion of our knowledge of the c - src kinase signaling network . the significance of tyrosine phosphorylation on each of the newly discovered sites remains to be determined . in any case , these results demonstrate that c - src - mediated tyrosine phosphorylation is extensive and implicates a number of hitherto unrecognized proteins as c - src kinase substrates .

worsening chronic heart failure ( chf ) is largely characterised by disabling symptoms , poor quality of life , frequent hospital admissions and need of specialist care . in a pilot study evaluate home care ( hc ) versus conventional care ( cc ) in relation to medical safety , health - related quality of life ( hrql ) and cost - utility in patients with worsening chf . thirty - one patients with deteriorating chf were randomised to hc or cc when seeking medical attention at hospital . patients in the hc group were discharged from the hospital and were followed - up in their homes by a specialist nurse . follow - ups were conducted for both groups , 1 , 4 , 8 and 12 months after inclusion in the study . health - related quality of life assessed by euroqol-5d vas , standard gamble technique , sf-36 and kansas city cardiomyopathy questionnaire . all health care related costs were assessed and cost utility analysis was performed to compare cost / qalys between groups . the total cost related to chf was lower in the hc group after 12 months . median direct health care related costs in hc were 1122 and in cc 5670 ( p 0.05 ) . cost / qalys ranged 74580 in hc compared to cc 2891013 , calculated from each follow - up . the cost utility ratio was ( cc / hc ) 2.55 ( sg ) and 2.65 ( vas ) . reductions in cost of care for selected patients with chf eligible for hospital care might be achieved by a very early discharge from hospital followed by home visits . more importantly , hc seems to be safe and no difference was found in hrql between two groups . this pilot study provides clinicians with useful information in their decisions concerning chf patient management , who are reluctant to hospitalisation .

diabetes mellitus is a universal health problem and may occur at any age . the traditional biochemical measurements for initially detecting patients with diabetes mellitus are random estimations of blood and urine glucose concentration . despite their common use , both tests are nonspecific , being influenced by a wide variety of drugs and conditions . measurement of the glycosylated hemoglobin concentration in random blood specimens is a simpler screening test for diabetes and has the advantage that it reflects blood glucose control under physiologic conditions . and it is an accepted method for assessing long - term diabetic control . since the measurement of fructosamine as an index of diabetic control , jerntorp et al . and other investigators have reported that fructosamine concentrations were correlated with hbalc and other measures of glycemia , and fructosamine appeared more useful than hbalc for monitoring short - term changes of glycemia . the present study was undertaken to determine the clinical usefulness of fructosamine estimations in monitoring short - term glycemic control during the management of patients with niddm . serum fructosamine , fasting plasma glucose and hbalc were measured in 50 normal controls and 36 patients with niddm . we studied four men and six women selected at random among the 36 patients with niddm , aged 2173 years ( median 47 ) , who had been admitted to our hospital from nov . three patients have been associated with peripheral neuropathy and four patients had retinopathy ( table 1 ) . references for serum fructosamine , fasting plasma glucose and hbalc values were determined in 50 non - diabetic volunteers aged 2065 years ( median 43 ) . diabetes was excluded from this reference population using the fasting plasma glucose , postprandial 2 hour glucose and urinary glucose results . the patients studied were on a diet regimen and had taken oral hypoglycemic agents or insulin injection . in these patients , fasting plasma glucose , serum fructosamine and hbalc were measured on admission and at biweekly intervals to 8 weeks . fructosamine was measured using an abbott vp automated analyzer by colourimetric test based on the ability of ketoamines to reduce nitroblue tetrazolium in alkaline medium ( fructosamine test , roche ) . hbalc was measured by a mini - column method using a perkin - elmer junior model 35 spectrophotometer . fasting plasma glucose concentrations in the same subjects were 70109 mg / dl ( mean 90 mg / dl ) and hbalc values were 4.46.4% ( mean 5.3% ) , which are compatible with normal ranges of other laboratory criteria . fasting plasma glucose concentrations in patients with niddm were 174374 mg / dl ( mean 235 mg / dl ) . the levels of serum fructosamine and hbalc were significantly higher in niddm patients ( 2.44.5 mmol / l : mean 3.27 mmol / l , 7.212.5% : mean 8.8% , respectively , p<0.005 ) compared with those in the normal controls ( table 2 ) . the correlation of serum fructosamine to fasting plasma glucose and to hbalc was examined in normal controls and diabetic patients . correlation coefficients were 0.78 and 0.76 respectively and p values indicate statistical significance ( p<0.001 , p<0.005 respectively ) ( table 3 , fig . also , in ten patients studied , retrograde assessment for the correlation of serum fructosamine to fasting plasma glucose was periodically performed after management of diabetes for 8 weeks . the serum fructosamine concentration had a significant correlation to the fasting plasma glucose level determined 2 weeks before ( r = 0.72 , p<0.002 ) and 4 weeks before ( r = 0.54 , p<0.005 ) , but there were no significant correlations to the fasting plasma glucose levels 6 weeks before ( r = 0.16 , ns ) and 8 weeks before ( r = 0.18 , ns ) ( table 4 ) . we studied the changes and regression rates of fasting plasma glucose , serum fructosamine and hbalc during the management of diabetes for 8 weeks . ii indicates that the change of serum fructosamine shows a sharper downward slope than that of hbalc until 6 weeks ( fig . the regression rates of serum fructosamine increased more than those of hbalc at 2 weeks , 4 weeks and 6 weeks ( 17.5% , 22% and 21% ; 10% , 9.8% and 16% , respectively ) . that is to say , until 6 weeks conversely , at 8 weeks , hbalc decreased more sensitively than serum fructosamine ( table 5 , fig . while the value of plasma glucose measurement at standard times throughout the day is established as a guide to modifying treatment , an accurate measure of mean blood glucose concentrations over several days or weeks would prove extremely useful in judging overall diabetic control and the response to changes in management . fructosamine provides such a measurement , reflecting mainly the basal or fasting plasma glucose concentration with little contribution from transient postprandial hyperglycemia . measurement of the glycosylated hemoglobin concentration is an accepted method for assessing long - term diabetic control , many studies reported that fructosamine , as an index of short - term diabetic control , correlated with hbalc and other measures of glycemia and appeared more useful than hbalc for monitoring short - term changes . other previous reports showed that the fasting plasma glucose concentration varies in response to stress or exercise , urinary glucose is modified by the renal threshold , the hbalc value depends on red cell turnover and fructosamine is influenced by alterations in serum protein metabolism . in this study , all patients had normal liver and renal function and normal hematologic profiles . in controls , serum fructosamine and hbalc levels were significantly higher in niddm patients compared with those in normal controls and serum fructosamine was significantly correlated with fasting plasma glucose and hbalc . also , in retrograde study , serum fructosamine concentration had a significant correlation to fasting plasma glucose determined 2 weeks before and 4 weeks before . in patients , hyperglycemia was well controlled with improved symptoms , without newly developed complications including hypoglycemia . during the management of diabetes for 8 weeks conversely , at 8 weeks , hbalc decreased more sensitively than serum fructosamine . with these results , we believe that serum fructosamine and hbalc measurement may be helpful in screening for diabetes mellitus , and serum fructosamine measurement may be useful in monitoring short - term control of plasma glucose in patients with niddm . further more , as an ongoing study , we will continue to monitor the serum fructosamine and hbalc levels in patients with niddm .

ocular nocardia infections are usually considered refractory to conventional topical antibiotics , resulting in a protracted clinical course and progressive extension of the disease . if proper therapy is initiated , nocardia keratitis resolves with minimal scarring , with or without vascularization . the main indication for amniotic membrane transplantation ( amt ) is ocular surface reconstruction in persistent epithelial defects such as noninfectious corneal ulcers . this case report describes a patient with nocardia corneal ulcer unresponsive to conventional topical medication that was successfully treated with amt . a 20-year - old man presented to the emergency room with left eye pain from 4 days ago . there were corneal edema and infiltration and an epithelial defect of 2 mm 2 mm with ring - like superficial infiltrate in a wreath pattern ( fig . 1 ) . three days after plating , minute white colonies appeared along the culture streak on the blood agar plate . performed in the nocardia isolates from the patient revealed resistance to gentamicin , ciprofloxacin , and chloramphenicol , but sensitivity to amikacin . sulfamethoxazole , and amikacin did not control the infection , and the keratitis progressed with epithelial defect , which extended to temporal limbus after 1 month ( fig . 2 ) . persistent epithelial defect ( ped ) with new zone of keratitis and vascularization occurred after 2 months ( fig . the entire cornea , including the limbus , was covered with one layer of amniotic membrane , epithelial side - down , as a patch . after 1 week , there was some pain relief , and as soon as the membrane was largely dissolved , we removed the nylon sutures and looked for ped and persistent irregular epithelium . keratitis decreased and ped resolved , but deep vascularization did not decrease , and corneal scar persisted ( fig . 4 ) . 4decreased keratitis after amniotic membrane transplantation progressive keratitis after 1 month new zone of keratitis after 2 months decreased keratitis after amniotic membrane transplantation the typical clinical picture described by various authors in nocardia keratitis is a well - defined epithelial defect with scalloped margins . have inferred that the corneal nocardiosis like our case is longer lasting , slowly progressive , and may present with ped . however , srinivasan and sharma have described four patients of nocardia keratitis healing in 710 days with topical 10% sodium sulphacetamide and ampicillin trihydrate . in this case , our patient presented with non - healing keratitis with very prolonged clinical course , so we use adjuvant amt . the epithelial healing observed in our patient with amt is consistent with reports for other corneal diseases . the amniotic membrane acts as a substrate for epithelial growth in vivo in case of ped . a similar effect , with the promotion of epithelial growth , has been shown in vitro . it has also been reported that the amniotic basement membrane facilitates the migration of epithelial cells , strengthens the adhesion of basal epithelial cells , and promotes epithelial differentiation . in this case , we covered the entire surface of the cornea with the amniotic membrane to optimize the limbal and corneal epithelial cell microenvironments . the reservoir effect of drug absorption , and the bandage properties are shared with amt by hydrogel contact lenses . in addition , amt has anti - inflammatory or antiscarring capability , although vascularization and scaring did not decrease in this case . in conclusion , this case report for the first time indicates that amt associated with topical amikacin application is safe for the treatment of severe nocardia keratitis .

marathon runners and other endurance athletes often compete in hot , humid environments throughout the spring , summer , and fall months . at the end of most endurance events each finisher is provided with food , water or other recovery fluids ( carbohydrate - electrolyte solution , chocolate milk , etc . ) , and a reflective blanket ( rb ) . although some of these items are scientifically sound choices , several recovery trends are not steeped in scientific evidence , one of which includes rbs . the intended use of the blankets is to retain the metabolic heat produced during running , but often they are used regardless of environmental temperature . the manufacturers of the mcr medical thermal blankets state the main benefit of the 87 59 blankets is to retain up to 90% of the body s heat ( 1 ) , and in cold - environments may be incredibly effective . however , these rbs are being used post - race despite the ambient temperatures reaching 100f throughout the racing season . when running , the core body temperature ( cbt ) naturally increases due to metabolic heat ( 3 ) . upon completion , we anticipate cbt decreases , simply due to a chance in intensity ( 3 ) , but some external factors ( clothing , rbs , etc . ) rbs seem to be counterintuitive to the need to return to baseline cbt , especially when used in hot , humid environments . if body heat is retained by a rb or additional clothing , the human body will have difficulty dissipating heat and returning to normal , resting cbt . when cbt remains elevated despite ending exercise , undesired stress is placed on the cardiovascular system as the body continues to use its cooling mechanisms , against resistance . the purpose of this study was to evaluate the effect of rbs on cooling rate . we hypothesized participants using the blankets would have a slower cbt cooling rate than the participants who did not use the blankets . after receiving irb approval , we recruited 19 healthy , endurance - trained individuals . participants included 14 males and 5 females ( age=255y ; mass=76.816.7 kg ; height=1779 cm ) who ran a minimum 10 mi / wk for at least the last three months . exclusion criteria included history of stroke , cardiovascular disease , recent incident of exertional heat stroke ( within 6 months ) , diabetes , recent history of lower extremity injury / surgery ( within 6 months ) , smoking , or taking medications that alter heart rate . we used a pilot , randomized control experimental design to observe the effect of rbs on cbt cooling rate . we used four conditions : no blanket , blanket , recovery walking with blanket , and recovery walking with no blanket . because some participants rarely sit , unless unable to ambulate , immediately following a race , we attempted to replicate the activities post - race . those walking on the treadmill were instructed to casually walk during the recovery period ( at no elevation ) . participants completed a health history questionnaire during the pre - screening session on the study prior to participation in the protocol . the participants completed the questionnaire to exclude anyone who may not have met the inclusion criteria and to gather demographic data . during the pre - screening session , we measured height in centimeters using a standard wall mounted tape measure ( novel products ; rockton , il ) , body mass using a standard physician scale ( transcell ti 500e ; koenig scale , terre haute , in ) , resting heart rate and blood pressure using an automatic sphygmomanometer ( hem- 780 , omron ; bannockburn , il ) . during the pre - screening session , we scheduled the participant for the exercise session . within 24 hours of the exercise session , we met with the participant to ensure they were in good health and provided them with the ingestible thermistor ( jonah capsule , minimitter company , inc ; bend , or ) . we randomly assigned participants into one of four groups for the cool down protocol : walk with blanket ( wb ) ( n=5 ) , sit with blanket ( sb ) ( n=5 ) , walk without blanket ( wnb ) ( n=5 ) , sit without blanket ( snb ) ( n=5 ) . on the day of the exercise session , participants reported to the environmental heat chamber in shorts , t - shirt , and athletic shoes . a self - selected warm - up was allowed for as long as desired prior to the official start of data collection . to monitor and maintain consistency between exercise sessions , we measured fluid consumed , environmental conditions , distance , heart rate , and rating of perceived exertion . after the entire protocol was complete , we measured the volume of the fluid ( ml ) consumed by using a metered water bottle ( mean=850270ml ; no significant differences between groups f3,15=1.497 , p=0.256 ; range=7001040ml ) . we measured wet bulb , dry bulb , and wet bulb globe temperature as well as relative humidity in the environmental heat chamber ( which includes radiant heat lamps to replicate sunshine ) . we measured environmental variables using an area heat stress monitor ( hs-32 metrosonics , quest technology ; oconomowoc , wi ) . we maintained a stable hot , humid environment between conditions ( wet bulb=22.841.28c ; dry bulb=29.851.35c ; wet bulb globe temperature=26.182.78c ; relative humidity=43.5711.15% ) comparable to similar studies in - vivo and in - vitro ( 4 , 5 ) . to monitor cbt , participants swallowed a small capsule , the jonah ingestible core temperature capsule , 58 hours before activity . the capsule measures cbt and transmits a signal to the vitalsense monitor ( minimitter company , inc ; bend , or ) . we used the capsule and monitor to gather data in real - time and we measured cbt throughout the data collection session . while on the treadmill , we monitored distance with the intention that all participants complete an 8 km run ( actual distance mean=7.51.1 km ) . due to some irb limitations , we asked that participants control their own intensity and for as long as they could without experiencing signs and symptoms of exertional heat stroke . because of the environmental conditions , some individuals were unable to complete the 8 km run , but achieved an elevated cbt . we used a heart rate ( hr ) monitor ( polar fti ; lake success , ny ) to measure the participants hr and we used the borg scale ( 620 ) to rate the perceived exertion ( rpe ) to monitor perceived intensity . as participants were running , we monitored cbt to ensure it reached the desired temperature of 103.5104f ( 39.2240c ) and to avoid risk of exertional heat illness ( > 104f ) . the cooling protocol began once the participants reached the target temperature , completed the 8 km run , or until they expressed a desire to stop running . we measured the athletes cbt , rpe , and hr every three minutes during the exercise protocol until the cbt reached 39c . , at which time we measured these three factors every minute for the remaining duration of the exercise protocol . if a participant was in the wb or sb groups , we immediately placed a rb over their shoulders when they finished running . the cooling data was recorded for a total of 62 minutes following the completion of the running protocol . cbt and hr measurements were taken every minute for the first 20 minutes of the protocol to ensure participant safety , and were then measured every three minutes for the remainder of the cooling protocol . at the conclusion , we calculated the rate of cooling ( 62 minutes used due to the 3 min intervals ) . we used separate kruskal - wallis non - parametric one - way anovas ( which is reported as a ) to evaluate peak cbt , hr , rpe , running pace , and environmental variables for each of the four conditions . because of the small sample size , we used the kruskal - wallis to compare the cooling rates in each condition . significance was achieved at p<0.05 and analysis was performed using spss for windows ( version 20 ) . after receiving irb approval , we recruited 19 healthy , endurance - trained individuals . participants included 14 males and 5 females ( age=255y ; mass=76.816.7 kg ; height=1779 cm ) who ran a minimum 10 mi / wk for at least the last three months . exclusion criteria included history of stroke , cardiovascular disease , recent incident of exertional heat stroke ( within 6 months ) , diabetes , recent history of lower extremity injury / surgery ( within 6 months ) , smoking , or taking medications that alter heart rate . we used a pilot , randomized control experimental design to observe the effect of rbs on cbt cooling rate . we used four conditions : no blanket , blanket , recovery walking with blanket , and recovery walking with no blanket . because some participants rarely sit , unless unable to ambulate , immediately following a race , we attempted to replicate the activities post - race . those walking on the treadmill were instructed to casually walk during the recovery period ( at no elevation ) . participants completed a health history questionnaire during the pre - screening session on the study prior to participation in the protocol . the participants completed the questionnaire to exclude anyone who may not have met the inclusion criteria and to gather demographic data . during the pre - screening session , we measured height in centimeters using a standard wall mounted tape measure ( novel products ; rockton , il ) , body mass using a standard physician scale ( transcell ti 500e ; koenig scale , terre haute , in ) , resting heart rate and blood pressure using an automatic sphygmomanometer ( hem- 780 , omron ; bannockburn , il ) . during the pre - screening session , we scheduled the participant for the exercise session . within 24 hours of the exercise session , we met with the participant to ensure they were in good health and provided them with the ingestible thermistor ( jonah capsule , minimitter company , inc ; bend , or ) . we randomly assigned participants into one of four groups for the cool down protocol : walk with blanket ( wb ) ( n=5 ) , sit with blanket ( sb ) ( n=5 ) , walk without blanket ( wnb ) ( n=5 ) , sit without blanket ( snb ) ( n=5 ) . on the day of the exercise session , participants reported to the environmental heat chamber in shorts , t - shirt , and athletic shoes . a self - selected warm - up was allowed for as long as desired prior to the official start of data collection . to monitor and maintain consistency between exercise sessions , we measured fluid consumed , environmental conditions , distance , heart rate , and rating of perceived exertion . after the entire protocol was complete , we measured the volume of the fluid ( ml ) consumed by using a metered water bottle ( mean=850270ml ; no significant differences between groups f3,15=1.497 , p=0.256 ; range=7001040ml ) . we measured wet bulb , dry bulb , and wet bulb globe temperature as well as relative humidity in the environmental heat chamber ( which includes radiant heat lamps to replicate sunshine ) . we measured environmental variables using an area heat stress monitor ( hs-32 metrosonics , quest technology ; oconomowoc , wi ) . we maintained a stable hot , humid environment between conditions ( wet bulb=22.841.28c ; dry bulb=29.851.35c ; wet bulb globe temperature=26.182.78c ; relative humidity=43.5711.15% ) comparable to similar studies in - vivo and in - vitro ( 4 , 5 ) . to monitor cbt , participants swallowed a small capsule , the jonah ingestible core temperature capsule , 58 hours before activity . the capsule measures cbt and transmits a signal to the vitalsense monitor ( minimitter company , inc ; bend , or ) . we used the capsule and monitor to gather data in real - time and we measured cbt throughout the data collection session . while on the treadmill , we monitored distance with the intention that all participants complete an 8 km run ( actual distance mean=7.51.1 km ) . due to some irb limitations , we asked that participants control their own intensity and for as long as they could without experiencing signs and symptoms of exertional heat stroke . because of the environmental conditions , some individuals were unable to complete the 8 km run , but achieved an elevated cbt . we used a heart rate ( hr ) monitor ( polar fti ; lake success , ny ) to measure the participants hr and we used the borg scale ( 620 ) to rate the perceived exertion ( rpe ) to monitor perceived intensity . as participants were running , we monitored cbt to ensure it reached the desired temperature of 103.5104f ( 39.2240c ) and to avoid risk of exertional heat illness ( > 104f ) . the cooling protocol began once the participants reached the target temperature , completed the 8 km run , or until they expressed a desire to stop running . we measured the athletes cbt , rpe , and hr every three minutes during the exercise protocol until the cbt reached 39c . , at which time we measured these three factors every minute for the remaining duration of the exercise protocol . if a participant was in the wb or sb groups , we immediately placed a rb over their shoulders when they finished running . the cooling data was recorded for a total of 62 minutes following the completion of the running protocol . cbt and hr measurements were taken every minute for the first 20 minutes of the protocol to ensure participant safety , and were then measured every three minutes for the remainder of the cooling protocol . at the conclusion , we calculated the rate of cooling ( 62 minutes used due to the 3 min intervals ) . we used separate kruskal - wallis non - parametric one - way anovas ( which is reported as a ) to evaluate peak cbt , hr , rpe , running pace , and environmental variables for each of the four conditions . because of the small sample size , we used the kruskal - wallis to compare the cooling rates in each condition . significance was achieved at p<0.05 and analysis was performed using spss for windows ( version 20 ) . participants completed the exercise sessions with similar intensity and under similar conditions , as none of the conditions demonstrated significant differences for cbt , hr , rpe , or environmental measures ( table 1 ) . we did not identify any significant differences between conditions on cooling rate ( df=3 , =2.301 , p=0.512 , 1-=0.512 , es=0.005 ) or at the end of the cooling period ( df=3 , =0.658 , p=0.883 , 1-=0.90 , es=0.117 ) , suggesting rbs neither cool nor heat the body , whether seated ( sb=0.0210.011deg / min , 37.90.3c ; snb=0.0290.002deg / min , 37.60.1c ) or walking ( wb=0.0150.025deg / min , 37.90.5c ; wnb=0.0210.011deg / min , 37.80.1c ) in a hot , humid environment ( figure 1 ) . our primary purpose was to determine the degree that rbs effect cooling rates when utilized by runners following an 8 km run in a hot , humid environment . in the four groups , we observed cooling rates of athletes while seated and walking in a hot , humid environment . we hypothesized the use of rbs post - race by distance runners would decrease the cooling rate . however , our results indicate the rbs neither impede nor expedite cbt cooling an 8 km run in a hot , humid environment . although not significant , the wb elicited the least amount of cooling while the snb showed the greatest amount of cooling . this observation may indicate a trend towards inhibited cooling when an athlete completes an active cool down with the use of a rb , due to maintained metabolic heat production , compared to wnb . we observed the largest amount of cooling when an athlete cooled passively without the use of a rb , yet not statistically different between groups . our power analysis indicated moderate to strong power , yet little effect . as compared to other methods of cooling , these methods would not be recommended for rapid cooling in the case of exertional heat stroke ( 7 ) . the amount of metabolic heat produced is unique to the individual and the environmental circumstances . adenosine triphosphate is produced and metabolized in the body , and used for mechanical energy accounting for no more than 25% of the energy utilized . when the hypothalamus is stimulated by thermoreceptors , indicating an increase in cbt , the sympathetic nervous system causes increased blood flow to the skin and increased sweat production to cool the body ( 9 ) . the four mechanisms of cbt cooling are evaporation , radiation , convection , and conduction ( 9 ) . during prolonged exercise , evaporation , radiation , and convection conduction only occurs when coming into contact with other materials ( 9 ) . during our investigation , conduction may have contributed to the cooling rate in the conditions with rbs , while evaporation would have been inhibited . on the contrary , in the snb and wnb conditions , other intrinsic factors playing a major role in metabolic heat production are the body size and metabolic rate of each athlete ( 810 ) . extrinsic factors that inhibit the amount of cbt cooling are the environmental temperature , humidity , air flow , intensity , clothing , and equipment ( 10 ) . each of these plays their own role in inhibiting natural methods of cooling by influencing the ability for radiation , evaporation , convection , and conduction to occur in the body . radiation occurs most when at rest and is caused by the body releasing infrared rays to all surrounding objects as long as the body has a higher temperature than these objects . evaporation is the primary means of cooling while exercising where it can account for up to 80% of cooling ( 9 ) . when sweat is produced , it is converted to vapor which releases excess heat . in our study , radiation , evaporation , convection , and conduction should have occurred similarly because the environmental conditions were consistent between conditions . differences in cooling rates , although not significant , could be attributed to the athletes clothing and/or the rbs that impede natural cooling mechanisms of the human body . states changes occur in hr and cbt up to an increase of 0.12c to 0.25c and 35 beats / min for every 1% decrease of an individual s body mass , which is primarily body water loss ( 4 ) . maintaining hydration levels greatly assist in maintaining lower cbt and hr levels during endurance activities ( 2 , 4 , 6 ) . while completing aerobic activity for long periods of time , cardiovascular drift naturally rises placing increased demands on the heart to continue to produce the same cardiac output ( co ) . this cardiovascular drift is caused by an increased amount of blood needed at the skin , but not returning to the heart . therefore , the hr increases to maintain co. when hypohydrated the viscosity of blood increases because of reduced plasma volume . eventually , the body can not manage both the needed increase in hr to manage co ( 4 , 6 , 9 ) . when rbs are placed over the body s surface , the natural mechanism of evaporative cooling to dissipate sweat and the conduction of another surface on the skin may inhibit cooling . although not different between groups , the loss of natural mechanisms to cool and the continuation of metabolic heat production during active recovery may have equally contributed to slower cooling rates , as did the environmental conditions . our initial hypothesis was based on potential factors impeding cooling in association with rbs . due to the amount of metabolic heat likely to be retained when using the rbs , it seemed the temperature surrounding the body would remain elevated . as a result , the primary means of cooling would be evaporation , which would be limited by the blanket covering the athlete . with the increase in sweat produced during the cooling process , it seemed likely hr and temperatures would maintain their levels or even increase ( 2 , 4 , 6 ) . during the cooling protocols , the blanket conditions also limited convection from becoming a major cooling influence by limiting air flow . we expected intensity during the cooling protocol to play a major factor and cause the participants required to walk to have slower cooling rates throughout the data collection due to the metabolic heat being produced ( 4 ) . although our results were not shown to be significant , the cooling rates in this study were drastically slower than any other means discussed in the literature . to standardize our protocol , we could have evaluated vo2max values of the participants to fix both exercise and cooling intensity . also , we did not measure hypohydration including sweat rate , percent body mass loss , urine osmolality , and other clinical measures , which could have played a role in limiting cooling ( as the rb may have prohibited evaporative and convection cooling , while increasing conduction ) . anecdotally , participants disliked wearing the rb and felt hot and became irritable . providing a mechanism to collect qualitative data regarding how the rbs felt would have enhanced this investigation . this study represents the first to evaluate the effect of rbs on cbt cooling rates . we did not see any significant differences in cbt cooling rates that would have been caused specifically by the rbs retaining metabolic heat produced by the athletes whether they were walking or seated with or without a rb . this is contrary to our initial hypothesis that the retention of this metabolic heat would cause temperatures to stay elevated for increased durations following exercise . overall , rbs neither elongated nor expedited cooling after an 8 km run in a hot , humid environment . rbs are neither essential , nor necessary after the conclusion of a race in a hot , humid environment , similar to the one replicated in our study .

considerable progress has been made in increasing immunization coverage of children around the world through expanded immunization program ( epi ) of world health organization ( who ) . before the implantation of universal epi by who in 1974 , < 5% of children were vaccinated against the vaccine - preventable diseases worldwide . until 2013 , global coverage with the four core vaccines especially diphtheria - tetanus - pertussis ( dtp ) , polio and measles has reached more than 84% . therefore , a considerable success in eradication or noticeable reduction in the incidence and mortality of childhood diseases occurred . despite good achievement and increasing immunization coverage , timely vaccination has become an important challenge in many countries . to achieve the highest level of immunity against the target diseases evermore the only accomplished studies in iran showed that 42 - 67.6% of children received vaccines out of time and this rate in outskirt of iranian cities is 56.6 - 93.2% for hepatitis1 and measles , mumps , and rubella vaccines . delayed administration of vaccination is a powerful risk factor for fatal childhood diseases and can create epidemic and menace international elimination disease programs . therefore , after the high immunization coverage in iran recognizing the predictor of delayed time vaccination is necessary due to little information about timely immunization . delayed vaccination against the pertussis increases the risk of disease and up to four - fold increased the risk of hospitalization . moreover , due to the importance of third dose of dtp vaccine ( dtp3 ) as one of the most important indicators of public health , the current study aimed to evaluate the predictors of untimely dtp3 vaccination in children of suburbs of five big iranian cities . a historical cohort study was conducted in suburbs of iran 's cities including tehran , isfahan , mashhad , zahedan , and arak in june 2013.the target population was children 24 - 47 months old that lived in these areas at the time of the survey . the delay time in dtp3 vaccination was calculated according to the time of vaccination and national immunization schedule . due to the excluding children without the vaccination card , 3610 were analyzed in study finally . trained interviewers collected data using the who standard questionnaire and interviews conducted with the child 's mother or nurse . the more details about sampling schedule , study setting , and data collection described in another study . since dtp3 vaccine coverage is one of the most important indicators of the public health and for childhood immunization program , the delay time of third dose of dtp vaccine was considered as the outcome variable . independent variables were demographic characteristics including child sex , parent 's education and job , living city , prior rural residency , birth order , and nationality . life table approach used for the survival analysis to estimate the median and proportional probability of vaccination in each month periods intervals of delay in dtp vaccination . a total of 3610 children was entered into the analysis , of which 1754 ( 48.6% ) were female , and 1851 ( 51.3% ) were male . the overall delayed time for dtp3 was 38.52 days . the probability ( cumulative incidence ) of vaccination at the end of 1 , 2 , and 3 months after recommended age for dtp3 vaccination were 0.23 , 0.07 , and 0.02 , respectively . the wilcoxon test showed a significant difference , as presented in table 1 , in median delay time among under study living cities , nationality , education level of parents , birth order , and prior rural residency ( p < 0.001 ) . nevertheless , there was no significant difference between the delay time of dtp3 vaccination in children based on parents ' jobs and child gender . figures 1 and 2 showed a higher probability of delayed vaccination in non - iranian children based on life table and kaplan - mayer approaches . median delay time and proportional probability ( se ) of dtp vaccination at 1 , 3 and 6 months after immunization schedule according to related factors life table approach of cumulative proportional probability of diphtheria - tetanus - pertussis 3 vaccination for nationality after recommended age of vaccination based on immunization schedule kaplan - mayer approach of cumulative proportional probability of diphtheria - tetanus - pertussis 3 vaccination for nationality after the recommended age of vaccination based on immunization schedule according to the results , more than 95% of children are vaccinated 3 months after the recommended age for immunization according to the national schedule program . prior residency in the rural area , higher birth order , and more distance from the capital are the risk factors for increasing the delayed vaccination rate . nevertheless , iranian nationality and high parent 's education are protective factors for delayed vaccination rate that caused earlier vaccination . the results of our study showed a significant relationship among under study living cities and probability vaccination after recommended schedule time in specified intervals . it is due to the inequity in the distribution of health care facilities , socioeconomic , and cultural differences among different studied cities . so that , the pairwise comparison showed that the probability of delayed vaccination in children living in zahedan is higher than other cities . zahedan is away from the capital with lower socioeconomic standards and more concentrated illegal immigrant , especially from afghanistan . on the other hand , children in arak have the best condition with the lowest median of delayed vaccination time due to the small size of the town and easier access to health centers . in addition , according to the results , the probability of early vaccination in non - iranian children is lower than iranian . conducted a study on preschool children in athens , greece and showed that incomplete vaccination is more prevalent in immigrants and other minority . moreover , several studies similar to our study have shown that with increase family size , the timely vaccination decreases . because usually in crowded families , parents ' education level and socioeconomic status is low , and the attention of parent for immunization of children decreases . this study showed that the median of delay time for dtp3 vaccine in three intervals ( 1 month , 2 to 3 and 4 to 6 month ) is longer in children that lived in the rural area . it is shown that parent 's education have a significant effect on the probability of un - time vaccination and low levels of education inversely related to the increasing probability of delayed vaccination . pairwise comparison showed that illiterate parent had more delay time for dtp3 vaccination in all intervals . it has been shown in various studies that mother job and education is related to health care utilization and health disparities . since , literate mothers have more knowledge about the childhood diseases and are oriented regarding the importance of vaccination at the recommended age . in addition , gender difference did not observe regarding earlier vaccination in boys , unlike some studies . moreover , we did not find a significant association between parent job status and timely vaccination of children . but the two studies showed that women 's occupation related with vaccination status and children whose mothers work out of home were better vaccinated . however , in the view point of health policy , the probability of vaccination in each period after recommended age for dtp is calculated . therefore , more attempts are needed for increasing the timeliness of vaccination as high immunization coverage . the delay time in dtp vaccination is increased with distance from the capital , lower levels of parent education , increasing foreign population , and busy families . therefore , increasing awareness of parents by educational intervention programs should be considered especially for mothers in crowded families . in addition , more attention should be taken for an increase of welfare of families in poor areas .

profound vascular functional and structural changes occur in many disease states , and emerging evidence suggests that oxidative stress has a major role in mediating these changes . for example , increased oxidative stress has been shown in hypertension - induced vascular diseases , stroke and subcortical vascular dementia [ 24 ] . moreover , oxidative stress from various sources has been implicated in endothelial dysfunction and structural remodeling in the cerebral vasculature [ 5 , 6 ] . although there are many sources of reactive oxygen species ( ros ) , the primary source of superoxide production in the vascular wall is thought to be nadph oxidase . nadph oxidase consists of a membrane - bound b - type cytochrome composed of 91 and 22 kda subunits ( referred to as gp91 ( also known as nox2 ) and p22 , resp . ) , and three cytosolic proteins ( p47 , p67 , and p21 ) . while functional forms of nadph oxidase have been demonstrated throughout the vasculature , there are subtle , but important , structural differences with respect to its subunits depending on vessel size and region . for example , it appears that nox2 may play a role in nadph oxidase activity in vascular muscle of small resistance arteries , whereas homologues of nox2 , such as nox1 and nox4 , may be more important in large cerebral arteries [ 8 , 9 ] . furthermore , a number of studies showed that angiotensin ii - induced impairment of endothelial function and reduced cerebral blood flow are restored in nox2-deficient ( -/y ) mice [ 10 , 11 ] . these findings highlight the importance of nox2-containing nadph oxidase in the pathology of hypertension in the cerebral circulation . it is , therefore , important to understand the mechanism for functional and structural changes during chronic hypertension in these small brain vessels . in an effort to determine the source of ros in cerebral arterioles during hypertension , the first goal of this study was to examine the hypothesis that nox2-derived ros results in vascular dysfunction and hypertrophy in cerebral arterioles during hypertension . we used a transverse aortic banding procedure to increase cerebral vascular pressure and oxidative stress in the right side of the brain [ 13 , 14 ] . an advantage of this model is that the left cerebral hemisphere remains normotensive relative to the right side , and thus vessels in the left hemisphere can be used as normotensive controls . nitric oxide ( no ) is a major mediator of endothelium - dependent dilation and inhibits mitogenesis and proliferation of vascular smooth muscle cells . no readily reacts with superoxide ; thus , the local concentration of superoxide is an important determinant of the biological availability of no . a previous study has demonstrated the relevance of the no - dependent pathway in endothelial dysfunction and hypertrophy in cerebral vasculature . thus , our second goal was to examine the hypothesis that the no - dependent pathway plays a role in nox2-derived ros - induced dysfunction and hypertrophy of cerebral arterioles during chronic hypertension . nox2-/y and wild - type ( wt ) mice were purchased from jackson laboratory ( bar harbor , me , usa ) . animals were housed in pathogen - free facility at 24c , exposed to 12 hours of light ( lights on at 06:00 , off at 18:00 ) and allowed free access of food and fluid . procedures followed in this study were approved by the institutional animal care and use committee of the university of iowa . increased pressure in the proximal aorta in all animals was induced by means of thoracic aortic banding using the method described previously . briefly , mice were anesthetized with ketamine ( 100 mg / kg , i.p . ) plus xylazine ( 5 mg / kg , i.p . ) , intubated with 20-gauge tubing and ventilated ( harvard apparatus rodent ventilator , model 687 ) at 100 breaths per minute ( 0.1 ml tidal volume ) . the transverse aortic arch was ligated ( 70 prolene ) between the innominate and left common carotid arteries with an overlying 27-gauge needle , and then the needle was removed , leaving a discrete region of stenosis . l - name ( 10 mg / kg / day , 4 weeks ) was given in drinking water to wt ( n = 16 ) and nox2-/y ( n = 16 ) mice . we adjusted the concentration of l - name every time based on the volume an individual mouse drank . systemic arterial blood pressures were measured in 6 mice from each group using an automated tail - cuff device ( visitech systems bp-2000 , apex , nc , usa ) . mice were placed in specifically designed mouse holders that allow measurement of systolic blood pressure under resting conditions . mice were trained for 5 days , and then blood pressure was measured at days 0 ( baseline ) , 7 , 14 , 21 , and 28 of treatment . each day , four weeks after aortic banding , we measured diameter in first - order arterioles on the surfaces of the right and left cerebral hemispheres through an open skull preparation as described in detail previously ( n = 8 in each group ) [ 19 , 20 ] . cerebral arterioles were monitored through a microscope connected to a closed - circuit video system with a final magnification of 356 . arteriolar diameter was measured from digitized images of arterioles using nih image version 1.62 ( national institute of health , usa ) . about 30 minutes after completion of craniotomy , cerebral arterioles were exposed to acetylcholine ( ach , 10 m ) dissolved in artificial cerebral spinal fluid ( csf ) for 5 min . in addition , systemic arterial pressure was measured continuously via catheters inserted into the right and left common carotid arteries . to determine whether increases in arterial pressure that result from transverse aortic banding are limited to the right side of the brain , pressure was measured in right- and left - sided first - order cerebral arterioles in a separate group of anesthetized wt mice ( n = 6 ) using a servo - null system as described in detail previously [ 19 , 20 ] . the mice had undergone the transverse aortic banding procedure 4 weeks before measuring arteriolar pressure . systolic ( sp ) , diastolic ( dp ) , mean ( mp ) , and pulse ( pp ) pressures were significantly higher ( p < 0.05 ) in right - sided ( 62 6 , 35 2 , 44 3 , and 28 4 mmhg ; sp , dp , mp , and pp , resp . ) than in left - sided cerebral arterioles ( 39 3 , 28 2 , 32 2 , and 10 1 mmhg ; sp , dp , mp , and pp , resp . ) . furthermore , the levels of sp , dp , mp , and pp in left - sided arterioles in aortic banded mice were similar to those we observed previously in normotensive wt mice [ 5 , 6 ] . in another set of animals ( n = 8 in each group ) after the baseline diameters were measured , arterioles then were suffused with csf - containing edta ( 67 mm ) , which produces maximal dilation of cerebral arterioles . arterioles were fixed at physiological pressure in vivo by suffusion of vessels with glutaraldehyde fixative ( 2.25% glutaraldehyde in 0.1 m cacodylate buffer ) while maintaining cerebral arteriolar pressure at baseline levels . after the anesthetized animal was euthanized using overdose sodium pentobarbital , cerebral arteriolar segments and carotid arteries were removed , processed , and embedded in spurr 's low - viscosity resin while maintaining cross - sectional orientation . cross - sectional area ( csa ) of the vessel wall was determined histologically using a method described previously . in another set of animals ( n = 8 in each group ) , superoxide levels were evaluated in vitro in 68 m thick frozen sections of unfixed right- and left - sided cerebral arterioles using hydroethidine - based ( 2 m hydroethidine ) confocal microscopy as described previously . laser settings were identical for the acquisition of all images , and vessels from wt and nox2-/y mice were processed and imaged in parallel . relative increases in ethidium fluorescence were determined and normalized to the cross - sectional area of the vessel wall . ach , snp , and l - name were purchased from sigma ( st . louis , mo , usa ) . analysis of variance was used to compare blood pressure , cerebral arteriolar diameters , cross - sectional areas , and superoxide levels of the vessel wall . probability values were calculated using graph pad prism 5 ( graph pad software , inc . , san diego , ca , usa ) . values were presented in mean sem and were considered different when p < 0.05 using post hoc bonferroni test . to determine if nox2-containing nadph oxidase is responsible for hypertension - induced superoxide production , levels of superoxide were determined in cerebral arterioles from wt and nox2-/y mice by ethidium fluorescence . representative micrographs show that fluorescence of ethidium was higher in right- than left - sided cerebral arterioles in wt mice ( figure 1(a ) , left panel ) . in contrast , fluorescence in right - sided arterioles did not appear to be increased in nox2-deficient mice ( figure 1(a ) , right panel ) , and fluorescence in left - sided arterioles appeared to be lower in nox2-deficient mice than in wt mice . semiquantification of ethidium signal confirmed that levels of fluorescence were higher in right- than left - sided cerebral arterioles in wt mice ( figure 1(b ) ) and similar in right - and left - sided arterioles in nox2-/y mice ( figure 1(c ) ) . these findings suggest that nox2 is the major source of hypertension - induced superoxide in cerebral arterioles . sds measured under conscious conditions by a tail - cuff method prior to l - name treatment were similar in wt and nox2-/y mice ( figure 2 ) . blood pressure of wt and nox2-/y mice was increased in the last two weeks of l - name treatment . pressures were measured in right and left carotid arteries in anesthetized mice to confirm that transverse aortic banding was successful . sp and pp , but not dp and mp , were significantly increased by similar levels in right- compared to left - sided carotids in untreated wt and untreated nox2-/y mice ( table 1 ) . to test whether endothelial dysfunction induced by hypertension is nox2-dependent , dilator response to ach was significantly decreased in right - sided cerebral arterioles relative to left - sided arterioles in untreated wt mice , suggesting an endothelial dysfunction on the hypertensive side ( figure 3(a ) ) . response to snp was similar in both sides of wt mice , indicating that aortic banding did not affect smooth muscle contractility ( figure 3(b ) ) . in untreated nox2-/y mice , responses to ach were restored in right - sided cerebral arterioles comparable to that of the left side , suggesting normal endothelial function . treatment with l - name reduced dilator responses in left - sided cerebral arterioles to ach , but not snp , in both wt and nox2-/y mice . moreover , l - name blunted dilator responses to ach in right - sided arterioles in nox2-/y mice . to determine whether nox2 contributes to hypertension - induced hypertrophy in cerebral arterioles , we measured csa of the arteriolar wall . csa of the arteriolar wall was greater in right- , than in left - sided , cerebral arterioles in untreated wt mice , but not in untreated nox2-/y mice ( figure 4(a ) ) . treatment with l - name increased csa in left - sided , but not right - sided cerebral arterioles , in wt mice . in contrast , l - name did not produce hypertrophy in either right- or left - sided arterioles in nox2-/y mice , which suggests an important role for nox2-dependent production of ros in the development of hypertension - induced cerebral arteriolar hypertrophy . in contrast to cerebral arterioles , nox2 deficiency did not prevent increases in csa of the vessel wall in carotid arteries ( figure 4(b ) ) , suggesting that nox2 does not contribute to hypertension - induced hypertrophy in larger conduit arteries . chronic hypertension has profound impacts on the vasculature and is a known risk factor for stroke and dementia . it is important to understand the mechanism of vascular changes during chronic hypertension , particularly in smaller resistance arterioles because they provide substantial vascular resistance and are important in controlling local blood flow . in this study , we used a transverse aortic banding model to increase blood pressure to the right , but not the left , side of the brain to study mechanisms of vascular dysfunction and structural remodeling in chronic hypertension . first , superoxide levels were increased in the hypertensive side of the brain in wt , but not in nox2-/y mice . this result suggests that nox2-containing nadph oxidase is the major source of superoxide in cerebral arterioles during hypertension . second , deficiency of nox2 prevented hypertension - induced impairment of endothelium - dependent dilatation in cerebral arterioles . moreover , l - name treatment eliminated the normalized endothelial function in hypertensive cerebral arterioles of nox2-/y mice , suggesting an no - dependent mechanism . third , hypertension caused hypertrophy in cerebral arterioles from wt mice , but not in nox2-/y mice . this suggests that ros derived from nox2-containing nadph oxidase play a key role in hypertension - induced hypertrophy in cerebral arterioles . taken together , this study provides in vivo evidence that chronic hypertension induces cerebral arteriolar dysfunction and hypertrophy via increased production of ros derived from nox2-containing nadph oxidase . we used ethidium fluorescence to examine the effects of nox2 deficiency on hypertension - induced production of superoxide in cerebral arterioles . being aware of potential problems with this method , matched pairs of hypertensive ( right - sided ) and normotensive ( left - sided ) cerebral arterioles from each mouse were examined in parallel using the same reagents and laser settings . in addition , we have shown previously that incubation with peg - sod , a scavenger of superoxide , abolishes ethidium fluorescence in aorta of mice that overexpresses human renin and human angiotensinogen . using a model of abdominal aortic banding , it was shown previously that superoxide levels are elevated in noncerebral vessels [ 13 , 14 ] . in the present study , transverse aortic banding was used to increase pressure in right - sided ( hypertensive ) cerebral arterioles relative to left - sided ( normotensive ) cerebral arterioles . we found in wt mice that the production of superoxide was elevated in right - sided cerebral arterioles relative to left - sided arterioles , whereas in nox2-/y mice levels of superoxide were essentially the same in right- and left - sided arterioles . this finding indicates that nox2-containing nadph is the major source of increased production of ros in cerebral arterioles during hypertension . it is still debatable as to whether nox2-derived ros play a role in the development of hypertension . for example , the pressor response to angiotensin ii was found to be reduced in nox2-/y mice in one study and unaffected in another . the finding in this study that l - name increased systemic blood pressure in nox2-/y mice to levels similar to those found in wt mice supports the idea that nox2-derived ros do not contribute significantly to hypertension . endothelial dysfunction caused by hypertension in the cerebral circulation has been previously demonstrated in various animal models [ 10 , 26 ] . to our knowledge , this is the first study to examine endothelium - dependent function in cerebral arterioles using the transverse aortic banding model in mice . one of the advantages of this model is that the contralateral side can be used as a normotensive control . previous studies found disparate effects of hypertension induced by aortic banding on endothelium - dependent function in aorta or coronary arteries [ 13 , 27 ] , probably due to the differences in the vascular beds studied and the location of the band . in this study , our finding that transverse aortic banding impairs endothelium - dependent dilatation in right - sided ( hypertensive ) cerebral arterioles in wt mice , but not in nox2-/y mice , suggests that superoxide derived from nox2-containing nadph oxidase may play an important role in altered endothelium - dependent function in cerebral arterioles during hypertension . hypertension - induced impairment of endothelium - dependent dilatation is thought to result from reduced availability of no due either to its destruction by nadph - derived superoxide or to an uncoupling of enos , which results in the production of superoxide instead of no . our finding that nox2 deficiency protected against impairment in cerebral arteriolar dilatation during hypertension produced with transverse aortic banding , but not l - name , supports the concept that hypertension impairs endothelial - dependent dilatation of cerebral arterioles through the destruction of no by nadph - derived superoxide , and not by uncoupling of enos . cerebral vascular hypertrophy is a well - known consequence of hypertension [ 17 , 19 , 21 ] . a role for superoxide in the development of vascular hypertrophy is implicated by the finding that mice deficient in copper - zinc superoxide dismutase develop cerebral arteriolar hypertrophy while remaining normotensive . additional support for this concept is provided by the finding in this study that whereas transverse aortic banding resulted in hypertrophy of hypertensive cerebral arterioles in wt mice , hypertensive arterioles did not undergo hypertrophy in nox2-/y mice . one mechanism by which superoxide may promote vascular hypertrophy is through the destruction of no . no has been shown to inhibit mitogenesis and proliferation of cultured smooth muscle , and treatment with l - name , as well as deficiency of enos , has been shown to induce hypertrophy of cerebral arterioles in mice . further support for this concept would appear to be provided by our finding in this study that treatment of aortic banded wt mice with l - name tended to cause hypertrophy in normotensive ( left - sided ) , as well as hypertensive ( right - sided ) , cerebral arterioles ( p = 0.063 versus untreated wt mice ) . however , the possibility that destruction of no may not be a critical factor in the development of ros - induced hypertrophy of cerebral arterioles is suggested by our finding that the treatment of nox2-dieficient mice with l - name did not induce hypertrophy in either hypertensive or normotensive cerebral arterioles . instead , this finding suggests that ros derived from nox2-containing nadph oxidase may play a central role in the development of cerebral arteriolar hypertrophy . while we can not exclude the possibility that ros downstream of superoxide , such as peroxynitrite or hydrogen peroxide , contribute to the hypertrophic process , we believe that superoxide plays a more important and direct role in causing cerebral vascular hypertrophy . we base this speculation on two observations . first , we showed in this study that l - name inhibition , which supposedly limits the interaction of superoxide and no to form peroxynitrite , does not attenuate the degree of cerebral arteriolar hypertrophy induced by transverse aortic banding in wt mice . second , we showed in a previous study that the deficiency of copper - zinc superoxide dismutase , which leads to reduced conversion of superoxide to hydrogen peroxide , nevertheless causes hypertrophy in cerebral arterioles . the present study demonstrated that ros derived from nox2-containing nadph oxidase are critical in hypertension - mediated cerebral arteriolar vascular dysfunction and hypertrophy . this may lead to reduction of dilator capacity and the ability to control local cerebral blood flow during hypertension .

small bowel obstruction is a common clinical problem resulting from congenital [ 13 ] or acquired causes [ 47 ] . small intestinal mechanical obstruction can be reproduced in laboratory animals using rings of different materials . proximal to the partial obstruction site , the intestinal segment remodels morphologically and biomechanically [ 814 ] . partial obstruction causes intestinal smooth muscle hyperplasia and hypertrophy [ 8 , 9 , 11 , 12 , 1517 ] . increased contraction force of remodeled smooth muscle layer has been reported , but the force was decreased when normalized to the tissue wet weight . furthermore , the obstructed intestinal wall became stiffer during longstanding partial obstruction [ 11 , 12 , 14 ] . therefore , it is important to study the function of remodeled intestinal smooth muscle due to partial obstruction . it is possible to obtain isometric length - tension diagrams of phasic and tonic smooth muscle contraction in vitro . tools have now been developed for studying the active ( phasic and tonic contractions ) and passive length - tension behavior in the human gut in vivo using impedance planimetric distension [ 1921 ] . from a biomechanical standpoint , muscle mechanical properties must be described in terms of stress and strain , that is , the force per area and tissue deformation . computation of the stress depends on the wall thickness which can not be directly measured in vivo . however , it is possible to measure the wall thickness in vitro and thus obtain the stress - strain relationship of the intestinal wall with reference to the zero - stress state . furthermore , the passive , tonic , and phasic stress - strain curves can be obtained when using papaverine to abolish smooth muscle activity . in the present study we computed stress - strain data for assessment of the smooth muscle function of we hypothesized that the stress - strain properties of the remodeled small intestine change and that the remodeling is determined by the stress similar to the cardiovascular system . male guinea pigs ( 600800 g ) were divided into four obstructed and four sham - obstructed groups living for 2 , 4 , 7 , and 14 days , respectively . ten age - matched guinea pigs not exposed to surgery were used as normal controls . we have long - term experience with the operative procedures [ 11 , 12 , 14 ] , which is likely the reason for a mortality rate below 20% . the final number of animals was 6 in each obstructed group and 4 in each of the sham - obstructed group . the guinea pigs had access to water but were restricted from food intake from the night before the operations and experiments . the animals were weighted daily . the surgical procedure for partially obstructing the small intestine is well established [ 11 , 14 ] . atropine ( atropin dak , denmark ) 0.3 mg kg s.c . was given 30 minutes prior to anesthesia with hypnorm 0.5 mg and dormicum 0.25 mg per 100 g body weight ( hypnorm : dormicum : sterile water = 1 : 1 : 2 ; subcutaneous injection ) . a small midline laparotomy was done when surgical anesthesia was achieved . a loop of the midjejunum was selected , and the mesenterium was carefully incised close to the intestine to create a small window . a 3.5 mm wide polyurethane band was passed through the mesenteric window and closed antimesenterically with a 60 silk suture at a circumferential length about one mm longer than the outer circumference of the small intestine . hence , a loose fit around the intestine was obtained without any apparent compression of the tissue . in the sham - obstructed group , the mesenteric incision was made and marked with a 60 silk suture but no band was placed . buprenorphine ( temgesic , reckitt & colman , uk ) 0.05 mg kg was given subcutaneously to counter postoperative pain along with 10 ml saline to prevent dehydration . the guinea pigs were anesthetized with hypnorm and dormicum when the scheduled time had arrived . a ten cm intestinal segment proximal to the band in the obstructed animals and segments from the corresponding location in sham - obstructed and normal animals were removed . a 0.5 cm long intestinal piece from the proximal end of the excised segment was cut and used for histological analysis . further two short ring - shaped pieces perpendicular to the longitudinal axis were cut and used for zero - stress state analysis . the remaining segment was immediately put into the organ bath containing krebs solution of the following composition ( mmol l ) : nacl , 118 ; kcl , 4.7 ; nahco3 , 25 ; nah2po4 , 1.0 ; mgcl , 1.2 ; cacl2h2o , 2.5 ; glucose , 11 ; ascorbic acid , 0.11 . the 37c krebs solution was aerated with a gas mixture ( 95% o2 and 5% co2 , ph 7.4 ) . the proximal end of the intestinal segment was tied on the cannula with silk threads . the cannula was via a tube connected to a syringe containing krebs solution for applying luminal pressures ( 0.8 ml min ) . the lumen was pressurized by a pump ( genie programmable syringe pump , world precision instrument , stevenage , uk ) . the distal end of the intestinal segment was tied by a silk thread on the three - way tube connected to a micromanipulator that could stretch the intestinal segment in longitudinal direction . the ramp distension experiment with pressure up to 10.0 cm h2o was done on the intestinal segment at the longitudinal stretch ratios 0% , 10% , and 20% . the intestinal diameters at the locations where the pressure was recorded in the intestinal segments were videotaped by a ccd camera ( sony , japan ) on a stereomicroscope . the data sampling frequency for pressure and diameter data was 10/second . the method for determination of the gastrointestinal zero - stress state has been described in detail previously [ 24 , 25 ] . one - two mm wide rings were transferred to a calcium - free krebs solution with egta and papaverine . then , each ring - shaped segment was cut radially under the microscope resulting in an open sector geometry . photographs representing the zero - stress state were taken ~60 min after the radial cutting to allow viscoelastic creep to occur . the segment was fixed in 10% buffered formalin over 24 hours followed by dehydration in a series of graded ethanol ( 70% , 96% , and 99% ) and embedding in paraffin . five - micron sections were cut perpendicular to the mucosa surface , and the paraffin was cleared from the slides with coconut oil ( over 15 min . redehydration occurred in 99% , 96% , and 70% ethanol followed by staining with hematoxylin and eosin . calculation was the assessment of the no - load state , zero - stress state dimensions and the outer diameters of the specimen at varying pressures . the kirchhoff stress and green 's strain in the intestinal wall at a given pressure were computed assuming circular geometry as follows : circumferential kirchhoff 's stress : ( 1)s=priphp2 . circumferential midwall green 's strain : ( 2)e=212 , where p is the transmural pressure difference , r is the luminal radius h is the wall thickness and is the circumferential stretch ratio . stress and strain data immediately before the contraction ( stress and strain thresholds ) and at maximal contraction ( maximal amplitude of stress and strain ) were used for further analysis . the total phasic stress and the total tonic stresses ( both composed of active and passive tissue properties ) were extracted from the top points during contraction and the baseline between the contractions during the distension ( figure 2(a ) ) . the passive stress was extracted from the data obtained during distension after administration of papaverine . the strain points were not fixed ; hence , it was not possible to directly compare between different samples and groups . the total tonic and passive stresses increased in an exponential - like way as function of strain . consequently the stress - strain curves were fitted to the exponential function equation ( 3)s=(s+)e(ee) , where s * and e * are the stress and strain at a physiological reference level . the total phasic circumferential stress - strain curves increased in a polynomial way as function of strain . as a consequence the stress - strain curves were fitted to the polynomial equation ( 4)s = s0+a1e3+a2e2+a3e1 , where a1 , a2 , and a3 are constants . the active phasic and tonic stresses were defined as the total phasic and tonic stresses minus the passive stress ( figure 2(b ) ) ( 5)active phasic stress = total phasic stresspassive stress , active tonic stress = total tonic stresspassive stress . the active phasic and tonic stresses were normalized to muscle layer thickness as follows : ( 6)normalized active phasic stress = active phasic stressmuscle thickness , normalized active tonic stress = active tonic stressmuscle thickness the muscle layer thickness ( um ) was obtained by histological measurement . the total phasic , total tonic , active phasic , and active tonic stresses were compared between different groups by anova analysis . the normalized active phasic and tonic stresses as function of strain were also compared between groups . the peritoneum had no signs of inflammation or adhesions that could potentially influence the mechanical properties and intestinal contraction . the intestinal segments from the animals obstructed for 2 days and 4 days were clearly dilated . after seven days , histological analysis showed that the thickness of both the submucosa layer and muscle layer increased during the development of the obstruction ( table 1 ) . muscle layer thickening was observed after 4 days of obstruction whereas submucosal thickening was observed after 7 days of obstruction . seven days postobstruction the intestinal segments were also visibly hypertrophied . a typical pattern of the muscle layer proliferation is shown in figure 3 . figure 4 shows the pressure and diameter changes during the distension from a normal jejunal segment without longitudinal stretch applied . waves of peristaltic contraction were clearly observed both from pressure and diameter curves ( figure 4(a ) ) . the pressures , stresses , and strains at the contraction threshold and maximal contraction points in different groups are shown in figure 5 . during ramp distension - induced contractions , the threshold and maximal pressure amplitude ( figure 5(a ) ) and stresses ( figure 5(b ) ) increased after obstruction . however , the strains at the contraction threshold and maximal contraction ( figure 5(c ) ) decreased during the development of the obstruction . significant differences were found after 4 days of obstruction compared with normal and sham - obstructed controls ( p < 0.05 , p < 0.01 ) . thus the remodeled intestine due to obstruction required higher pressure and stress levels to induce contractions , and the induced contractions produced high pressures and stresses during ramp distension . however , the deformation of the intestinal wall became smaller indicating wall stiffening . in accordance with our previous study , however , longitudinal stretch did not influence the differences of tonic and phasic stress - strain curves among the groups . figure 6 illustrates the total phasic ( a ) , total tonic ( b ) , active phasic ( c ) , and active tonic ( d ) stresses as function of strain in the different groups . the total phasic stress increased in a polynomial way as function of strain whereas the total tonic stress increased in an exponential - like way . at corresponding circumferential strains , the amplitude of total phasic , total tonic , active phasic , and active tonic circumferential stresses increased after 7 days of obstruction compared with normal and sham - obstructed controls ( p < 0.05 and 0.01 ) . however , when normalized to the muscle layer thickness , the amplitude of active phasic and tonic stresses as function of strain did not differ among the groups ( figure 7 , p > 0.05 ) . the length - tension diagrams known from physiological and pharmacological studies of smooth muscle strips in vitro [ 19 , 26 ] can be reproduced in intact segment of intestine in vitro as shown in the present and previous studies . from a biomechanical standpoint , phasic and tonic stress - strain data are required for evaluation of smooth muscle mechanical function in intact intestinal segments . the main findings in the current study were that the amplitude of total phasic , total tonic , active phasic , and active tonic circumferential stresses increased after 7 days of obstruction . however , when normalized to muscle layer thickness , the amplitude of active stresses did not differ among the groups . the mechanical properties of the small intestine can be divided into properties arising from a passive or connective tissue element , an active ( tonic ) element , reflecting baseline muscle activity , and an active ( phasic ) element , reflecting the effects of distension - induced neuromuscular function . the passive and the active stress - strain curves depend on the wall structure , the wall mechanical properties , and the smooth muscle contractile properties . longstanding nonoccluding intestinal obstruction results in structural changes with marked dilatation , increased collagen content , and hypertrophy of especially the muscle layer proximal to the obstruction site [ 9 , 27 ] . thus , stress - strain data are important for understanding the mechanical function of remodeled smooth muscle in the partially obstructed intestine . length - tension diagrams have been derived from the human gastric antrum and duodenum [ 21 , 28 ] , though butylscopolamine may not have abolished all phasic activity . the present and previous in vitro studies produced tonic and phasic stress - strain curves referenced to the passive stress - strain curve because papaverine completely abolished all smooth muscle activity . computation of the stress depends on the wall thickness which can not be directly measured in vivo . the partial obstruction narrows the intestine which will increase the resistance to flow during bolus passage . to compensate experimental evidence demonstrated that longstanding partial intestinal obstruction results in marked proliferation of the intestinal muscle layers [ 8 , 9 , 11 , 12 , 1417 ] . in the present study the intestinal muscle layer , especially the circumferential muscle layer , thickness markedly increased during obstruction . previous studies on pressure contraction curves of obstructed intestinal segments have demonstrated that the total contractile ability of remodeled smooth muscle layer increased [ 12 , 29 ] . in the present study the pressure curve ( figure 5(a ) ) confirmed that the maximal contraction pressure increased after obstruction . in agreement with the proliferation of the smooth muscle layer after 4 days of obstruction , both muscle proliferation and stress increase in a time - dependent manner as function of obstruction time , indicating that muscle remodeling is determined by the stress . however , the maximal contraction strain significantly decreased during the development of obstruction indicating the wall deformation became smaller due to stiffening . further stress - strain analysis demonstrated that the total phasic , total tonic , active phasic , and active tonic stresses as function of strain also significantly increased after 7 days of obstruction . therefore , the increased total contraction force is mainly due to smooth muscle proliferation . as a result , increased total contraction force can compensate to push the content through the partial obstruction site . furthermore , it is worthwhile to notice that the maximum of active phasic and active tonic stress differ . this probably means that they are regulated in different ways . despite the fact that the total contraction force increased after obstruction it is also interesting to notice at the present study that when normalized to muscle layer thickness , the active phasic and tonic stresses as function of strain did not differ between obstruction groups and sham - obstructed and normal groups . therefore the remodeled smooth muscle cells may be somehow damaged due to long - term partial obstruction . earlier studies demonstrated that the hypertrophied muscle cells exhibit ultrastructural changes of sarcoplasmic reticulum , gap connections , and cytoplasmic content and decreased ratio of myofilament to intermediate filament [ 3032 ] . the relative decrease in myofilament content suggests a loss of contractile machinery in remodeled smooth muscle cells due to partial obstruction . furthermore , it was reported that the stiffness of collagen fibrils could influence vascular smooth muscle cell phenotype and function . the extracellular matrix including collagen was reported to increase due to the obstruction [ 11 , 12 ] . therefore , the altered extracellular matrix may also affect the function of smooth muscle cells . furthermore , we noticed in the present study that the pressure and stress thresholds to induce contraction increased after obstruction . this can be caused by altered mechano - sensory function such as resetting of the mechanoreceptors . the partially obstructed intestinal model has been validated previously [ 10 , 11 , 15 ] and demonstrated that it is a good model for studying histomorphological and biomechanical intestinal remodeling . the size of the band relative to the circumference of the intestine is important for successful obstruction . if the band is too tight , the animal dies within 48 hours ; if it is too loose , no changes will occur . in the present study , the ring was one millimetre bigger than the outer diameter of the intestinal segment . although the band does not compress the resting intestine , during bolus passage the band would increase the resistance during the propagation of contractions by compressing the intestinal wall . the total active phasic and tonic stress increased in long - term - obstructed intestine which is likely related to increased total contraction force to push the content through the partial obstruction site . however , the contraction force per smooth muscle unit did not increase in obstructed intestine when compared to normal intestine .

in europe the breeding of sheep and goats is a marginal agricultural activity ( 3.6% of the total value of livestock production ) but , in some countries such as uk , ireland , spain , romania and italy , the production of sheep milk and goat milk has been growing since 1995 by about 10% ( and international , 2011 ) . maturation or ripening of cheese from goat milk is governed by many many factors including the wide variety of microrganisms used in culturing , and the forming and pressing techniques . the characterization of an artisanal cheese includes the study of the technological process of manufacture , the chemical and biochemical study of ripening process and the microbiological changes which take place . in northen italy ( insubria area ) , a traditional goat cheese namely formaggella di capra is produced with whole raw milk added with mesophilic and termophilic lactic acid bacteria as starter culture ( www.ars-alimentaria.it ) . the manufacturing of this cheese with raw milk linked with the short ripening ( about 30 days ) , could be considered as risk factors for the health of consumers . the aims of this research were i ) to study the dynamics of different bacteria species ( as a function of technological parameters ) in a traditional italian goat cheese during three cheesemaking replicate made at three different time from september to october 2012 and ii ) to study the behaviour of pathogenic bacteria in naturally contaminated milk during the cheesemaking . to evaluate the microbiological variability between milk collected in three different days , a total of three cheesemaking replicates were performed from september to october 2012 . the cheese , namely formaggella di capra , was produced according to producer s specifications ( cosciani - cunico et al . , 2014 ) in the same farm located in insubria region with whole raw goat milk . during the process , three samples ( n=3 ) of raw milk , curd and cheese at 3 , 7 , 11 , 14 , 21 and 30 days of ripening were taken from three cheesemaking replicates ( n=3 ) . the samples were collected into sterile containers and brought refrigerated to the institute for experimental veterinary medicine of lombardy and emilia romagna regions ( istituto zooprofilattico sperimentale della lombardia e dellemilia romagna ; izsler ) laboratory . for microbiological analysis on milk , counts were estimated by direct plate count . for curd and cheese , 25 g of samples were homogenized 1:3 ( w : v ) in sterile peptone water ( pw ) ( conda , madrid , spain ) for 3 min using a stomacher blender . total mesophilic bacteria ( tmb ) were determined according to iso 4833 method ( iso , 2003 ) ; the number of enterobacteriaceae ( ent ) was determined according to iso 21528 - 2 method ( iso , 2004 ) . for the enumeration of mesophilic lactobacilli and lactococci ( mlb and mlc ) , the appropriate dilution were pour plating ( 1 ml ) in de man , rogosa and sharpe agar ( mrs ) and m17 agar ( microbiol diagnostici , uta , italy ) respectively and incubating at 37c for 48 - 72 h. escherichia coli -glucuronidase - positive counts ( ec ) were determined according to iso 16649 - 2 method ( iso , 2001 ) , and coagulase - positive staphylococci ( cps ) were determined following the iso 6888 - 2 method ( iso , 1999 ) . the ph value was measured on approximately 10 g of samples using a hi 223 calibration checktm microprocessor ph meter ( hanna instrument , woonsocket , ri , usa ) equipped with a gel - glass electrode ( hamilton , switzerland ) . during the cheese manufacture the temperatures of milk , curd and cheese were monitored using electronic data loggers ( cox tracer ; cox technologies , belmont nc , usa ) . mean of bacterial counts and ph values were averaged from three replicates samples ( n=3 ) for each sampling time for three cheesemaking replicate ( n=3 ) . boxplot analysis was performed to evaluate the variability of each parameter during three cheesemaking replicates : the bottom of the box is at the first quartile , that corresponding to 25th percentile , and the top is at the third , that corresponding to 75 percentile . the whiskers are the lines that extend from the top and bottom of the box to the lowest and highest observations that are inside the region . the horizontal line within the box corresponds to the median value and the vertical lines are an index of data variability outliers are points outside of the lower and upper limits and are plotted with open circles . to evaluate the microbiological variability between milk collected in three different days , a total of three cheesemaking replicates were performed from september to october 2012 . the cheese , namely formaggella di capra , was produced according to producer s specifications ( cosciani - cunico et al . , 2014 ) in the same farm located in insubria region with whole raw goat milk . during the process , three samples ( n=3 ) of raw milk , curd and cheese at 3 , 7 , 11 , 14 , 21 and 30 days of ripening were taken from three cheesemaking replicates ( n=3 ) . the samples were collected into sterile containers and brought refrigerated to the institute for experimental veterinary medicine of lombardy and emilia romagna regions ( istituto zooprofilattico sperimentale della lombardia e dellemilia romagna ; izsler ) laboratory . for microbiological analysis on milk , counts were estimated by direct plate count . for curd and cheese , 25 g of samples were homogenized 1:3 ( w : v ) in sterile peptone water ( pw ) ( conda , madrid , spain ) for 3 min using a stomacher blender . total mesophilic bacteria ( tmb ) were determined according to iso 4833 method ( iso , 2003 ) ; the number of enterobacteriaceae ( ent ) was determined according to iso 21528 - 2 method ( iso , 2004 ) . for the enumeration of mesophilic lactobacilli and lactococci ( mlb and mlc ) , the appropriate dilution were pour plating ( 1 ml ) in de man , rogosa and sharpe agar ( mrs ) and m17 agar ( microbiol diagnostici , uta , italy ) respectively and incubating at 37c for 48 - 72 h. escherichia coli -glucuronidase - positive counts ( ec ) were determined according to iso 16649 - 2 method ( iso , 2001 ) , and coagulase - positive staphylococci ( cps ) were determined following the iso 6888 - 2 method ( iso , 1999 ) . the ph value was measured on approximately 10 g of samples using a hi 223 calibration checktm microprocessor ph meter ( hanna instrument , woonsocket , ri , usa ) equipped with a gel - glass electrode ( hamilton , switzerland ) . during the cheese manufacture the temperatures of milk , curd and cheese were monitored using electronic data loggers ( cox tracer ; cox technologies , belmont nc , usa ) . mean of bacterial counts and ph values were averaged from three replicates samples ( n=3 ) for each sampling time for three cheesemaking replicate ( n=3 ) . boxplot analysis was performed to evaluate the variability of each parameter during three cheesemaking replicates : the bottom of the box is at the first quartile , that corresponding to 25th percentile , and the top is at the third , that corresponding to 75 percentile . the whiskers are the lines that extend from the top and bottom of the box to the lowest and highest observations that are inside the region . the horizontal line within the box corresponds to the median value and the vertical lines are an index of data variability outliers are points outside of the lower and upper limits and are plotted with open circles . temperature distribution registered during three cheesemaking replicates was extremely narrow throughout the process : during the fermentation step the milk was heated up to 33 - 35c for a time of 1 - 1.5 h and subsequent temperature ranged from 12 and 15c during the cheese ripening ( data not shown ) . the development of the different microbial groups throughout the cheesemaking and ripening are shown in figure 1 . the average of total mesophilic bacteria and enterobacteriaceae count in raw milk was 5.270.57 and 3.81.02 log cfu / ml respectively and grew during the cheesemaking . lactic acid bacteria were the predominant bacterial group during the manufacture of formaggella di capra cheese . in the first days of the process a more rapid growth of mesophilic lactococci respect to lactobacilli group was observed : on m17 agar , lactococci showed an increase of over 4 logarithms in three days of process . the lactic acid bacteria developed in different ways in each of the media used in this study also during the ripening process . microbial concentration variability was observed between three cheesemaking replicates ( figure 1 ) , however this variability did not affect the ph values that remains very similar in all the cheesemaking replicates ( table 1 ) . the evolution of ph and the different microbial group counts throughout the process of formaggella di capra cheese in each replicates are shown in table 1 . in our study , e. coli and coagulase - positive staphylococci were detected in raw milk , with a large variability of microbial concentrations ( figure 1 ) . in figure 2 the contamination levels of milk and the behaviour of e. coli and coagulase - positive staphylococci during the cheese manufacture are indicated . even if the milk collected and used for cheesemaking replicates n. 1 and n. 3 , has not be found contaminated at countable levels , during the ripening a growth of e. coli and coagulase - positive staphylococci was observed . the presence of these bacteria allows us to study their natural behaviour within the food matrix , at difference of the challenge test in which the pathogens were artificially inoculated in the food , and to know their behaviour as a function of the production process . in the milk used for the cheesemaking replicate n. 2 , the e. coli level was 5.070.03 log cfu / ml , and increased by about 1 log in the curd . during the ripening the e. coli concentration ranged from 6.14 to 6.89 log cfu / ml until the last week of ripening , when the level decreased to 5.690.2 log cfu / ml ( figure 2 ) . the milk was found to be contaminated also by coagulase - positive staphylococci ( 3.180.06 log cfu / ml ) , but the behaviour of this group appeared to be very changeable . goat milk production is a dynamic and developing industry that is fundamental to the wellbeing of hundreds of millions of people worldwide and is an important part of the economy in many countries ( silanikove et al . , 2010 ) . the microbiological quality of goat milk was assessed and high levels of total bacteria and enterobacteriaceae were enumerated ( > 5 log cfu / ml ) . since the enterobacteriaceae counts are widely used as a contamination index , an elevated number in milk indicates deficient handling during milking and collection . a high number of enterobacteriaceae may not be considered as pathogenic , but their presence might indicate a potential contamination by more dangerous organisms . in fact , in our study we observed a correlation between the presences of higher levels of enterobacteriaceae in milk collected during the cheesemaking replicate 2 and the presence of other contaminants bacteria such as e. coli and coagulase - positive staphy lococci . the count of each bacterial group increased in curd and this was considered a normal process in the cheesemaking . this was due to the physical retention of bacteria in the coagulum and , in part , to the microbial growing during the coagulation and whey drainage ( fontn et al . , 2001 ) . the m17 agar is a selective medium for lactococcus , a very active genus starting to ferment the lactose and to proliferate in the initial stages of the ripening process . the lactococci decrease , observed in the later stages of ripening , can be related to their low ability to compete with other more acid - resistant microbial populations such as lactobacilli . in fact , lactobacilli have a slower metabolism than lactococci , and initially growth slower . however , since lactobacilli are aciduric bacteria they are more tolerant to unfavorable conditions , concentration increase during the ripening process , till they are high to the end , when the ph values reach the optimum for growth ( 5.5 ) . lactobacilli include secondary flora which generally play a significant role during the ripening ( beresford et al . , 2001 ) . in agreement with cogan ( 2000 ) high densities of microorganisms were observed in cheese throughout ripening and they can play a significant role in the ripening process . in our study we observed the contamination of milk with e. coli and coagulase - positive staphylococci at different level between the replicates . leedom ( 2006 ) reported that occasionally a lactating animal s udder becomes infected with haemolytic streptococci of human origin , and this may result in milk - borne epidemics of scarlet fever or septic sore throat . the most prevalent trouble with a goat milk - borne bacterial contamination is the foodborne disease and the most prevalent cause is the presence of staphylococcus aureus and its enterotoxin ( cremonesi et al . , 2007 ) . there are occasional reports or even outbreaks of alimentary toxicosis involving other pathogens , including e. coli ( espie et al . , 2006 ) . the hygienic quality of milk herd should be of primary importance to any producer , especially where raw milk is used to make fresh cheese . based on the potential pathogenic issues previously described , there is reason to implement more stringent food safety control systems in the dairy goat industry . an effective programme for prevention of zoonoses and pathogenic bacteria in food products can be assured if this includes regular monitoring of bacterial infections of herds by the appropriate national veterinarian authorities . also , routinary testing of products by the dairies prior to release to the market is to be considered as necessary . in this work a first step of process control and microbial groups study was performed and the cheesemaking process was registered in website www.ars-alimentaria.it , the italian site on quality and safety of italian food supported by the italian board of health .

modern drug discovery programs typically begin with a screening campaign ( e.g. , biochemical , virtual , or biophysical ) for agonists , antagonists , or inhibitors of a nominated target associated with a particular disease . after hit identification , the generation of high - affinity ligands ( so - called lead compounds ) is followed by chemical refinement into derivatives of superior potency , selectivity , and desirable pharmacokinetic properties . selected drug candidates are then validated in vivo and , upon verification of safety and efficacy , progressed to human trials . while the merits of this well - defined process are undeniable , including several major breakthroughs in anticancer therapy , this strategy is also associated with declining productivity in the pharmaceutical industry and limited success to tackle the most aggressive cancers of unmet therapeutic need . high attrition rates at late stages of drug development underlines that cancer heterogeneity across patients and adaptive drug resistance mechanisms are major obstacles for the development of effective and long - lasting anticancer targeted therapies . these challenges have stimulated out - of - the - box thinking in pharmacotherapy research ( e.g. , targeted polypharmacology , antibody - drug conjugates , innovative prodrug approaches , etc . ) and the re - examination of the core principles of drug discovery in complex diseases . the rise of modern phenotypic drug discovery together with the use of more clinically relevant disease models to guide early drug development are representative examples of the paradigm shift initiated in the field to trigger a positive inflection point . they govern a wide range of basic intracellular functions and coordinate cell - to - cell and extracellular matrix - to - cell communication to modulate cell and tissue physiology . consequently , their malfunctioning is directly linked to progressive diseases including cancer and inflammation . the success in the clinic of several anticancer kinase inhibitors has validated a number of kinases as oncotargets , while the increasing understanding of cancer cell biology has demonstrated the essential role of different kinases in tumor suppressor pathways ( antitargets ) . the vast majority of kinase inhibitors bind to the kinase adenosine triphosphate ( atp ) pocket . since all kinases ( > 500 ) necessarily possess this relatively well - conserved catalytic site , there is a great potential for cross - reactivity . in fact , even though most kinase inhibitors are developed from single target hypotheses , they typically display broad selectivity profiles which , in some cases , have resulted in unanticipated clinical applications ( e.g. , sorafenib ) . inhibitor promiscuity may also be advantageous for anticancer therapy when off - target activities assist to address bioactivity issues related to pathway redundancies , molecular heterogeneity , or resistance mechanisms . however , if these activities result in the inhibition of antioncogenic pathways or lead to severe side effects , drug promiscuity becomes a major drawback . paradoxically , some kinases may behave as a target or an antitarget depending on the cancer context . the expression of the activated fusion oncoprotein bcr - abl is a genetic abnormality associated with chronic myeloid leukemia ( cml ) , and abl inhibitors ( imatinib , dasatinib ) are clinically used in chronic phase cml treatment . also , abl family kinases are abnormally activated in various solid tumors , supporting their involvement in oncogenesis . however , abl ( abl1 ) and arg ( abl2 ) have been found to negatively modulate breast cancer progression in vivo , indicating that abl inhibition could be counterproductive for breast cancer treatment (= antitarget ) . this example serves to delineate the complexity of cancer etiology and highlights the necessity of developing kinase inhibitors with tailor - made pharmacodynamic profiles for the effective targeting of each cancer subtype . unfortunately , despite significant investments in the development of kinase inhibitors and the biomedical knowledge compiled over several decades , our still limited understanding of cancer biology prevents us from anticipating and optimally targeting the complex orchestrated actions that generate , maintain , and progress most neoplastic processes . acknowledging these limitations , many research groups including ours are frontloading the collection of robust empirical data to progress anticancer drug development programs away from classical black - and - white anticancer target hypotheses to more unbiased and evidence - led strategies for hit selection and lead generation . following that principle , in this manuscript we show that cooperative ligand - based design and phenotypic screening , complemented with biochemical assays and the use of published data ( literature , patents , etc . ) , can be effectively applied to accelerate the generation of preclinical drug candidates . our strategy builds on three wide - ranging hypotheses : ( i ) targeting the kinase atp pocket with compounds derived from promiscuous kinase inhibitors can enable rationally - biased serendipitous discoveries ; ( ii ) early optimization of drug - likeness can be concurrently applied to explore pharmacodynamic diversity ; and ( iii ) phenotypic screening of chemically related compounds in designated models of cancer can generate target - agnostic structure bioactivity relationships and tailor ligand optimization to particular cancer types / subtypes . by means of this pragmatic approach to anticancer kinase inhibitor discovery , target deconvolution of identified hits and leads is largely simplified , thereby assisting the mechanistic elucidation of the molecular targets and antitargets involved in the observed phenotype . in this work we describe how the implementation of such an approach led to the discovery of a kinase inhibitor with potent activity against breast cancer cells and a unique selectivity profile : the first small molecule able to inhibit src at subnanomolar levels with a 1000-fold selectivity over abl . pp1 is a promiscuous inhibitor that indiscriminately targets protein tyrosine kinases , many of which are involved in oncogenesis such as the src family kinases ( sfk ) , ret , kit , and abl . moreover , related derivatives developed thereafter have shown strong inhibition of a variety of kinases with relevance in cancer including igf-1r , egfr , btk , vegfr , pdgfr , pi3k , and mtor . according to the co - crystal structure of pp1 with hck and ret kinases , this small molecule is an archetypical type i kinase inhibitor , with its n5 and 4-nh2 groups forming multiple h - bonds with the hinge region of the kinase atp site ( figure 1 ) . the c3 p - tolyl group is positioned toward a hydrophobic region well - conserved across tyrosine kinases , thus being responsible for the partial selectivity of pp1 over other kinase families . although pp1 s potent inhibition of disease - associated kinases make it a valuable biological tool , its use is limited by low solubility in water and poor selectivity , major limiting factors for the clinical translation of many drug candidates . we envisaged that the substitution of pp1 s tert - butyl group at the n1 position with flexible water - solubilizing groups could be used to improve drug - like properties and , at the same time , explore the accessible sugar / phosphate regions occupied by the natural ligand atp in the search for novel binding affinity profiles . as shown in figure 1 , compounds were designed to display a cyclic tertiary amine connected to the n1 position of the pyrazolopyrimidine ring through an ethylene linker . following the route described in scheme 1 ( see full details in the supporting information ) , a highly focused 12-member library was generated by coupling a selection of cyclic secondary amines to an aldehyde - modified derivative of pp1 ( 6 ) via reductive amination . l by reductive amination using 12 commercially available cyclic secondary amines ( piperidines , morpholine , and piperazines ) . the 12-member library was then tested against human mammary adenocarcinoma mcf7 cells , using the inhibition of cell growth as the primary output of the screening . live - cell high - content image - based phenotypic assays were carried out to kinetically profile the antiproliferative response of mcf7 breast cancer cells following treatment with a single dose ( 100 m ) of compounds 7a l for 5 d. experiments were performed in triplicate , using cells treated with pp1 ( 100 m ) or dmso as a positive and negative control , respectively . cell proliferation was monitored by time - lapse imaging using an incucyte zoom microscope and analyzed by its software , enabling determination of cell density ( % confluence ) over time after compound addition . as shown in figure 2a , compounds 7d and 7i l ( colored lines ) led to a strong inhibition of cell proliferation , maintaining confluence levels equal or below those imaged at time zero ( point of compound addition ) . interestingly , all these hits presented at least two tertiary amines in their n1 motifs , providing the first insights into structure / antiproliferative activity relationships . ( a ) time - lapse imaging analysis of breast cancer mcf7 cell proliferation over 5 d. cells were imaged every 3 h using an incucyte zoom microscope and growth measured by its integrated software . curves represent % confluence of cells treated with compounds 7a l ( 100 m , 0.1% dmso ) over 5 d of incubation . dmso ( 0.1% , v / v ) was used as untreated cell control ( in black ) . ( b ) dose response curves and calculated ec50 values determined by prestoblue cell viability assay after incubation of mcf7 cells with compounds 7d and 7i l and the positive control pp1 ( dose range : 0.03100 m ) . half - maximal effective concentration ( ec50 ) values were then calculated for the five hits and pp1 in mcf7 cells using an 8-point half - log dose response study ( 0.03 to 100 m ) . based on the mcf7 growth kinetics data provided by the incucyte assay , cell viability was determined at day 5 using the prestoblue reagent . figure 2b shows that derivatives 7j and 7k were the most potent among the novel compounds , with ec50 values of 12 m . due to its potency and lower molecular weight , the dimethylamino - containing piperidinyl group of compound 7j was chosen as the n1 motif for the preparation of a second library of pyrazolopyrimidines . several investigations have reported that substitutions of the p - tolyl group at the c3 position of pp1 by different aryl moieties ( even closely related ones ) have a major impact on protein ligand binding . medicinal chemistry at that position has generated inhibitors for a variety of kinases , including receptor and nonreceptor tyrosine kinases ( e.g. , src , abl , ret , egfr , btk , pdgfrs , vegfrs , kit ) and nontyrosine kinases ( e.g. , pi3ks , mtor ) . to exploit this feature as an opportunity to expand the prospective pharmacodynamic scope of 7j , a selection of five arylboronic acids and phenylacetylene were employed to functionalize the c3 position of the heterocyclic ring by palladium - catalyzed cross - coupling chemistry to synthesize derivatives 9a f ( scheme 2 ) . together with the piperidine - containing compounds 9a f , the corresponding acetal intermediates 8a f were selected for testing against breast cancer cells to increase the chemical diversity of the screening . antiproliferative properties of compounds 8a f and 9a f were evaluated in mcf7 cells ( dose response studies ) , using compound 5 ( acetal intermediate of library 1 ) , 7j ( most potent derivative of library 1 ) , and pp1 as controls . as shown in figure 3 , compounds 8d and 9d ( both having a 7-azaindol-5-yl group at c3 ) exhibited superior antiproliferative properties ( ec50 < 2 m ) . remarkably , time - lapse imaging of compounds 8d and 9d ( 1100 m ) revealed patently distinct antiproliferative mode of actions ( figure 3b and movies s1 and s2 ) . compound 9d containing the piperidinyl moiety of 7j induced cell death , while its acetal precursor 8d halted cell division . ( a ) ec50 values calculated after treatment of mcf7 cells with acetal - functionalized compounds 5 and 8a f ( top panel ) and piperidine - functionalized derivatives 7j and 9a f ( bottom panel ) . ( b ) incucyte zoom images of mcf7 cells treated with 8d , 9d , and dmso at 0 , 48 , and 120 h after compound addition . inhibitor dose : 100 m . to identify the target / s that could be responsible of the phenotype induced by hits 8d and 9d in mcf7 cells and thereby shed some light over further optimization campaigns , inhibition activities were tested for pp1 , 7i k , 8d , and 9d against a selection of kinases involved in human cancer . kinase profiling ( reaction biology corp . , usa ) was performed by measuring p incorporation on the substrate ( poly [ glu , tyr ] 4:1 ) relative to dmso . dose response curves and calculated half - maximal inhibitory concentration ( ic50 ) values are shown in figure s1 and table 1 , respectively . derivatives 7i k and 9d strongly inhibited sfk members , whereas acetal - functionalized compound 8d preferentially inhibited mtor ( > 28-fold more potent than pp1 ) . the most potent inhibitor was compound 9d , which displayed similar potency to pp1 against src but higher selectivity over ret , pdgfr , and kit . interestingly , according to the inhibition profile of compounds 7i k ( only varying on the spacer between the dimethylamino group and the piperidine ) , potency and selectivity for src were enhanced by the proximity of the dimethylamino to the ring ( ic50(abl)/ic50(src ) for 7i = 50 ) . using the information provided by the phenotypic screening and the kinase inhibition profiling of leads 8d and 9d , two independent optimization campaigns were implemented toward the generation of anticancer drug candidates inhibiting mtor and sfk , respectively . optimization of lead 8d into the highly selective mtor inhibitor ecf309 has been reported elsewhere . herein is described the generation and structure activity relationships ( sar ) of pyrazolopyrimidine derivatives with high potency and selectivity for sfk . the data collected from the kinase profiling study indicated that the 2-[4-(dimethylamino)-1-piperidyl]ethyl group at the n1 position was optimal to generate sfk inhibitors of high selectivity , while the superior potency of 9d against src suggested that on - target potency could be further enhanced by optimization of the substituent at the c3 position . a literature survey on src inhibitors was thus performed to assist in the design of derivatives with improved src binding . we focused our attention on the c3 substituent of pp20 , a potent dual src / abl inhibitor developed by apsel et al . novel derivatives were designed by substituting the 7-azaindol-5-yl group at c3 of 9d with the functionalized phenyl ring found in pp20 , whereas the piperidines used for 7i and 7j were introduced at the n1 position . acetal deprotection of compound 4 in tfa / water ( 1:1 ) followed by reductive amination with either 4-(dimethylamino ) or 4-(dimethylaminomethyl)piperidine generated iodo intermediates 10a , b , respectively , which afforded derivatives 11a ( also known as ecf506 ) and 11b ( figure 4a ) by suzuki cross - coupling with 4-(n - boc - amino)-3-methoxyphenylboronic acid ( see synthesis in the supporting information ) . ( a ) structures of pp20 and compounds 11a and 11b . ( b ) ec50 values calculated after treating mcf7 and mda - mb-231 cells with compounds 11a , 11b , pp20 , and dasatinib ( dose range : 110,000 nm ) for 5 d. cell viability was determined using the prestoblue reagent . ( c ) western blot analysis of src and fak activity in mda - mb-231 cells treated with 11a and dasatinib for 1.5 h. ( d ) scratch - wound migration assay . mda - mb-231 cells were treated with 11a or dasatinib ( 10 nm ) , and cell migration compared with untreated cell control ( dmso , 0.1% , v / v ) at 6 , 12 , and 24 h. cells were imaged and analyzed using an incucyte - zoom microscope with integrated scratch - wound migration software module . error bars : sd from n = 3 ; p < 0.001 , * * * ; p < 0.01 , * * . ( e ) snapshots of the scratch - wound area of mda - mb-231 cells at 0 and 24 h. yellow lines highlight the gap formed by the scratch . blue lines indicate the advance of cells into the wound after 24 h incubation in the absence and presence of 11a ( 10 nm ) . antiproliferative properties of compounds 11a , b were then tested in cells using pp20 and dasatinib ( a clinically approved dual src / abl inhibitor ) as positive controls . along with mcf7 cells , breast adenocarcinoma mda - mb-231 cells were tested as a model of triple - negative breast cancer known to be particularly sensitive to src inhibitors . as shown in figure 4b , compound 11a induced a very potent antiproliferative effect in both mcf7 and mda - mb-231 cells , significantly superior to the activity displayed by derivatives 9d 11b , and pp20 . notably , 11a also outperformed the gold - standard src inhibitor dasatinib . inhibition of src kinase activity in cells was studied by western blot . upon activation study of phospho - src levels can then serve both as evidence for the presence of active src and to evaluate direct inhibition of src kinase activity . phosphorylation of the focal adhesion kinase ( fak ) , a downstream substrate of src with high relevance in cancer progression , was also studied in both mda - mb-231 ( figure 4c ) and mcf7 ( figure s2 ) cells . cell lysates were prepared following 24 h of serum starvation and 1.5 h exposure to each inhibitor and serum stimulation for 1 h. analyses demonstrated that 11a inhibits phosphorylation of src and fak at low nanomolar levels , with complete inhibition observed at 100 nm . dasatinib activity was close to that of 11a , although it also induced a dose - dependent increment of total src ( figure 4c ) . having demonstrated that 11a targets src in cells , we investigated fak activity and cell viability in cells that lack expression of src . syf murine embryonic fibroblasts ( src/ , yes/ , fyn/ ) were treated with either 11a or dasatinib and levels of total / phospho - fak analyzed by protein immunoblot . as expected ( figure s3 ) , fak activity was found to be unaffected by drug treatments , indicating that the inhibition of phospho - fak in the breast cancer cell lines was a consequence of src inhibition . compounds 11a and 11b displayed significantly less antiproliferative activity in syf cells than dasatinib , suggesting increased selectivity against src family kinases ( figure s3 ) . an automated 96-well scratch - wound cell migration assay ( analyzed in real - time by the incucyte zoom system ) was then set up to determine whether compound 11a could halt migration of mda - mb-231 cells , as would be expected for a src inhibitor . cells treated with 11a were monitored for 24 h at a dose range ( 110,000 nm ) and compared with untreated cells ( 0.1% v / v dmso ) and dasatinib treatment . compound 11a significantly reduced cell motility at 10 nm as early as 6 h into the study , with equivalent efficacy to dasatinib ( figure 4d , e ) . 11a treatments at higher concentrations induced greater inhibition of cell migration ( figure s4 ) , although cell viability was also significantly reduced . image - based measurement of caspase 3/7 activity in mcf7 breast cancer cells demonstrated significant levels of apoptotic cell death after 5 d treatment with 11a at concentrations 100 nm ( figure s4 ) , in accordance with the ec50 values observed for this cell line . ic50 values were determined for 11a and dasatinib against the panel of recombinant kinases used in previous screenings ( see ic50 values in table 1 and dose response curves in figure s5 ) . as expected , dasatinib induced potent inhibition of nonreceptor tyrosine kinases , such as abl and src , but also high potency against receptor tyrosine kinases ( e.g. , pdgfr , kit ) . in contrast , 11a exclusively inhibited sfk , with subnanomolar ic50 values against src and yes ( activities for the rest of the sfk members are shown in table s1 ) . it is important to highlight that 11a displayed a vast difference in activity ( > 950-fold difference ) between abl and its primary target src . while small molecule inhibitors with reverse binding capabilities have been developed ( e.g. , imatinib strongly targets abl without inhibiting src ) , this is , to the best of our knowledge , the first case of a small molecule with subnanomolar ic50 for src that requires a concentration 3 orders of magnitude greater to reach the same level of inhibition in abl . such properties have only been previously reported in peptide - based bisubstrate inhibitors of src . to shed light over the structural features responsible for the high antiproliferative properties and unique selectivity profile of 11a , a library of 24 closely related analogs was developed ( see syntheses in the supporting information ) by introducing small changes on key functional groups of 11a ( see figure 5 ) . mda - mb-231 was the cell line chosen for the screening due to its superior sensitivity to 11a treatment . cell viability assays were performed as previously described , and ec50 values calculated for each compound . ic50 values were subsequently determined against src and abl for those compounds exhibiting high antiproliferative activity ( 12d , 12e , 12 m , 12w , and 12x ) and for selected compounds with low activity ( 12a , 12i , 12j , 12 t , and 12v ) . structural motifs modified on 11a for the generation of compounds 12a x are highlighted as colored circles . ec50 values against mda - mb-231 breast cancer cells are shown below each structure . src and abl inhibition values ( as ic50 ) are reported for selected compounds . analysis of the properties of compounds 12a e ( figure 5 ) indicates that the presence of a tertiary amino group at the position 4 of the pyperidinyl ring is essential for the activity . substitution of the methyl groups by larger aliphatic rings was tolerated , however , with compounds 12d and 12e being among the most potent src inhibitors of the series . removal of the pyrimidine ring ( see compound 12f ) resulted in > 3000-fold decrease in antiproliferative activity . screening of compounds 12g x evidenced the limited structural variations permitted at the top of the molecule to achieve high bioactivity . most modifications of the boc and the methoxy groups of the phenyl moiety at the c3 position led to a significant reduction in bioactivity . even minor chemical modifications such as the substitution of the carbamate group by urea ( 12i ) or amide ( 12j ) , or the change of the methoxy by oh ( 12s ) , resulted in > 200-fold decrease in both antiproliferative properties and src inhibitory activity . remarkably , introduction of a benzylamino group instead of the boc was tolerated well , as observed from the bioactivity exhibited by compound 12 m . however , introduction of endocyclic nitrogen atoms in the ring ( see 12n and 12o ) dramatically reduced compounds activity , particularly in the ortho position . interestingly , while the methoxy group is required for maintaining high levels of activity when the boc group is present in the structure ( see 12s u ) , substitution of the boc group by an ester tolerated the elimination of the methoxy group ( see 12w and 12x ) . most kinase inhibitors , especially adenine analogues , bind to the catalytic domain of the enzyme in its active conformation , thus competing with the natural substrate atp . to determine whether this was the mode of binding of 11a to its primary target src , experiments were designed to obtain km values for atp in the absence and presence of different concentrations of inhibitor 11a . the reactions were monitored every 10 min to obtain progress curves with time course ( figure s6 ) . these were found to be linear regardless of the compound s concentration , suggesting that src inhibition by 11a is not time - dependent (= reversible ) . the slopes ( m / min ) were represented against atp concentration for a michaelis burk plot ( double - reciprocal plot ) , using graphpad prism software ( figure s6 ) . apparent km increased when inhibitor concentration increased and all lines converged on the y - axis in the double - reciprocal plot , thus suggesting that 11a is competitive with respect to atp against src . response curves of src inhibition by 11a at different concentrations of atp are plotted in figure s7 . calculated ic50 values increased in direct proportion to atp concentration , further confirming that 11a is a type i kinase inhibitor and therefore binds to the src atp site in its active conformation . global fit analysis using grafit software provided a ki of 0.8 nm ( figure s7 ) . to gain insight on the selective binding affinity of 11a for src over abl , in silico docking studies were performed with both the dual abl / src inhibitor pp20 and the src - selective inhibitor 11a . docking of pp20 into src and abl produced a plausible predicted binding mode in both cases ( figure 6a , b ) , with the adenine - mimicking pyrazolopyrimidine making h - bonds to the hinge region of the proteins . the tert - butyl group of the carbamate occupies a hydrophobic pocket formed by several lipophilic residues in src and abl , including the phe of their aspartate - phenylalanine - glycine ( dfg ) motif . free energy of binding was predicted by autodock to be approximately the same for both proteins ( 8.5 and 8.9 kcal / mol , respectively ) . docking of 11a into src also produced a plausible predicted binding mode ( figure 6c ) and a higher predicted affinity than pp20 ( calculated free energy of binding of 10.6 kcal / mol ) . in src , the dimethylamino group of the ethylpiperidinyl moiety on n1 interacts with asp404 , one of the protein s phosphate binding residues ( see figure 6c , e ) . in contrast , the large n1 group of 11a ( compared to the isopropyl on pp20 ) clashes with tyr253 in the catalytic site of abl . this resulted in autodock being unable to find a position in the atp binding site of abl where 11a could mimic the interactions made by the adenine moiety of atp ( figure 6d ) . this study indicates that the flexible polyamine linker on n1 is responsible for the low affinity of compound 11a for abl . ( a - d ) predicted binding modes of pp20 and 11a in src and abl kinases . ( a ) pp20 in src , ( b ) pp20 in abl , ( c ) 11a in src , and ( d ) 11a in abl . the p - loop , c helix , and dfg motif are in red , orange , and magenta , respectively . pp20 and 11a are represented by sticks , where carbon atoms are in black , nitrogen atoms are in blue , and oxygen atoms are in red . ( e ) 11a in src active site compared to adenosine monophosphate ( amp ) ( from pdb 3dqx ) . amp is represented by sticks , where carbon atoms are in green , nitrogen atoms are in blue , oxygen atoms are in red , and the phosphorus atom in orange . predicted binding mode indicates that the dimethylamino group at the piperidine ring is optimally placed to interact with the carboxylic group of asp404 ( dfg motif , in pink ) . developing zebrafish provides a rapid phenotypic assay to simultaneously test safety and efficacy of novel compounds in a living vertebrate . small molecule phenotypic - based screens in zebrafish have recently implicated src kinase in the migration of the posterior lateral line primordium , a cohesive cluster of cells that migrates horizontally under the skin along the myoseptum to the end of the tail , periodically depositing neuromasts . to determine the effects of 11a on cell migration in vivo , we treated tg(brn3c : mgfp ) transgenic zebrafish that express green fluorescent protein ( gfp ) in the mechanosensory hair cells of the lateral line ( which form part of the neuromasts ) with 11a for 2 d and measured the distance of the last neuromast to the tip of the tail ( marked by the end of the notochord and the presence of black melanocytes , figure 7a , in red ) . 11a significantly reduced neuromast migration ( > 100 m in average ) with minimal effect on the development of the embryos ( figure 7a c ) . in contrast , dasatinib treatment at > 10 m resulted in severe cardiotoxicity and death of most embryos . at concentrations that were compatible with embryo survival ( 110 m ) , dasatinib did not inhibit the migration of neuromasts , whereas it did still induce a patent cardiotoxic phenotype ( note heart enlargement in figure 7c ) . further safety studies ( see figure s8 ) showed that dual abl / src inhibitor pp20 also induces severe cardiotoxicity in zebrafish even after short treatment . these results , which correlate with the essential role of abl in heart development and healing , suggests that the selectivity of 11a over abl might be advantageous for therapy when abl inhibition is not required . fresh e3 media with dmso or 11a ( 500 m ) was added to zebrafish embryos at 20 hpf , 36 hpf , and 48 hpf and imaged at 72 hpf . ( a ) representative images of the tail of a 3 dpf zebrafish without ( top ) and with 11a treatment ( bottom ) . neuromasts are identified by gfp expression ( green ) and the tip of the notochord as a red line . ( b ) imaging analysis of the distance between the last neuromast and the tip of the tail ( n = 3 ) under treatment with dmso ( negative control ) or 11a ( 500 m ) . ( c ) study of zebrafish heart development under short treatment with 11a ( 500 m ) and dasatinib ( 10 m ) . compounds were added to 2 dpf zebrafish embryos and incubated for 4 h ( n = 3 ) . subsequently , fresh media was added , and the fish imaged after 48 h incubation . compound 11a displays very high solubility in water ( > 100 mg / ml , ph = 7.4 ) , a significant advantage over dasatinib , whose solubility in water ranges from moderate ( 18 mg / ml at ph = 2.6 ) to extremely low ( < 0.001 mg / ml at ph = 7 ) . notably , analysis of clogp with the osiris property explorer provided an estimated value of 2.2 for 11a , while experimental log d at ph = 7.4 was determined to be 0.04 . inhibition of herg channel and cytochrome p450 ( cyp ) enzymes was determined for 11a to assess off - target liabilities associated with cardiotoxicity ( qt interval prolongation ) and potential drug drug metabolic interactions . results demonstrated weak herg inhibition ( 50.1% inhibition at 25 m ) and marginal inhibition of cyp enzymes at 10 m ( figure s9 ) . a plasma protein binding assay was performed with compound 11a to test to what extent the compound can bind to proteins in the blood , an important factor to predict free drug levels in the body . warfarin ( positive control , 99100% bound ) and 11a were incubated with human or rat plasma in duplicates at 37 c for 24 h and the free ( unbound ) fraction of compound determined by lc - ms / ms . percentages of unbound 11a were 9.4 and 19.1% in rat and human plasma , respectively . in vitro metabolic stability of 11a was systematically assessed in human liver microsomes ( hlm ) , blood plasma , and primary hepatocytes ( cyprotex , uk ) . lc - ms / ms analysis of 11a showed excellent compound stability ( 94% ) after 30 min incubation with hlm at 37 c . 11a was subsequently incubated ( 1 m , 2 h , 37 c ) with plasma from either human , mouse , or rat and analyzed by lc - ms / ms at different time points . regardless of the species , analyses showed full plasma stability ( figure s10 ) . lastly , compound 11a ( 3 m ) was incubated with mouse and human primary hepatocytes for 60 min and samples analyzed by lc - ms / ms at various time points . verapamil and umbelliferone were used as control compounds . in agreement with the hlm study , compound 11a showed high stability in human hepatocytes with a half - life of 751 min ( figure s10 ) . as expected , faster clearance was observed in mice hepatocytes ( 7-fold decrease in half - life ) . encouraged by the in vitro dmpk profile of 11a , an in vivo study was performed in mice to determine oral bioavailability and half - life ( cyprotex , u.k . ) . female cd1 mice were given a single dose of 10 mg / kg ( 0.25 mg/25 g mouse ) of compound 11a orally ( as a solution in nanopure water ) or via iv injection ( as a saline solution ) . blood / plasma samples were taken at different time points ( final sample taken after 8 h ) , and compound levels measured by lc - ms / ms ( figure s11 ) . the calculated half - life value for 11a was 2.9 h , substantially higher than that reported for dasatinib in mice ( 0.9 h ) . compound 11a showed moderate oral bioavailability ( 25.3% ) , although it is important to note that no excipients ( just pure water ) were used for its oral formulation . the presence of active ( phosphorylated ) src in human colorectal cancer hct116 cells and its inhibition under 11a treatment was verified by western blot ( figure s12 ) . subsequently , an in vivo pd study was performed in a xenograft model of hct116 cells in mice . cells were injected subcutaneously , and tumors were allowed to grow up to 34 mm in diameter . mg / kg , in nanopure water ) or vehicle ( nanopure water ) by oral gavage and culled 3 h after the last dose ( n = 4 ) . tumors were excised , fixed , and sections labeled for phospho - src and stained with hematoxylin . as shown in figure 8 , microscopy analysis demonstrated significant reduction of phospho - src in the xenograft sections from mice treated with 11a relative to the untreated animal controls . ( a ) images of representative sections ( low and high resolution ) of hct116 xenografts from ( left ) untreated mice and ( right ) mice treated with 11a ( n = 4 ) . quantification of immunohistochemistry across tumor tissue sections from untreated animals ( water ) and 11a treated groups performed in blinded fashion . to move away from orthodox target - centric strategies , a pragmatic approach for rapid discovery of novel high - quality anticancer kinase inhibitors was implemented through the generation and phenotypic screening of analogues of pp1 , a pyrazolopyrimidine known to inhibit multiple oncogenic pathways . derivatives of this promiscuous kinase inhibitor were designed to improve physicochemical properties and explore favorable pharmacological features through small chemical modifications . to accelerate the advance from hits to leads to drug candidates , compounds were screened by phenotypic assays in the search for derivatives with potent antiproliferative properties . chemical design was biased toward human breast cancer treatment ( rather than to a particular target ) by using the breast adenocarcinoma cell line mcf7 as a discriminating cell model . such a pseudotarget - agnostic strategy identified compounds that inhibited pathways involved in breast cancer survival and also disregarded compounds with low cell penetrability (= deficient drug - likeness ) . after two rounds of design , synthesis , and screening of highly focused libraries , novel candidates with low micromolar activity in mcf7 cells and high solubility in water were identified ( compounds 8d and 9d ) . elucidation of the potential target / s linked with their bioactivity was followed by a literature survey to explore chemical motifs that could further improve these phenotypic leads . from that point , this manuscript has focused on the development of src inhibitor 11a , a novel pyrazolopyrimidine analogue that exhibits potent antiproliferative properties against breast cancer cells , halts mda - mb-231 cell migration , and inhibits intracellular src signaling at low nanomolar concentrations . remarkably , kinase profiling revealed that 11a is the first small molecule that inhibits src at subnanomolar concentration ( ic50 < 0.5 nm ) with a 1000-fold selectivity over abl . this is of relevance because the manifestation of cardiac events , especially in elderly patients , is a well - established adverse effect of abl inhibition . synthesis and phenotypic screening of 24 closely related derivatives of 11a were carried out to gain further insight into the optimal chemical space for the generation of potent , selective , and cell - active src inhibitors . analysis of sar provided evidence of the strict structural requirements of the pyrazolopyrimidine scaffold required to create such a class of antiproliferative src / non - abl inhibitors . the relatively low molecular weight of 11a ( 510 da ) and its good physicochemical properties , including excellent water solubility and clogp of 2.2 , prompted us to investigate further the biological properties of this compound . biochemical and in silico studies confirm that 11a is a type i kinase inhibitor of src and provided rationale for its selective affinity for src over abl . screening of herg channel and cyp induction , together with in vitro and in vivo pk / pd assays ( including zebrafish pd / safety studies ) support the nomination of 11a as a src - targeting drug candidate that could potentially offer a superior therapeutic window than dual src / abl inhibitors . this novel src - targeting inhibitor belongs to a second generation of src inhibitors that do not target the abl kinase , being the first one to demonstrate suppression of src phosphorylation in vivo . finally , in concordance with the primary role of src in driving tumor invasion and drug resistance mechanisms , more in - depth preclinical proof - of - concept studies using appropriate in vivo models of disease progression and rational drug combination studies will be required to demonstrate the efficacy and therapeutic value of src / no - abl inhibitors in cancer treatment . nonmicrowave reactions were performed under an inert atmosphere of nitrogen using anhydrous solvents . all commercially available chemicals were obtained from either fisher scientific , matrix scientific , sigma - aldrich , or vwr international ltd . nmr spectra were recorded at ambient temperature on a 500 mhz bruker avance iii spectrometer . chemical shifts are reported in parts per million ( ppm ) relative to the peak of the solvent . the data are presented as follows : chemical shift , integration , multiplicity ( s = singlet , d = doublet , t = triplet , q = quartet , m = multiplet ) , coupling constants ( in hertz , hz ) and interpretation . tlcs were ran on merck tlc silica gel 60 f254 plates , typically 5 10 cm , and monitored using a 254 nm uv source or permanganate staining . purifications were carried out using flash column chromatography with commercially available silica gel and solvents . all compounds used in the biological screenings were determined to be > 95% pure by analytical hplc with evaporative light scattering detection ( agilent ) . 5-amino-1h - pyrazole-4-carbonitrile , 1 ( 3 g , 27.77 mmol ) , and formamide ( 15 ml ) were added to a 20 ml microwave vial , and the mixture heated at 180 c for 2 h using microwave radiation . the precipitate formed on cooling was filtered off and washed with water ( 50 ml ) and allowed to dry giving the product as a pale brown solid ( 3.5 g , 25.9 mmol , 93% ) . the experiment was repeated to give a second batch of product ( 3.44 g , 25.5 mmol , 92% ) . h nmr ( 500 mhz , dmso ) 13.34 ( s , 1h ) , 8.13 ( s , 1h ) , 8.07 ( s , 1h ) , 7.69 ( br . m , 2h ) ; c nmr ( 126 mhz , dmso ) 158.2 , 156.0 , 155.0 , 132.8 ( ch ) , 99.8 ; ms ( es + ve ) ( m + h ) : 136.0 , 157.9 ( + na ) , ( es ve ) ( m h ) : 133.9 . compound 2 ( 1.5 g , 11.11 mmol ) was suspended in 15 ml of dmf , and n - iodosuccinimide ( 1.2 equiv , 3.0 g , 13.3 mmol ) added . etoh ( 80 ml ) was added to the reaction , and a precipitate began to form , which was aided by sonication . the precipitate was filtered and washed with etoh ( 3 , 20 ml ) and allowed to dry in an oven at 40 c overnight to give a sand colored solid ( 2.115 g , 8.1 mmol , 73% ) . h nmr ( 500 mhz , dmso ) 13.80 ( s , 1h ) , 8.16 ( s , 1h ) , 7.796.44 ( m , 2h ) ; c nmr ( 126 mhz , dmso ) 157.6 , 156.1 , 155.0 , 102.5 , 89.8 ; ms ( es + ve ) ( m + h ) : 283.9 ( + na ) , ( es ve ) ( m h ) : 259.9 , 287.8 ( na ) . to a solution of 3 ( 500 mg , 1.9 mmol ) in dmf ( 15 ml ) was added sodium hydride ( 1.5 equiv , 2.9 mmol , 60% dispersion in mineral oil , 115.2 mg ) , and the solution allowed to stir for 30 min until the gas evolution stopped . bromoacetaldehyde diethyl acetal ( 1.5 eq 2.9 mmol , 0.435 ml ) was then added dropwise , and the mixture heated at 150 c in the microwave for 40 min . etoac and water ( 50 ml ) were added to the mixture , and the organics separated . the aqueous layer was washed with etoac ( 50 ml , 3 ) , and the organics combined and washed with water ( 3 , 30 ml ) , dried over anhydrous mgso4 , and concentrated in vacuo . the crude product was purified by column chromatography meoh / dcm ( 05% ) to give a light orange solid ( 461 mg , 1.2 mmol , 64% ) . h nmr ( 500 mhz , dmso ) 8.21 ( s , 1h ) , 7.906.30 ( m , 2h ) , 4.93 ( t , j = 5.7 , 1h ) , 4.33 ( d , j = 5.8 , 2h ) , 3.62 ( dq , j = 9.4 , 6.9 , 2h ) , 3.40 ( dq , j = 9.6 , 7.0 , 2h ) , 0.98 ( t , j = 7.0 , 6h ) ; c nmr ( 126 mhz , dmso ) 157.9 , 156.3 ( ch ) , 154.0 , 103.2 , 99.5 , 89.5 , 61.4 ( ch2 ) , 48.8 ( ch2 ) , 15.39 ( ch3 ) ; ms ( es + ve ) ( m + h ) : 377.8 , 400.0 ( na ) , ( es ve ) ( m h ) : 376.0 . to a solution of 4 ( 1.135 g , 3.0 mmol ) in dioxane / water ( 10 ml/1 ml ) were added 7-azaindole-5-boronic acid pinacol ester ( 1.5 equiv , 614 mg , 4.5 mmol ) , potassium carbonate ( 1.5 equiv , 624.7 mg , 4.5 mmol ) , and followed by palladium acetate ( 5 mol % , 33.8 mg ) , and the mixture heated in the microwave at 120 c for 1 h. etoac and water ( 50 ml ) were added to the mixture , and the organic layer separated , dried over anhydrous mgso4 , and concentrated in vacuo . purified by column chromatography , meoh / dcm ( 06% ) , to give a white solid ( 93 mg , 0.253 mmol , 96% ) . h nmr ( 500 mhz , cdcl3 ) 9.53 ( s , 1h ) , 8.62 ( d , j = 1.9 , 1h ) , 8.41 ( s , 1h ) , 8.24 ( d , j = 2.0 , 1h ) , 7.45 ( d , j = 3.4 , 1h ) , 6.62 ( d , j = 3.5 , 1h ) , 6.295.86 ( br . s , 2h ) , 5.14 ( t , j = 5.7 , 1h ) , 4.62 ( d , j = 5.7 , 2h ) , 3.79 ( dq , j = 9.4 , 7.0 , 2h ) , 3.55 ( dq , j = 9.4 , 7.0 , 2h ) , 1.14 ( t , j = 7.0 , 6h ) ; c nmr ( 126 mhz , cdcl3 ) 156.8 , 154.6 , 153.4 ( ch ) , 148.9 , 143.8 , 142.8 ( ch ) , 128.8 ( ch ) , 126.8 ( ch ) , 121.3 , 120.5 , 101.8 ( ch ) , 99.9 ( ch ) , 98.5 , 62.2 ( ch2 ) , 49.3 ( ch2 ) , 15.3 ( ch3 ) ; ms ( es + ve ) [ m + h ] : 368.2 , 390.2 ( na ) , ( es ve ) [ m h ] : 366.2 ; hrms ( es + ve ) , c18h22n7o2 ( m + h ) : calculated 368.18295 , found 368.18090 . 5 ml of water was added , followed by 5 ml of tfa , and the mixture heated at 100 c for 1 h. the mixture was concentrated in vacuo to give a light brown oil which was used without further purification . n , n - dimethyl-1-(4-piperidyl)methanamine ( 1.5 equiv , 0.245 mmol , 34.8 mg ) was added followed by a drop of acetic acid . the mixture was allowed to stir for 10 min , then sodium triacetoxyborohydride ( 1.5 equiv , 0.245 mmol , 51.9 mg ) was added , and the mixture allowed to stir for 2 h. the mixture was concentrated in vacuo , and the product purified by column chromatography , meoh / dcm ( 510% then 10% with 520 drops of nh3 aq./100 ml ) to give a light orange solid ( 15.3 mg , 0.0365 mmol , 15% ) . h nmr ( 500 mhz , meod ) 8.52 ( s , 1h ) , 8.29 ( d , j = 2.0 , 1h ) , 8.28 ( s , 1h ) , 7.52 ( d , j = 3.5 , 1h ) , 6.62 ( d , j = 3.5 , 1h ) , 4.64 ( t , j = 6.4 , 2h ) , 3.25 ( d , j = 11.7 , 2h ) , 3.13 ( t , j = 6.3 , 2h ) , 2.96 ( d , j = 7.2 , 2h ) , 2.84 ( s , 6h ) , 2.37 ( t , j = 11.4 , 2h ) , 1.88 ( m , 1h ) , 1.80 ( d , j = 13.1 , 2h ) , 1.361.31 ( m , 2h ) ; c nmr ( 126 mhz , meod ) 161.8 , 158.6 , 155.5 ( ch ) , 154.3 , 148.2 , 143.7 , 141.8 ( ch ) , 128.7 ( ch ) , 127.1 ( ch ) , 120.7 , 100.6 ( ch ) , 98.2 , 62.7 ( ch2 ) , 56.3 ( ch2 ) , 52.2 ( ch2 ) , 43.4 ( ch2 ) , 42.7 ( ch3 ) , 30.9 ( ch ) , 28.4 ( ch2 ) ; ms ( es + ve ) [ m + h ] : 420.2 ; hrms ( es + ve ) , c22h29n9 [ m + h ] : calculated 420.25404 , found 420.254249 . 150 mg ( 0.398 mmol ) of 4 was added to a 10 ml microwave tube . 2.5 ml of water and 2.5 ml of tfa were added , and the mixture heated to 100 c for 1 h. the mixture was concentrated in vacuo to give a white solid which was used without further purification . 4-(n , n - dimethylamino)piperidine ( 1.5 equiv , 0.598 mmol , 76.6 mg ) was added followed by a drop of acetic acid and the mixture allowed to stir for 10 min . sodium triacetoxyborohydride ( 1.5 equiv , 0.598 mmol , 126.8 mg ) was added , and the mixture allowed to stir for 17 h overnight . the mixture was concentrated in vacuo , and the product purified by column chromatography , meoh / dcm ( 010% then 520 drops of nh3 aq . per 100 ml ) to give a light orange / brown solid ( 163.8 mg , 0.405 mmol , 99% ) . h nmr ( 500 mhz , meod ) 8.22 ( s , 1h ) , 4.49 ( t , j = 6.4 , 2h ) , 3.32 ( s , 3h ) , 3.15 ( d , j = 12.1 , 2h ) , 2.96 ( ddd , j = 16.0 , 8.0 , 4.0 , 1h ) , 2.91 ( t , j = 6.4 , 2h ) , 2.72 ( s , 6h ) , 2.15 ( td , j = 12.0 , 2.0 , 2h ) , 2.041.96 ( m , 2h ) , 1.54 ( qd , j = 12.2 , 3.9 , 2h ) ; c nmr ( 126 mhz , meod ) 158.07 ( c ) , 155.67 ( ch ) , 153.70 ( c ) , 103.59 ( c ) , 86.97 ( c ) , 63.18 ( ch ) , 55.86 ( ch2 ) , 51.38 ( ch2 ) , 44.37 ( ch2 ) , 39.31 ( ch3 ) , 26.42 ( ch2 ) ; ms ( es + ve ) to a solution of 10a ( 50 mg , 0.1205 mmol ) in dioxane / water ( 4.5 ml/0.5 ml ) were added [ 4-(tert - butoxycarbonylamino)-3-methoxy - phenyl]boronic acid ( 1.5 equiv , 48.3 mg , 0.181 mmol ) , potassium carbonate ( 1.5 equiv , 25.0 mg , 0.181 mmol ) , and triphenylphosphine ( 20 mol % , 9.5 mg ) , followed by palladium acetate ( 5 mol % ) , and the mixture heated in the microwave at 120 c for 1 h. etoac ( 50 ml ) and water ( 50 ml ) were added to the mixture , and the organic layer separated . the aqueous layer was washed with etoac ( 20 ml , 2 ) , and the organics combined , dried over anhydrous mgso4 , and concentrated in vacuo . the crude product was purified by column chromatography , meoh / dcm ( 010% then 520 drops of nh3 aq . per 100 ml ) to give a light brown solid ( 23.1 mg , 0.0453 mmol , 38% ) . h nmr ( 500 mhz , meod ) 8.27 ( s , 1h ) , 8.08 ( d , j = 8.2 , 1h ) , 7.30 ( d , j = 1.8 , 1h ) , 7.26 ( dd , j = 8.2 , 1.9 , 1h ) , 4.56 ( t , j = 6.7 , 2h ) , 3.98 ( s , 3h ) , 3.14 ( d , j = 11.9 , 2h ) , 2.94 ( t , j = 6.7 , 2h ) , 2.39 ( m , 7h ) , 2.14 ( dd , j = 12.0 , 10.0 , 2h ) , 1.90 ( d , j = 12.5 , 2h ) , 1.57 ( s , 9h ) , 1.49 ( qd , j = 12.1 , 3.6 , 2h ) ; c nmr ( 126 mhz , meod ) 158.53 ( c ) , 155.40 ( ch ) , 154.12 ( c ) , 153.46 ( c ) , 149.24 ( c ) , 145.02 ( c ) , 128.78 ( c ) , 127.38 ( c ) , 120.43 ( ch ) , 119.56 ( ch ) , 110.28 ( ch ) , 97.73 ( c ) , 80.18 ( c ) , 62.29 ( ch ) , 56.22 ( ch2 ) , 55.07 ( ch3 ) , 52.20 ( ch2 ) , 44.00 ( ch2 ) , 40.06 ( ch3 ) , 27.24 ( ch2 ) , 27.21 ( ch3 ) ; ms ( es + ve ) [ m + h ] : 511.3 ; hrms ( es + ve ) , c26h38n8o3 [ m + h ] : calculated 511.31396 , found 511.3151 . mcf7 , mda - mb-231 , and syf cells were grown in dulbecco s modified eagle medium ( dmem ) , supplemented with serum ( 10% fetal bovine serum ) and l - glutamine ( 2 mm ) , and incubated in a heracell 240i tissue culture incubator at 37 c and 5% co2 . cells were plated in 96-well nunc black optical - bottom plates ( thermo scientific ) at 1000 cells / well in 100 l of dmem medium containing 10% fbs and 2 mm l - glutamine and incubated for 48 h in an incubator at 37 c and 5% co2 . the media was replaced with fresh media containing 100 m concentration of 7a l , pp1 , or dmso ( 0.1% v / v ) , and the plates imaged in the incucyte zoom system . cell growth was monitored at sequential time points over 5 d using the bright - field microscopic images acquired by the incucyte zoom system . cells were plated in 96-well plates at 2000 cells / well in 100 l of dmem medium containing 10% fbs and 2 mm l - glutamine and incubated for 48 h in an incubator at 37 c and 5% co2 . after 48 h , the media was aspirated from each well and replaced with 95 l of fresh medium . compounds , including dmso , were prepared at 20 in dmem medium in a separate 96-well intermediate plate . 5 l from the intermediate plate was then added to each well containing cells . after 5 d , prestoblue cell viability reagent ( 10 l ) was added to each well , and the plates incubated for 6090 min . fluorescence emission was detected using an envision fluorescence plate reader ( excitation 540 nm , emission 590 nm ) . all conditions were normalized to the untreated cells ( 100% ) , and curves were fitted using a four parameter logistic fit with minimum value constrained to zero using graphpad prism software , to calculate ec50 values . cells were plated in 96-well nunc black optical - bottom plates ( thermo scientific ) at 3000 cells / well in 100 l of dmem medium containing 10% fbs and 2 mm l - glutamine and incubated for 48h in an incubator at 37 c and 5% co2 . the media was replaced with 95 l of fresh media containing nucview 488 from biotium at 1 m concentration , and drugs or dmso added along a concentration gradient , as described in the cell viability assay , and the plates imaged in the incucyte . cell growth was monitored over 5 days using bright - field and nucview 488 fluorescence ( excitation 460 nm , emission 524 nm ) microscopy . cell confluence and apoptotic count ( positive nucview 488 emission signal threshold above background signal ) were performed by the incucyte software . the numbers generated from the applied confluence and nucview 488 labeled cells masks were divided so as to create a ratio of number of apoptotic cells to cell confluence . the data were then normalized to dmso to account for the decreased number of cells found in higher concentrations of drug treatment . cells were plated at 1 10 cells / well in 2 ml of dmem medium containing 10% fbs and 2 mm l - glutamine in 6-well plates and incubated at 37 c with 5% co2 . after 24 h , the media was aspirated and replaced with 2 ml of dmem medium containing 0.1% fbs and 2 mm l - glutamine and the cells incubated for a further 24 h. two l of compounds dissolved in dmso at appropriate concentration was then added to each well and plates incubated for 1.5 h. 222 l of fbs was then added to each well ( giving a final concentration of 10% ) , and cells incubated for 1 h. cell lysates were then prepared using 100 l of cell lysis buffer ( 1% triton x-100 , 50 mm hepes , ph 7.4 , 150 mm nacl , 1.5 mm mgcl2 , 1 mm egta , 100 mm naf , 10 mm sodium pyrophosphate , 1 mm na3vo4 , 10% glycerol and protease and phosphatase inhibitors ) per well . the total cell protein concentration in each lysate was determined using precision red advanced protein reagent # 2 from cytoskeleton . lysates were normalized to 2 mg / ml and boiled at 100 c for 3 min in sds - page sample buffer ( 40% glycerol , 8% sds , 0.1 m dtt , 0.25 m tris - hcl , ph 6.8 ) . samples were resolved by sds - page using biorad 415% precast gels over 60 min at 140 v and transferred to pvdf membranes over 150 min at 210 ma . nonspecific antibody binding was blocked by incubation of membranes for 1 h at room temperature using western blocking reagent solution ( roche ) prior to adding primary antibodies in 0.5% blocking buffer at 4 c overnight . membranes were washed with tbs / t ( 3 , 5 min ) and then secondary antibody linked to horseradish peroxidase ( hrp ) added for 1 h at room temperature . following further washing with tbs / t ( 3 , 5 min ) and tbs ( 2 , 5 min ) , hrp was detected by peroxidase enhanced chemiluminescence ( pod ecl from roche ) and bands visualized using x - ray film or the chemidoc mp imaging system from biorad . cells were plated at 50,000 cells / well in 100 l of dmem medium containing 10% fbs and 2 mm l - glutamine in a 96-well imagelock plate from essen bioscience and left overnight to adhere in an incubator at 37 c and 5% co2 . scratch wounds were created in each well using the woundmaker supplied by essen bioscience , and each well washed with media ( 100 l , 2 ) to remove floating cells . compounds , including dmso , were prepared at 20 in dmem medium in a separate plate , and 5 l was then added to each well containing cells . images were recorded every 30 min using the incucyte - zoom for 24 h. analysis of cell migration into the wound was performed using the incucyte software . radioisotope - based assay ( [ -p ] atp ) consisting of measuring p incorporation on the substrate ( poly [ glu , tyr ] 4:1 ) relative to dmso . compound ic50 values were determined from 10-point , 1:3 dilution curves starting at 10 m , by reaction biology corp , with 10 m atp . kinase reaction buffer : 20 mm hepes - hcl , ph 7.5 , 10 mm mgcl2 , 1 mm egta , 0.02% brij35 , 0.1 mm na3vo4 , 0.02 mg / ml bsa , 2 mm dtt , and 1% dmso . np_005408 ) , c - terminal his - tagged , expressed in insect cells , mw = 62.3 kda reaction conditions : 0.6 nm src , 0.2 mg / ml poly [ glu , tyr ] 4:1 , and varied atp . experimental procedure : the kinase assays were performed at room temperature . 11a was added as 10-point dose ic50 mode into the enzyme / substrate mixture using acoustic technology with 20 min preincubation . the atp , substrate , and 11a concentrations tested were as follows : ( i ) atp concentrations tested : 1 , 5 , 15 , 75 , and 150 m atp ; ( ii ) substrate concentration : constant at 0.2 mg / ml ; ( iii ) 11a concentrations tested : 10-point dose ic50 with 3-fold serial dilution started at 100 nm ; ( iv ) time points measured : 0 , 5 , 10 , 15 , 20 , 30 , 45 , 60 , 75 , 90 , 105 , and 120 min . pdb 3geq was selected to represent src as it contained pyrazolopyrimidine ligand pp2 , which most closely resembles compounds pp20 and 11a , and its dfg motif is in the active conformation . similarly , pdb 4twp was selected as the abl structure as its dfg motif is also flipped in the atp - interacting position . the side chain of asp381 in the abl dfg motif was manually rotated in pymol to match the atp - binding mode of the equivalent asp in src . abl in this structure also contains a t316i mutation which was reverted to wild type . models of pp20 and 11a were built using the structure and binding mode of pp2 as a template . water molecules and other heteroatoms were removed from the optimized structures , and the program pdb 2pqr 1.8 used to assigned position - optimized hydrogen atoms , utilizing the additional propka algorithm with a ph of 7.4 to predict protonation states . the mgltools 1.5.4 utility prepare_receptor4.py was used to assign gasteiger charges to atoms . hydrogen atoms were assigned to compound structures using openbabel 2.3.2 , utilizing the -p option to predict the protonation states of functional groups at ph 7.4 . autodock 4.2.3 was used to automatically dock the compounds into the atp binding pocket of the crystal structures . a grid box that encompassed the maximum dimensions of the cognate atp ligand plus 12 in each direction was used . the starting translation and orientation of the ligand and the torsion angles of all rotatable bonds were set to those of the modeled compounds . the autodock parameter file specified 10 lamarckian genetic algorithm runs , 15,000,000 energy evaluations and a population size of 300 . transgenic tg(brn3c : mgfp ) zebrafish embryos were collected from breeding pairs and reared at 28 c in e3 embryo media . one dpf embryos were treated with 11a at different doses ( 10750 m ) at 20 hpf , 36 hpf and 48 hpf , or dmso ( 0.1% v / v ) . safety assays : wild - type zebrafish embryos were collected from ab - tpl breeding pairs and reared at 28 c in e3 embryo media . one dpf embryos were treated with 11a or dasatinib at 100 m , and dmso ( 0.1% v / v ) as negative control , for 4 h before being washed off and replaced with fresh e3 media . for pp20 treatment , the fish were incubated for 2 h post - amputation then replaced with fresh e3 media . the embryos were left to develop in e3 media at 28 c for 2 d , after which , they were imaged by light microscopy . zebrafish husbandry was performed under home office license in compliance with the animals ( scientific procedures ) act 1986 and approved by the university of edinburgh ethics committee . three female cluster of differentiation 1 ( cd1 ) mice , 2530 g , were dosed per administration route per time point , per compound . 11a was administered either orally or intravenously ( typical dose level of 10 mg compound per kg body weight ) . at the following time points , the animals were anaesthetised , blood collected in heparinised tubes , and animals sacrificed : ( i ) oral dosing : 0.08 , 0.25 , 0.5 , 1 , 2 , 4 , and 8 h post - dose ; ( ii ) iv dosing : 0.08 , 0.25 , 0.5 , 1 , 2 , 4 , and 8 h post - dose . tumor xenografts were generated in mice by injection of 2 million hct116 cells subcutaneously . a daily dose of 50 mg / kg of 11a in pure water was administered by oral gavage . mice were sacrificed 3 h after the last dose and tumors excised , fixed in 4% formaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) , and embedded in paraffin . sections were cut using a reichert - jung 1150/autocut microtome to perform phospho - src immunochemistry . antigen retrieval was performed using heat treatment under pressure in a microwave oven for 10 min in 10 mm citrate buffer ph = 6 . sections were blocked for endogenous peroxidase followed by incubation with antiphospho - src antibody ( cell signaling technology ) ( 1:200 dilution ) at 4 c overnight . staining was developed using envision ( dako ) and diaminobenzidene ( dako ) before slides were counterstained in hematoxylin , dehydrated , and mounted in dibutyl phthalate xylene . staining was scored by a single experienced observer , blinded to treatment , using a weighted histoscore method .

lipomatous hypertrophy of the interatrial septum ( lhis ) is an exaggerated growth of normal fat existing within the septum and is not a true tumor . the septal hypertrophy may be as much as 2 cm in thickness and is seen primarily in older patients and in those who are obese . it has been suggested that this disorder is associated with the presence of coronary artery disease in proportion to the degree of atrial septal thickness . lipomatous hypertrophy of the interatrial septum is indistinguishable from lipoma except that the former occurs in the atrial septum with a typical distribution ( generally sparing the fossa ovalis ) . in the absence of symptoms of atrial arrhythmias , heart block or rare vena caval obstruction nowadays , with advanced imaging techniques , lhis are picked up incidentally in the course of work - up for other conditions . such a case is presented here and imaging findings and literature on this lesion are reviewed . a 70-year - old female with a history of hypertension , atrial fibrillation , pacer implantation for symptomatic bradycardia , and a prior cerebrovascular accident was transferred to our institution for further evaluation and management of a recently identified persistent methicillin - sensitive staphylococcus aureus bacteremia . during workup of her bacteremia although no vegetations were seen on either pacer wires or cardiac valves , a massive homogeneous thickening of the superior portion of the interatrial septum was noted [ figure 1a and b ] . this mass effect extended to the posterior and roof portions of the right atrial wall as well as to the superior vena cava , causing proximal compression of this vessel [ figure 1c and d ] . in view of these findings , a chest computed tomographic examination was requested for better definition of this mass density . following contrast administration , the mass effect noted by transesophageal echocardiography was identified as intrapericardial fat density that extended toward the interatrial septum without evidence of infiltration of the interatrial septum directly [ figure 2 ] . additionally , a nodular opacity was also found in the lateral left upper lobe with a greatest diameter of 1 cm that was thought to be a potential septic embolus given the recent history of bacteremia ( not shown ) . ( a ) standard mid - esophageal four - chamber transesophageal view showing right atrium ( ra ) , right ventricle ( rv ) , left atrium ( la ) , and left ventricle ( lv ) , as well as the pacer wire , ( b ) pulling up the transesophageal probe to interrogate posterior aspects of both right and left atria demonstrated the presence of a diffusely homogeneous mass effect noted in the posterior aspect of the right atrium as demarcated by the arrow heads , ( c ) at a 90 angle , bi - atrial mid - esophageal view , both left ( la ) and right ( ra ) atria are seen . the homogeneous mass is also seen compressing the superior vena cava ( svc ) with the pacer wires , ( d ) another view of the mass , demarcated by arrows , from the superior aspect of the right atria compressing the superior vena cava ( svc ) shown by the hatched arrow transverse chest computed tomographic view showing the fat density in relation to the pacer wires , left atrium ( la ) , right coronary artery ( rca ) , and aortic valve ( av ) the initial continuous infusion of nafcillin was later changed to oxacillin , as surveillance blood cultures were all negative . the plan was to remove her transvenous pacer and replace it once she had completed her antibiotic therapy and follow - up blood cultures off antibiotics remained negative . lipomatous hypertrophy of the interatrial septum ( lhis ) is a rare , but increasingly recognized benign cardiac lesion characterized by excessive deposition of fat in the interatrial septum . while the true incidence of lhis has not been determined ; the expanding use of non - invasive imaging techniques in recent years has quoted an incidence up to 8% , compared to an incidence of 1% based on autopsy reports . although the exact etiology of lhis remains unclear , some theories have suggested the existence of embryonal mesenchymal cells within the primitive atria that can develop into adipocytes with an appropriate stimulus , particularly obesity and advanced age . the resultant effect is adipocyte hyperplasia and fat accumulation occurring in the epicardium , that is an extracardiac deposition fat , rather than within the interatrial septum as recently illustrated by silbiger , et al . in this report , the authors demonstrated how the walls of the left and right atria fold inward toward each other , forming a fat - filled depression between them called waterston 's groove . fat contained in this region is not a true interatrial septal density , but in reality is fat that overlies the epicardial surface of the heart . transesophageal echocardiography is one of the most commonly used imaging modalities to interrogate cardiac structures , in which routine examination of the interatrial septum is performed . the characteristic echocardiographic appearance of lhis involves an interatrial septal thickness > 2 cm usually represented by an hourglass appearance denoted by fat mass superior as well as inferior , sparing the fossa ovalis in between . most patients with lhis are usually asymptomatic ; however , in some cases , this fatty infiltration can either cause intraatrial conduction disturbances and atrial arrhythmias or when this fatty infiltration extends beyond the atrial septum may cause right atrial inflow obstructive symptoms . however , there are times that echocardiography alone is not capable of characterizing myocardial tissue , particularly when the extent of the process as described in our case is so massive . in such cases , the spatial resolution of cardiac mri is ideal in providing detailed information about the size and location of such masses . furthermore , with its ability to evaluate t1 and t2 characteristics of the mass , as well as the ability to use fat - suppression techniques in addition to gadolinium , the histopathologic characteristics of a mass can be clearly evaluated by cardiac mri . alternatively , chest computed tomography can be also used to help make the diagnosis . in this case , however , careful recognition of some anatomical findings might be useful in distinguishing this rare , but increasingly recognized benign cardiac lesion with other cardiac pathology .

the data related with bombay duck and beltfish muscle trancriptomes are deposited to ncbi sra database with accession number of srx1706379 and srx1674471 , respectively . calcium ( ca ) is the major element in the body and involved in a number of cellular events , including cell motility , transmission of nerve impulses , excitation - contraction of muscles , release of neurotransmitters , cell secretory , and membrane permeability . the contraction and relaxation events of the muscle are the best investigated mechanisms , which is mediated by the coordination of many important calcium cycling proteins . the ryanodine receptor ( ryr ) can release calcium ions ( ca ) from sarcoplasmic reticulum ( sr ) into cytoplasm to induce a calcium spark . later , this ca can form complex structure with troponin c ( tnnc ) , which can be inducing the muscle contraction in the body . afterwards , parvalbumin ( pvalb ) , an acidic intracellular ca - binding protein plays an important role on the function of muscle in the body . afterwards , the retrieval of ca ions from the myofibril can be transfer to the sr . normally , high concentration of intracellular ca buffer is present in the fast - contracting skeletal muscles across the animals . the parvalbumin protein coding gene called pvalb is one of the members of this family for ca - binding molecules , which is always check on ca switching in a cell , . the loss of function of pvalb can prolong the contraction / relaxation cycle in the fast - twitch muscle of animals . accordingly the cytoplasmic ca move back to sr through sarcoendoplasmic reticulum calcium transport atpase ( serca ) . normally , the free ca ions in the sr bound with calsequestrin ( casq ) and acts as dual role in excitation it can hold to increase sr capacity and modulate the activity of ca release ryanodine receptor ( ryr ) channels ( fig . the expression level of those calcium cycling proteins are positively correlated to the muscle contraction / relaxation ability of the cell . for example , the mutations of calcium cycling genes in human can lead to form many health problems like familial ventricular arrhythmias , cardiomyopathy and others , , . the same time , previous study clears that the overexpression of fast - twitch skeletal muscle type of serca in transgenic mouse heart can enhance myocardial contractility and increased ca transport function in the body .fig . 1schematic picture demonstrate the function of five calcium cycling genes ( casq , pvalb , ryr , serca and tnnc ) identified in bombay duck muscle transcriptome.fig . 1 schematic picture demonstrate the function of five calcium cycling genes ( casq , pvalb , ryr , serca and tnnc ) identified in bombay duck muscle transcriptome . bombay duck ( harpadon nehereus ) is a kind of lizardfish , which can inhabit at the tropical areas of the indo - pacific region . the little number of this fish can observe at bay of bengal and in the east and south china sea too . most of the seasons , it can observe at the deep water offshore on sandy mud bottom . the same time , it can also gathers in large shoals at deltas of rivers to feed during monsoons . the bombay duck can spawn six batches of breeds per year in the life and the adults usually have 25 cm in size . various observations suggested that the fish can also reach at maximum length of 40 cm in the life ( not all fishes ) . . normally fresh fish can usually fried and served as a starter in regional shops and homes . in mumbai , konkan , and the western coastal areas in india , this dish is popularly known as bombil fry . the previous studies say that , 90% of this fish includes moisture content , whereas the fewer amounts of protein and fat in muscle comparing with other species . in addition , the swimming ability of bombay duck is very poor and move only with the tidal oscillations . the slow swimming activity mainly helps the muscle fibers to perform basic aerobic metabolic functions including , circulatory and respiratory systems to supply needful substrates and oxygen . as swimming speed increases , the maximum performance can achieved throughout fast - starts associated with predation , escape responses and involves the mobilization of the entire white muscle mass . these white muscle masses are typically arranged by single fiber type that expresses fast isotypes of the myofibrillar proteins and containing high concentrations of the cytoplasmic ca - binding protein parvalbumin . it also has larger average diameters than red fibers and contains higher volume densities of myofibrils . and a more sarcoplasmic reticulum for faster ca cycling . in the previous work , we performed rnaseq to explore the muscle transcriptome in beltfish ( trichiurus lepturus ) with a robust swimming ability ( rnaseq data are deposited as srx1674471 ) . this study , we performed the rnaseq to explore the muscle transcriptome in bombay duck with a poor swimming ability . we have studied the hypothesis whether the expression level of calcium cycling proteins are positive correlated to fish swimming ability . the muscle tissue dissected from the wild juvenile bombay duck ( body length around 2.0 cm ) and stored in rnalater ( qiagen , hilden , germany ) at 80 c prior to rna extraction . the total rnas were extracted by trizol kit ( invitrogen , carlsbad , ca , usa ) and samples digested by dnase i to prevent the genomic dna contamination . the integrity and size distribution of rna was checked with bioanalyzer 2100 ( agilent technologies , santa clara , ca , usa ) . 2.5 g of rnas were used to synthesize the cdna libraries by illumina truseq rna sample preparation kit . after qpcr quantitation and dilution , the library was sequenced with illumina nextseq500 through 150 bp paired - end reads . the total of 45,944,846 raw paired - end reads were generated from the sample and the adaptor sequences were trimmed . further , the low quality reads removed by cutadapt software . finally , the removal of ambiguous nucleotides , duplicates and low - quality sequences ( phred quality scores < 20 ) from the data can get total of 47,752,240 cleaned reads ( 99.6% ) from the sample . the raw transcriptome sequences in the present study were deposited in the ncbi sra database ( srx1706379 ) . the cleaned reads were de novo assembled into contigs by trinity software with default parameters settings . the transcriptome was assembled into 26,288 unigenes with the n50 length of 486 bp . the assembled transcriptomic unigenes subjected to the similarity search against non - redundant ( nr ) protein , gene ontology ( go ) , kegg , eggnog and swissprot databases using blastx with an e - value cut off of 1e 5 . gene ontology ( go ) analysis was then conducted on the assembled transcriptome by using blast2go . kegg pathways were assigned to assembled unigenes using the online kegg automatic annotation server ( kaas ) ( http://www.genome.jp/tools/kaas/ ) . the bi - directional best hit ( bbh ) method was used to obtain kegg orthology ( ko ) assignment . blastx against nr , go , kegg , eggnog and swissprot databases show 100% , 65% , 26% , 94% and 88% annotation rate , respectively . 2comparison of the gene annotation rate of unigene against nr , go , kegg , eggnor and swissprot databases of bombay duck muscle transcriptome.fig . 2 comparison of the gene annotation rate of unigene against nr , go , kegg , eggnor and swissprot databases of bombay duck muscle transcriptome . the high - quality cleaned reads of each rnaseq library were mapped to the assembled transcripts with bowtie2 program . the unigene with rpkm ( reads per kilobase of exon per million reads mapped ) 100 was defined as abundant expressed genes . for the validation of protein identity of calcium cycling homologs , we downloaded the data of fish species includes zebrafish ( danio rerio ) , medaka ( oryzias latipes ) , fugu ( takifugu rubripes ) , tilapia ( oreochromis niloticus ) and large yellow croaker ( larimichthys crocea ) from ncbi ftp sites ( ftp://ftp.ncbi.nlm.nih.gov/genomes/ ) . we made a single protein database to perform in - house blast and constructed the gene - specific phylogenetic tree by using geneious software ( http://www.geneious.com/ ) with 1000 bootstrap neighbor - joining calculation ( fig . phylogenic tree topology analysis revealed the strong evidences to support the molecular identity of those calcium cycling genes identified in bombay duck muscle transcriptome . the comparison of gene expression level in fishes shows that 3 casq , 13 pvalb , 17 ryr , 15 serca and 7 tnnc unigenes in bombay duck . the same time , 2 casq , 11 pvalb , 19 ryr , 20 serca and 4 tnnc unigenes in beltfish muscle transcriptomes . the relative expression by rpkm method found that the 2 pvalb unigenes ( hne_c22292_g1_i1 and hne_c52631_g1_i1 ) , 1 serca unigene ( hne_c4397_g1_i1 ) and 1 tnnc unigene ( hne_c52572_g1_i1 ) have high relative expression level in bombay duck . in the beltfish , 3 pvalb unigenes ( tle_c47515_g1_i1 , tle_c18933_g1_i1 and tle_c18968_g1_i1 ) , 1 serca unigene ( tle_c13632_g1_i1 ) and 1 tnnc unigene ( tle_c22302_g1_i1 ) shows high relative expression level ( rpkm > 1000 , summarized in table 1 ) . the statistical comparison of all expressed calcium cycling unigenes in muscle has no significant difference between bombay duck and beltfish . however , the comparison of dominantly expressed unigenes in fish muscle shows that the calcium cycling gene expression in beltfish is 1.4- to 51.6-fold higher than bombay duck . among five calcium cycling genes , the fold change results are very significant in casq ( 51.6 fold ) and pvalb ( 9.1 fold ) and both of them are responsive for calcium binding to reduce free calcium concentration in the sr and cytoplasm ( see fig . , literature reported that the robust swimming fish species like ( pacific bluefin tuna ) and pacific cod have abundant expression of glycolytic enzyme genes . in this study , the results confirmed that the high abundant expression rate of calcium cycling genes in robust swimming fish species . the current muscle transcriptome and identified calcium cycling gene data can provide more insights into the muscle physiology of fish.fig . 3comparison of the gene expression level of calcium cycling genes between bombay duck ( red ) and beltfish ( blue ) . 3 comparison of the gene expression level of calcium cycling genes between bombay duck ( red ) and beltfish ( blue ) . s1phylogenetic tree for casq , pvalb , ryr , serca and tnnc based on protein sequences . the contigs from bombay duck and beltfish are labeled in red and blue , respectively . s1 phylogenetic tree for casq , pvalb , ryr , serca and tnnc based on protein sequences . the contigs from bombay duck and beltfish are labeled in red and blue , respectively . the muscle tissue dissected from the wild juvenile bombay duck ( body length around 2.0 cm ) and stored in rnalater ( qiagen , hilden , germany ) at 80 c prior to rna extraction . the total rnas were extracted by trizol kit ( invitrogen , carlsbad , ca , usa ) and samples digested by dnase i to prevent the genomic dna contamination . the integrity and size distribution of rna was checked with bioanalyzer 2100 ( agilent technologies , santa clara , ca , usa ) . 2.5 g of rnas were used to synthesize the cdna libraries by illumina truseq rna sample preparation kit . after qpcr quantitation and dilution , the library was sequenced with illumina nextseq500 through 150 bp paired - end reads . the total of 45,944,846 raw paired - end reads were generated from the sample and the adaptor sequences were trimmed . further , the low quality reads removed by cutadapt software . finally , the removal of ambiguous nucleotides , duplicates and low - quality sequences ( phred quality scores < 20 ) from the data can get total of 47,752,240 cleaned reads ( 99.6% ) from the sample . the raw transcriptome sequences in the present study were deposited in the ncbi sra database ( srx1706379 ) . the cleaned reads were de novo assembled into contigs by trinity software with default parameters settings . the transcriptome was assembled into 26,288 unigenes with the n50 length of 486 bp . the assembled transcriptomic unigenes subjected to the similarity search against non - redundant ( nr ) protein , gene ontology ( go ) , kegg , eggnog and swissprot databases using blastx with an e - value cut off of 1e 5 . gene ontology ( go ) analysis was then conducted on the assembled transcriptome by using blast2go . kegg pathways were assigned to assembled unigenes using the online kegg automatic annotation server ( kaas ) ( http://www.genome.jp/tools/kaas/ ) . the bi - directional best hit ( bbh ) method was used to obtain kegg orthology ( ko ) assignment . blastx against nr , go , kegg , eggnog and swissprot databases show 100% , 65% , 26% , 94% and 88% annotation rate , respectively . 2comparison of the gene annotation rate of unigene against nr , go , kegg , eggnor and swissprot databases of bombay duck muscle transcriptome.fig . 2 comparison of the gene annotation rate of unigene against nr , go , kegg , eggnor and swissprot databases of bombay duck muscle transcriptome . the high - quality cleaned reads of each rnaseq library were mapped to the assembled transcripts with bowtie2 program . the unigene with rpkm ( reads per kilobase of exon per million reads mapped ) 100 was defined as abundant expressed genes . for the validation of protein identity of calcium cycling homologs , we downloaded the data of fish species includes zebrafish ( danio rerio ) , medaka ( oryzias latipes ) , fugu ( takifugu rubripes ) , tilapia ( oreochromis niloticus ) and large yellow croaker ( larimichthys crocea ) from ncbi ftp sites ( ftp://ftp.ncbi.nlm.nih.gov/genomes/ ) . we made a single protein database to perform in - house blast and constructed the gene - specific phylogenetic tree by using geneious software ( http://www.geneious.com/ ) with 1000 bootstrap neighbor - joining calculation ( fig . phylogenic tree topology analysis revealed the strong evidences to support the molecular identity of those calcium cycling genes identified in bombay duck muscle transcriptome . the comparison of gene expression level in fishes shows that 3 casq , 13 pvalb , 17 ryr , 15 serca and 7 tnnc unigenes in bombay duck . the same time , 2 casq , 11 pvalb , 19 ryr , 20 serca and 4 tnnc unigenes in beltfish muscle transcriptomes . the relative expression by rpkm method found that the 2 pvalb unigenes ( hne_c22292_g1_i1 and hne_c52631_g1_i1 ) , 1 serca unigene ( hne_c4397_g1_i1 ) and 1 tnnc unigene ( hne_c52572_g1_i1 ) have high relative expression level in bombay duck . in the beltfish , 3 pvalb unigenes ( tle_c47515_g1_i1 , tle_c18933_g1_i1 and tle_c18968_g1_i1 ) , 1 serca unigene ( tle_c13632_g1_i1 ) and 1 tnnc unigene ( tle_c22302_g1_i1 ) shows high relative expression level ( rpkm > 1000 , summarized in table 1 ) . the statistical comparison of all expressed calcium cycling unigenes in muscle has no significant difference between bombay duck and beltfish . however , the comparison of dominantly expressed unigenes in fish muscle shows that the calcium cycling gene expression in beltfish is 1.4- to 51.6-fold higher than bombay duck . among five calcium cycling genes , the fold change results are very significant in casq ( 51.6 fold ) and pvalb ( 9.1 fold ) and both of them are responsive for calcium binding to reduce free calcium concentration in the sr and cytoplasm ( see fig . , literature reported that the robust swimming fish species like ( pacific bluefin tuna ) and pacific cod have abundant expression of glycolytic enzyme genes . in this study , the results confirmed that the high abundant expression rate of calcium cycling genes in robust swimming fish species . the current muscle transcriptome and identified calcium cycling gene data can provide more insights into the muscle physiology of fish.fig . 3comparison of the gene expression level of calcium cycling genes between bombay duck ( red ) and beltfish ( blue ) . 3 comparison of the gene expression level of calcium cycling genes between bombay duck ( red ) and beltfish ( blue ) . s1phylogenetic tree for casq , pvalb , ryr , serca and tnnc based on protein sequences . the contigs from bombay duck and beltfish are labeled in red and blue , respectively . s1 phylogenetic tree for casq , pvalb , ryr , serca and tnnc based on protein sequences . the contigs from bombay duck and beltfish are labeled in red and blue , respectively .

the utility of clickers and peer discussion in large - lecture introductory biology courses is well established . typically , instructors pose multiple - choice questions requiring application of a recently presented concept at several points during a class . in one commonly used approach called peer instruction , students first answer a concept question individually , discuss the question with their peers , and then revote before the answer to the question is revealed ( mazur , 1997 ) . the instructor then displays histograms of student responses , which give immediate feedback to both instructors and students on how well a concept is understood . several studies have shown that students enjoy using clickers , feel that this form of interactive engagement is useful for their learning , and learn from discussing questions with their peers ( e.g. , knight and wood , 2005 ; caldwell , 2007 ; preszler et al . however , despite the evidence about the positive impact of clickers in biology courses , we frequently encounter faculty members who say that there is no benefit to using them in small - enrollment ( fewer than 25 students ) seminar - style biology courses . when asked why , they commonly say that in small - enrollment courses the instructor already spends a lot of time interacting with students and asking questions . in short , these instructors feel that adding clickers to peer discussion would not add value to the way they teach these types of courses . however , we considered the possibility that clickers offer the additional benefit of instantaneous feedback , while allowing individuals to maintain anonymity . the latter benefit may create a situation that is conducive to encouraging all students to participate in a course , even if it is a small - enrollment course . to find out if this perception is correct , we investigated adding clickers to peer discussion in an 11-student upper - division embryology course at the university of colorado , boulder ( course demographics in table 1 ) . all of the students in this course had experience using clickers in their large - enrollment lower - division biology courses , but this was their first time using clickers in a small - enrollment upper - division biology course . in this embryology course , each student gave two presentations , one on a classic embryology paper chosen by the instructor and another on a recent paper of their choosing . ( the distribution of grading points for this course is described in table 3 , and a list of all the papers presented is given in supplemental material a ) . the students prepared powerpoint slides for use with their presentation and had the option to have it critiqued by the instructor ( t.t.s . ) ahead of time . the morning before each student 's presentation , the instructor added clicker questions to the powerpoint slides . the students , including the student presenter , did not see the clicker questions ahead of time . distribution of grading points in the embryology course two types of questions were added to the student presentations . the first type ( hereafter called reading quiz questions ) consisted of mostly fact - based questions to determine whether students read the paper before class . students answered these clicker questions individually without peer discussion and received a point only if they got the answer correct . the reading quiz questions were asked throughout the presentation and were designed to cover the key methods and results in the article . the second question type ( hereafter called application questions ) asked students to apply what they learned from the paper and presentation . students answered the application questions individually , discussed the answer with their peers , and then answered the same question again . the histograms of student responses were not revealed to students until after they answered the questions a second time , because when class responses to clicker questions are shown , students are inclined to move to the most common answer , which likely diminishes the value of peer discussion ( perez et al . , 2010 ) . participation points were awarded for these types of questions because the instructor wanted to emphasize discussion , scientific argument , and articulation of thought over just getting the right answer . students and the instructor sat around a single large table . for the peer - discussion portion of the application questions , our observations revealed that the 11 students tended to divide into two groups based on where they were sitting . a typical discussion would begin with one student saying what he or she answered and then the other students in the group would say if they agreed or disagreed with the answer . even if students all agreed on a particular answer , they would often discuss why the other answers were incorrect . a transcript of a student discussion is given in supplemental material b. the instructor removed herself from the peer discussion as much as possible and would ask clarifying questions only if the students started to go off track with their discussion . in addition to examining student performance results and observing peer discussion , a researcher not associated with the course ( m.k.s . ) interviewed 8 of the 11 students about their experience using clickers in this small - enrollment course . the interview results revealed that clickers added value to this embryology course in three important ways . first , all of the interviewed students stated that adding clicker questions to the student presentations encouraged them to read the papers before class . i doubt i would have read the papers if there were no clicker questions , or at least i would have tried to get by without reading them . the average score on the individually answered reading quiz questions was 69% ( sem = 3.21% , n = 79 questions ) . although that may not seem impressive at first , some studies have shown that only 2030% of the students read assignments before class when requested by their instructor ( reviewed by hobson , 2004 ) . also , several students felt that the way they read scientific papers improved because of the clicker questions . for example , one student said : as i am reading the paper i think , what will tin tin ask about ? it forces me to think about the organism used , the type of experiment , etc . of course , there are other ways to encourage students to read papers before class . during the previous five times teaching this class , the instructor used preclass reading quizzes , which consisted of factual questions only . however , the instructor observed that , even if students read the paper and were able to answer quiz questions correctly , they often tuned out the subsequent student presentation . in dispersing the reading quiz questions throughout the presentation with the facilitation of clickers , the instructor hypothesized that students would remain more engaged . during the interviews , seven of the eight students commented that having the clicker questions helped them pay attention to the student presentations . according to one student : i would still read the papers in other classes but for this class i highlight key things and keep the paper open while the presentation is going on , so i can keep track of what is going on . second , students felt that asking clicker questions creates an environment where every student , not just one or two , thinks through and answers each question being asked . in many small - enrollment biology courses , instructors ask open - ended questions and call on a student to answer . however , even with such efforts , there is potential for the remaining students to be left out of the discussion , especially if they are not confident about the answer . during the interviews , students talked about how they liked contributing to the class even when they were not speaking . three such descriptions follow : if we did n't have clicker questions then 23 people would dominate the conversation and tin tin might think because they understand what is going on , we all understand it . clickers give people a voice , even if it is not a verbal one . if you did n't have clickers and tin tin asked open - ended questions to us , most of the time i would just think : well i do not know and just sit there and listen to what she says is correct . they get students thinking and their brains moving . finally , the application questions gave students an opportunity to articulate their thinking and learn from their peers during class . when students voted individually on the application questions , the average percent correct equaled 53.9% ( sem 4.6 , n = 22 questions ) . but after peer discussion , the average percent correct increased to 91.5% ( sem 3.7 ) . therefore , just as has been reported in large - enrollment biology courses ( e.g. , knight and wood , 2005 ; smith et al . , 2009 ) , peer discussion in small - enrollment courses also improves student performance on clicker questions . one student described the peer discussion experience this way : it is like going to a coffee shop and talking about science over coffee . i like hearing what my classmates think and they often bring up something i did n't think of . we work together as a whole to figure out the answer . while all eight of the students we interviewed thought that clickers were a useful learning tool in small - enrollment courses , five of the eight students mentioned suggestions for improving their use . first , students wanted to have meet - and - greet time during the first week of class , so they could feel more comfortable talking with each other . second , students expressed that they wanted a chance to write some of their own clicker questions . they thought that writing a subset of the clicker questions sounded like a lot of fun and would help them organize their presentation . the performance , observation , and interview data provide encouraging information for instructors who are considering using clickers in their small - enrollment seminar - style biology courses . importantly , adding clicker use to peer discussion enhanced a small - enrollment biology course by increasing the chance students will read before class , helping the instructor engage all students , and giving students a focused opportunity to share thinking and to learn from their peers . there are many future research questions that can be asked regarding the use of clickers in small - enrollment settings . for example , do students who have not used clickers in previous courses find that clickers are an effective tool in a small lecture setting ? are there other small - enrollment settings such as laboratory meetings and departmental seminars where clickers could be an effective tool for engaging the audience ? for instructors interested in including in - class concept questions in their courses , several resources are available at http://stemclickers.colorado.edu . 0603.08 ) was granted by the institutional review board , university of colorado , boulder .

the world over bladder cancer is the seventh most common malignancy in men and the eighth most common malignancy in females . in india bladder cancer is the fifth most common cancer in men according to the delhi - based registry with age - adjusted incidence rate of 5.8/100,000 person years . in india it is predominantly the disease of the male population with a male to female ratio of 8.6:1 with median age at presentation of 60 years ( range : 18 - 90 years ) . transurethral resection of bladder tumor ( turbt ) is the cornerstone in the management of bladder cancer . goals of turbt are to look for detrusor muscle invasion and complete resection of tumor . even though conventional turbt , where tumor is removed in piecemeal , has been in practice for many years , issues like absence of detrusor in histopathology report and incomplete resection continue to plague the adequacy of turbt . presence of detrusor is a surrogate for the completeness of resection but various series have reported absence of detrusor musclein turbt specimen in up to 50% of the cases . we aim to assess performance of en - bloc turbt as compared to conventional turbt for adequacy of initial resection of the tumor judged by the presence of the detrusor muscle in the specimen . in a prospective nonrandomized interventional design a pilot study was conducted between september 2007 and december 2010 , to see the safety and efficacy of en - bloc turbt as compared to the conventional turbt . en - bloc turbt was done by a single surgeon and to avoid bias towards deliberate attempt to resect the tumor completely two other urologists did the conventional turbt . clinical data on the size ( measured by ultrasonography in 22 patients and by cect in 24 patients ) and the location of the tumor were recorded . patients presenting for the first time with single tumor on imaging and with tumor size from 2 - 4 cm were included for the study . patients with recurrences , multiple tumors and those with tumor size of less than 2 and more than 4 cm were excluded . tumors with prior history of turbt and pedunculated tumors were also excluded . the usual tungsten loop electrode and 26fr resectoscope ( storz , germany ) with 30 lens and monopolar cautery was used in all the cases under regional anesthesia and general anesthesia if tumor was located in the lateral wall . the angle of the loop electrode was changed manually from 90 to 45 [ figure 1 ] for en - bloc turbt . the feasibility of en - bloc turbt was initially tried in tumors less than 2 cm in size in six cases and then this study was started . ( a ) normal right - angled loop and ( b ) electrocautery loop bent at 45 ( for en - bloc turbt ) after identifying the tumor , normal mucosa , 1 cm away from the tumor base was marked all around it by using coagulation current at setting 60 - 80w ( valley lab , usa ) . blood vessels entering the base of the tumor were coagulated before starting the procedure [ figure 2 ] . glycine 1.5% was used as an irrigant during the resection . marking the periphery of the tumor and at the same time coagulating the feeding blood vessels by using cutting current setting at 100 - 115w , incision from the normal mucosa was deepened till detrusor muscle plane was reached and then retrograde resection was started in the deep layer of the detrusor seeing and coagulating the feeding vessels . angle of the loop and beak of the resectoscope ( not seen in the picture ) helped in elevating the tumor and going deep to its base till the whole tumor was resected [ figure 3 ] . angled loop helped in elevating the tumor and dissection at the base once the tumor was detached , then the larger tumors were cut into two to three pieces in the bladder itself . after stopping the inflow and resting the tumor in the trigone and base area the largest tumor removed was 3 cm in size in a female [ figure 4 ] . histopathological examination was done according to the 2004 world health organization ( who)international society of urological pathological grading system . blood loss was calculated in terms of fall in hemoglobin levels measured before and a day after turbt but it was not significant . statistical analysis was done using pearson chi square test and p value of < .05 was considered significant . en bloc turbt gives all three layers i.e. detrusor muscle , lamina and urothelium in the tumor as one specimen . photomicrograph showing all three layers with inked outer margin ; h and e stain , 20 patients presenting for the first time with single tumor on imaging and with tumor size from 2 - 4 cm were included for the study . patients with recurrences , multiple tumors and those with tumor size of less than 2 and more than 4 cm were excluded . the usual tungsten loop electrode and 26fr resectoscope ( storz , germany ) with 30 lens and monopolar cautery was used in all the cases under regional anesthesia and general anesthesia if tumor was located in the lateral wall . the angle of the loop electrode was changed manually from 90 to 45 [ figure 1 ] for en - bloc turbt . the feasibility of en - bloc turbt was initially tried in tumors less than 2 cm in size in six cases and then this study was started . ( a ) normal right - angled loop and ( b ) electrocautery loop bent at 45 ( for en - bloc turbt ) after identifying the tumor , normal mucosa , 1 cm away from the tumor base was marked all around it by using coagulation current at setting 60 - 80w ( valley lab , usa ) . blood vessels entering the base of the tumor were coagulated before starting the procedure [ figure 2 ] . glycine 1.5% was used as an irrigant during the resection . marking the periphery of the tumor and at the same time coagulating the feeding blood vessels by using cutting current setting at 100 - 115w , incision from the normal mucosa was deepened till detrusor muscle plane was reached and then retrograde resection was started in the deep layer of the detrusor seeing and coagulating the feeding vessels . angle of the loop and beak of the resectoscope ( not seen in the picture ) helped in elevating the tumor and going deep to its base till the whole tumor was resected [ figure 3 ] . angled loop helped in elevating the tumor and dissection at the base once the tumor was detached , then the larger tumors were cut into two to three pieces in the bladder itself . after stopping the inflow and resting the tumor in the trigone and base area the largest tumor removed was 3 cm in size in a female [ figure 4 ] . histopathological examination was done according to the 2004 world health organization ( who)international society of urological pathological grading system . blood loss was calculated in terms of fall in hemoglobin levels measured before and a day after turbt but it was not significant . statistical analysis was done using pearson chi square test and p value of < .05 was considered significant . en bloc turbt gives all three layers i.e. detrusor muscle , lamina and urothelium in the tumor as one specimen . photomicrograph showing all three layers with inked outer margin ; h and e stain , 20 three patients had multiple tumors so were excluded from the study . of 21 patients in the en - bloc group , 20 ( 94.4% ) had detrusor muscle in their initial specimen . in the en - bloc group , the procedure could be completed without any bladder perforation as vision was much better due to better hemostasis . one patient who did not have detrusor muscle in the resected specimen , had turbt in the initial part of the study . in conventional turbt , only 15 of 25 ( 60% ) had detrusor muscle in biopsy report . one patient had bladder perforation who had a 4-cm tumor on the lateral wall and was managed conservatively with a peritoneal drain . location of the tumor , stage and grade were comparable in both the groups [ table 1 ] . stage and grade of tumors in the two groups on first turbt though there was no significant difference in the blood loss , hemostasis was better in the en - bloc group as it was easy to coagulate blood vessels at the base at the time of cutting and lifting the tumor up . the second advantage was that the depth of resection could easily be controlled in comparison to the conventional turbt . of 10 patients who did not show muscle on an initial turbt , three patients refused to undergo second - look turbt , two had radical cystectomy for high - grade lamina - invasive transitional cell carcinoma and out of five who had second look , one had muscle - invasive tcc and the remaining four had detrusor muscle free of tumor . transurethral resection of bladder tumor remains a surgery in which the end point is the presence of muscle but complete resection is not always achieved . transurethral resection of the bladder tumor bladder tumor is the most underrated surgery in urology . its adequacy is a hallmark of a good local control but there is no yardstick available clinically to adjudge complete resection of the tumor . this is evident by the absence of the detrusor muscle in a significant number of patients in initial turbt . there are some surrogate markers to assess adequate resection described in the literature and those are , presence of detrusor muscle in the specimen and the rate of subsequent recurrence.[79 ] an ideal turbt would mean complete resection of the visible tumor , resection of the surrounding healthy looking mucosa for up to 1 cm and then the removal of the detrusor muscle . herr described three ways to measure the quality of a good turbt , i.e. complete resection , presence of deep muscle in the specimen and the rate of recurrence at the site of previous turbt . he also suggested classifying tumor resection as r0 , microscopic negative margin , r1 microscopic positive margin and r2 that is macroscopic positive margin . this kind of assessment is not practical in conventional turbt but could be possible in en - bloc resection , where we can have a complete piece of tumor tissue including lamina propria and detrusor muscle with it . the innermost surface of the detrusor muscle could then be inked thereby examining all three layers in one piece that would give us a better understanding of the tumor stage and a true perspective of the level of resection i.e. r0-r1 [ figure 4 ] . inadequate turbt in a conventional way is not only judged by the absence of muscle in an initial specimen but also by the rate of recurrence at the same site . it is common knowledge that recurrence is seen in 50 - 80% of non - muscle - invasive bladder cancer mostly during the first year . reasons that have been described for recurrences are incomplete resection , cell implantation or the tumor biology itself . european organization for research and treatment of cancer on review of seven randomized controlled trials noted significant difference in the recurrence rate among different institutions and after controlling established factors for recurrence like tumor size , multiplicity , stage and grade , it was concluded that a wide range in the rate of recurrence i.e. 0 - 46% , is due to the difference in the quality of resection . inadequate resection leading to a higher rate of recurrence at the same site is supported by another study where 81% of recurred tumors occurred at the site of previous resection . there are many ways described in the literature to improve the quality of turbt with the use of different kinds of loops and laser . en - bloc dissection has been described to have a better pathological evaluation for ta and t1 tumors . in that study a flat loop electrode was used to resect the tumors less than 2.5 cm in size . though the authors did not describe the presence or absence of the detrusor muscle , they concluded that invasion of the lamina was better delineated with en - bloc resection . a limitation of this technique described was the inability to use a flat loop for tumors located at the anterior and upper posterior wall . another limitation was that tumor of more than 2.5 cm was considered a contraindication . for a small - size papillary tumor it really does not make any difference whether an en bloc or conventional turbt is done ? but for doing en bloc for large tumors , there are indeed no reports in the literature . we used a 45 angle loop electrode with a 30 lens , that helped in scooping out the tumor in retrograde fashion . removing a large tumor resected en bloc though the urethra would be a difficult proposition therefore once the tumor was free , it was cut into two to three pieces for the removal from the bladder . the detrusor muscle has been described to be absent in 30 - 50% of turbt specimens at an initial resection . absence of the detrusor muscle not only affects the recurrence rate as previously described but also brings in the need for second - look turbt . the presence of the detrusor muscle lessens the rate of upstaging in second - look turbt . in one study , tumors were upstaged by second resection in 15% of 421 patients who had detrusor muscle at initial resection as compared to 45% of 280 patients with no muscle . therefore with the present method a higher incidence of finding the detrusor muscle in the specimen of an initial turbt would not only provide adequacy of the turbt but also reduce the need for second - look turbt . second - look surgery is a significant deterrent on the part of the patients , which not only adds to the mental anguish but also increases the cost of the procedure . another possible advantage of en - bloc turbt is that it could eliminate crush artefact of cold cup deep biopsy and diathermy artefacts are less likely to happen as the tissue piece obtained by it is too large to be affected by cautery . though the surgical expertise is an important denominator for the yield of detrusor muscle , even an experienced urologist did not fare well in getting complete removal of the bladder tumor with conventional turbt . the adequacy of an initial turbt that is judged by the presence of the detrusor muscle in the initial specimen is much better with en - bloc turbt than conventional turbt . thought no method described till date has resulted in the presence of detrusor muscle in 100% cases in initial turbt , en - bloc turbt helped in achieving adequate resection in 94% of cases . we have not used intravesical mitomycin in our study protocol . though there is level 1 evidence to suggest that intravesical mitomycin c ( mmc ) should be used , its role in preventing recurrences in high - grade or multiple tumors is not clear . it does not change surveillance cystoscopic protocol and the cost of follow - up remains high . the presence of the detrusor muscle which is considered as a surrogate marker for complete resection in turbt determines the rate of recurrence . en - bloc turbt is feasible and safe and has a better yield of the presence of the detrusor muscle in the initial resection specimen of non - muscle - invasive bladder cancer . rate of presence of muscle with en bloc was significantly higher than the conventional turbt . en - bloc turbt provides a better hemostasis and vision to control the resection limits . though our initial experience of en - bloc turbt is small the results are encouraging and it can be extended to a larger number of patients . in future it will be interesting to see the effect of en - bloc turbt on recurrence .

the world health organization ( who ) estimates that in the developing countries one woman dies every eight minutes due to unsafe abortions . these clandestine abortions are among the five leading causes of maternal mortality . in the developing world , an estimated five million women who undergo unsafe abortions require hospitalization annually . the fertility rate in pakistan has dropped by almost two births per couple , in the last two decades . however , the desire to limit births has not been accompanied by a parallel increase in usage of contraceptives . this gap between the aspiration to reduce births and use of contraceptives represents an unmet need in family planning. it has resulted in an exceptionally high prevalence of unwanted pregnancies . almost one third of the currently married women have an unmet need for contraception . it is not surprising then , to find that almost a million women in pakistan opt for induced abortion , annually . emergency contraceptive pills ( ecps ) are the only form of hormonal contraceptives that provide women a last chance to prevent pregnancy after unprotected sex[1214 ] . they may be used when a condom breaks ; when oral contraceptive pills have been missed or when a woman is raped or coerced into having sex . ecps contain higher doses of the hormones used in oral contraceptive pills and are effective if taken within third and fifth day ( 72 - 120 hrs ) after unprotected sex[1416 ] . furthermore , who confirms the safety of ecps which meet all criteria for over - the - counter sale . by themselves objections to the use of ecps include concerns that ; they may promote promiscuity ; they may increase prevalence of sexually transmitted diseases ; they may be abortificant[2428 ] ; they may not be cost effective ; they may be teratogenic or cause ectopic pregnancies ; that women having easy access to ecps may become less diligent when using regular contraceptives ; and that they may not have a significant impact on reducing the rate of unwanted pregnancies . this has the potential to lead to an increase in unwanted pregnancies.furthermore , the reduction in pregnancies and abortion rate caused by increased access to ecps has yet to be established conclusively through extensive field trials . other studies have shown that , ecps are cost effective , do not increase the incidence of sexually transmitted diseases , are medically safe , and do not adversely affect regular contraceptive usage[3437 ] . additionally they provide women a last chance of avoiding pregnancy after unsafe sex . in pakistan family planning services are provided to the people by the national program for maternal neonatal and child health ( mnch ) , the national program for family planning ( np ) and ministry of population welfare in collaboration with non governmental organizations ( ngos ) . the mnch program under the direction of the prime minister has been setup to provide the full range of contraceptives at all health facilities in order to reduce the unmet need ( 33% ) of contraceptives . the np , with its force of lady health workers ( lhws ) and lady health supervisors ( lhss ) , provides family planning services at the doorsteps of the people . despite efforts of ministry of health , ecps are currently not available to the population that is served by the np . it is expected that lhss will play a vital role when ecps are finally made available to this population . this study attempts to identify the knowledge gaps and attitudes of lhss of rawalpindi district towards ecp . this cross sectional survey was carried out in january 2010 at the rawalpindi district program implementation unit ( dpiu ) of np . a total of sixty seven lhss work in this dpiu , overseeing a force of 1841 lady health workers ( lhws ) . the lhss supervise the delivery of modern family planning services to an estimated population of 1,895,770 women of child - bearing age , at their doorstep . this study was conducted to explore the knowledge , attitudes and practices of these lhss , before ecps are included in the arsenal of contraceptives , delivered by the np . they were informed that participation was voluntary and anyone who was not willing to answer any particular question or did not want to contribute to the survey was free not to do so . they were asked to request for clarification if need be . at the end of the survey a 17 item questionnaire was used . at the start of the survey tool , ecps were defined as contraceptive pills that are used to avoid pregnancies after unprotected sexual intercourse . examples of an unforeseen visit of a spouse after protracted absence or unexpected breakage of the condom were also given . the next part collected the demographic characteristics of the participants including the age , marital status , years of education and rural or urban area of work . finally , they were asked if they perceived a need of emergency contraceptive pills for the clients of family planning services of np . the study instrument was based on similar surveys that have been carried out in some other countries . for example , although the prior use of ecps was enquired into , the associated sexual risk practices were not . currently , at least three different organizations are promoting their own brands of ecps in pakistan . these organizations have not made any significant attempt to educate the lhss or lhws about their products or their use . consequently , although the health workers are aware of ecps , their knowledge about them is rudimentary . we could not ask the participants to select an ecp from a list of several non - ecps . a participant might have known another brand and might not recognize the one mentioned in the questionnaire . they were asked to ; ( 1 ) identify from a list of several medicines , the one that was not an ecp , ( 2 ) recognize the maximum acceptable period after unprotected sex during which ecps remained affective and ( 3 ) , to confirm if ecps were abortive drugs or not . the attitudes of participants were measured using four items on a four point likert scale , ranging from strongly disagree to strongly agree . they were asked if they believed whether ; ( 1 ) ecps can lead to evil practices in society , ( 2 ) ecps can be harmful for health , ( 3 ) they would themselves use ecp in absolute need and ( 4 ) they would recommend it to a friend . using this four point scale finally , on the same scale they were asked if they believed that their family planning clients would benefit from introduction of ecps in the np statistical analysis was performed using the statistical package for social sciences ( spss 12.0 ) . this cross sectional survey was carried out in january 2010 at the rawalpindi district program implementation unit ( dpiu ) of np . a total of sixty seven lhss work in this dpiu , overseeing a force of 1841 lady health workers ( lhws ) . the lhss supervise the delivery of modern family planning services to an estimated population of 1,895,770 women of child - bearing age , at their doorstep . this study was conducted to explore the knowledge , attitudes and practices of these lhss , before ecps are included in the arsenal of contraceptives , delivered by the np . they were informed that participation was voluntary and anyone who was not willing to answer any particular question or did not want to contribute to the survey was free not to do so . they were asked to request for clarification if need be . at the end of the survey a 17 item questionnaire was used . at the start of the survey tool , ecps were defined as contraceptive pills that are used to avoid pregnancies after unprotected sexual intercourse . examples of an unforeseen visit of a spouse after protracted absence or unexpected breakage of the condom were also given . the next part collected the demographic characteristics of the participants including the age , marital status , years of education and rural or urban area of work . finally , they were asked if they perceived a need of emergency contraceptive pills for the clients of family planning services of np . the study instrument was based on similar surveys that have been carried out in some other countries . for example , although the prior use of ecps was enquired into , the associated sexual risk practices were not . currently , at least three different organizations are promoting their own brands of ecps in pakistan . these organizations have not made any significant attempt to educate the lhss or lhws about their products or their use . consequently , although the health workers are aware of ecps , their knowledge about them is rudimentary . we could not ask the participants to select an ecp from a list of several non - ecps . a participant might have known another brand and might not recognize the one mentioned in the questionnaire . they were asked to ; ( 1 ) identify from a list of several medicines , the one that was not an ecp , ( 2 ) recognize the maximum acceptable period after unprotected sex during which ecps remained affective and ( 3 ) , to confirm if ecps were abortive drugs or not . the attitudes of participants were measured using four items on a four point likert scale , ranging from strongly disagree to strongly agree . they were asked if they believed whether ; ( 1 ) ecps can lead to evil practices in society , ( 2 ) ecps can be harmful for health , ( 3 ) they would themselves use ecp in absolute need and ( 4 ) they would recommend it to a friend . using this four point scale finally , on the same scale they were asked if they believed that their family planning clients would benefit from introduction of ecps in the np statistical analysis was performed using the statistical package for social sciences ( spss 12.0 ) . this cross sectional survey was carried out in january 2010 at the rawalpindi district program implementation unit ( dpiu ) of np . a total of sixty seven lhss work in this dpiu , overseeing a force of 1841 lady health workers ( lhws ) . the lhss supervise the delivery of modern family planning services to an estimated population of 1,895,770 women of child - bearing age , at their doorstep . this study was conducted to explore the knowledge , attitudes and practices of these lhss , before ecps are included in the arsenal of contraceptives , delivered by the np . they were informed that participation was voluntary and anyone who was not willing to answer any particular question or did not want to contribute to the survey was free not to do so . they were asked to request for clarification if need be . at the end of the survey a 17 item questionnaire was used . at the start of the survey tool , ecps were defined as contraceptive pills that are used to avoid pregnancies after unprotected sexual intercourse . examples of an unforeseen visit of a spouse after protracted absence or unexpected breakage of the condom were also given . the next part collected the demographic characteristics of the participants including the age , marital status , years of education and rural or urban area of work . finally , they were asked if they perceived a need of emergency contraceptive pills for the clients of family planning services of np . the study instrument was based on similar surveys that have been carried out in some other countries . for example , although the prior use of ecps was enquired into , the associated sexual risk practices were not . currently , at least three different organizations are promoting their own brands of ecps in pakistan . these organizations have not made any significant attempt to educate the lhss or lhws about their products or their use . consequently , although the health workers are aware of ecps , their knowledge about them is rudimentary . we could not ask the participants to select an ecp from a list of several non - ecps . a participant might have known another brand and might not recognize the one mentioned in the questionnaire . they were asked to ; ( 1 ) identify from a list of several medicines , the one that was not an ecp , ( 2 ) recognize the maximum acceptable period after unprotected sex during which ecps remained affective and ( 3 ) , to confirm if ecps were abortive drugs or not . the attitudes of participants were measured using four items on a four point likert scale , ranging from strongly disagree to strongly agree . they were asked if they believed whether ; ( 1 ) ecps can lead to evil practices in society , ( 2 ) ecps can be harmful for health , ( 3 ) they would themselves use ecp in absolute need and ( 4 ) they would recommend it to a friend . using this four point scale finally , on the same scale they were asked if they believed that their family planning clients would benefit from introduction of ecps in the np statistical analysis was performed using the statistical package for social sciences ( spss 12.0 ) . 12 , 14 and 16 years of education correspond to matriculate , faculty of arts ( f.a ) , bachelor of arts ( b.a ) and master of arts ( m.a ) , respectively . only 3.8% ( n=2 ) and 11.3% ( n=6 ) had completed only 10 years and 16 years of education , respectively . majority ( 56% , n=30 ) ) had obtained a bachelors degree . majority ( 70 % , n=37 ) of the supervisors were resident of and serving in rural areas of rawalpindi district . general awareness of ecps was high ( 75.5% , n=40 ) but practical use was low ( 17% , n=9 ) . green star , a social marketing company was the major source ( 24.5% , n=13 ) of awareness . lady health visitors and doctors working at the designated health facility of lhss led to awareness of 18.9 % ( n= 10 ) and 11.3% ( n= 6 ) , respectively . similarly , population welfare organization and nurses were the source of information of 11.3% ( n= 6 ) and 3.8% ( n= 2 ) of the participants , respectively . awareness of ecps and source as seen in table 3 , almost half of the participants ( 45% n= 24 ) could identify ecps from a list of medicine . the duration after unprotected sex in which ecps may be helpful in preventing an unwanted pregnancy , was correctly identified by 24% ( n=13 ) of the respondents . almost 64% ( n=34 ) either thought that ecps were abortive drugs or were not sure . more than half ( 53 % n=28)of the participants thought ecps had harmful effects on future pregnancies . lady health supervisors knowledge about ecps the awareness and knowledge of the participants was not found to be associated with their age , marital status , educational background or past medical experience . however , the question regarding the mode of action of ecps being abortive was associated with the area of their service and residence being urban or rural ( mann - whitney u = 114.5 , p=.001 ) . more than 81% ( n=43 ) either strongly agreed or agreed that ecps can lead to evil practices in society . almost 30 % ( n = 15 ) would not consider using or prescribing them to a friend , even in case of need . however , more than 83% ( n=44 ) either strongly agreed or agreed that there is a need for ecps for the family planning clients of the np . using chi square test , attitudes were found to be significantly associated with knowledge of the participants ( p=0.034 , fisher 's exact test ) . relationship was sought between attitudes and other variables like marital status , education , rural or urban area of residence , past medical professional background . however , no statistically significantly association was found with any variable other than urban / rural residence of the participants . majority ( 81% , n=43 ) of the participants believed that ecps can lead to this attitude was found to be associated with the rural / urban residence of the lhs ( mann - whitney u = 159 , p=0.012 ) . 12 , 14 and 16 years of education correspond to matriculate , faculty of arts ( f.a ) , bachelor of arts ( b.a ) and master of arts ( m.a ) , respectively . only 3.8% ( n=2 ) and 11.3% ( n=6 ) had completed only 10 years and 16 years of education , respectively . majority ( 56% , n=30 ) ) had obtained a bachelors degree . majority ( 70 % , n=37 ) of the supervisors were resident of and serving in rural areas of rawalpindi district . general awareness of ecps was high ( 75.5% , n=40 ) but practical use was low ( 17% , n=9 ) . green star , a social marketing company was the major source ( 24.5% , n=13 ) of awareness . lady health visitors and doctors working at the designated health facility of lhss led to awareness of 18.9 % ( n= 10 ) and 11.3% ( n= 6 ) , respectively . similarly , population welfare organization and nurses were the source of information of 11.3% ( n= 6 ) and 3.8% ( n= 2 ) of the participants , respectively . awareness of ecps and source as seen in table 3 , almost half of the participants ( 45% n= 24 ) could identify ecps from a list of medicine . the duration after unprotected sex in which ecps may be helpful in preventing an unwanted pregnancy , was correctly identified by 24% ( n=13 ) of the respondents . almost 64% ( n=34 ) either thought that ecps were abortive drugs or were not sure . more than half ( 53 % n=28)of the participants thought ecps had harmful effects on future pregnancies . lady health supervisors knowledge about ecps the awareness and knowledge of the participants was not found to be associated with their age , marital status , educational background or past medical experience . however , the question regarding the mode of action of ecps being abortive was associated with the area of their service and residence being urban or rural ( mann - whitney u = 114.5 , p=.001 ) . more than 81% ( n=43 ) either strongly agreed or agreed that ecps can lead to evil practices in society . almost 30 % ( n = 15 ) would not consider using or prescribing them to a friend , even in case of need . however , more than 83% ( n=44 ) either strongly agreed or agreed that there is a need for ecps for the family planning clients of the np . using chi square test , attitudes were found to be significantly associated with knowledge of the participants ( p=0.034 , fisher 's exact test ) . relationship was sought between attitudes and other variables like marital status , education , rural or urban area of residence , past medical professional background . however , no statistically significantly association was found with any variable other than urban / rural residence of the participants . majority ( 81% , n=43 ) of the participants believed that ecps can lead to this attitude was found to be associated with the rural / urban residence of the lhs ( mann - whitney u = 159 , p=0.012 ) . 12 , 14 and 16 years of education correspond to matriculate , faculty of arts ( f.a ) , bachelor of arts ( b.a ) and master of arts ( m.a ) , respectively . only 3.8% ( n=2 ) and 11.3% ( n=6 ) had completed only 10 years and 16 years of education , respectively . majority ( 56% , n=30 ) ) had obtained a bachelors degree . majority ( 70 % , n=37 ) of the supervisors were resident of and serving in rural areas of rawalpindi district . general awareness of ecps was high ( 75.5% , n=40 ) but practical use was low ( 17% , n=9 ) . green star , a social marketing company was the major source ( 24.5% , n=13 ) of awareness . lady health visitors and doctors working at the designated health facility of lhss led to awareness of 18.9 % ( n= 10 ) and 11.3% ( n= 6 ) , respectively . similarly , population welfare organization and nurses were the source of information of 11.3% ( n= 6 ) and 3.8% ( n= 2 ) of the participants , respectively . awareness of ecps and source as seen in table 3 , almost half of the participants ( 45% n= 24 ) could identify ecps from a list of medicine . the duration after unprotected sex in which ecps may be helpful in preventing an unwanted pregnancy , was correctly identified by 24% ( n=13 ) of the respondents . almost 64% ( n=34 ) either thought that ecps were abortive drugs or were not sure . more than half ( 53 % n=28)of the participants thought ecps had harmful effects on future pregnancies . lady health supervisors knowledge about ecps the awareness and knowledge of the participants was not found to be associated with their age , marital status , educational background or past medical experience . however , the question regarding the mode of action of ecps being abortive was associated with the area of their service and residence being urban or rural ( mann - whitney u = 114.5 , p=.001 ) . more than 81% ( n=43 ) either strongly agreed or agreed that ecps can lead to evil practices in society . almost 30 % ( n = 15 ) would not consider using or prescribing them to a friend , even in case of need . however , more than 83% ( n=44 ) either strongly agreed or agreed that there is a need for ecps for the family planning clients of the np . using chi square test , attitudes were found to be significantly associated with knowledge of the participants ( p=0.034 , fisher 's exact test ) . relationship was sought between attitudes and other variables like marital status , education , rural or urban area of residence , past medical professional background . however , no statistically significantly association was found with any variable other than urban / rural residence of the participants . majority ( 81% , n=43 ) of the participants believed that ecps can lead to this attitude was found to be associated with the rural / urban residence of the lhs ( mann - whitney u = 159 , p=0.012 ) . even though ecps are not included among the methods adopted by np , the general awareness about them was high ( 75% ) among its lhss . this 75% awareness level is more than that found among graduate students in india ( 7.3% ) , kenya ( 39% ) , ghana ( 43.2% ) , clients for abortion in cape town sa ( 35.4% ) and usa ( 31% ) . this is understandable as their primary area of work is family planning , even though no formal ecp - related training is provided . open discussions on media are widely accepted as being a major source of awareness for the general population . almost all ( 95% n=37 ) of the participants had heard of ecps from formal sources . this is different from the findings from cameroon where formal sources were associated with adequate knowledge and from jamaica where even informal sources were associated with correct knowledge . this is a relatively larger percentage as compared to 7.4% in cameroon , 7.5% in kenya and 10% in jamaica . the majority of participants did not know the mechanism of action and deemed ecps as abortive drug . in pakistan as in other islamic countries , abortion is illegal and considered immoral . the use and prescription of ecps that are perceived to be abortive drugs may be resisted on these grounds . these religious and cultural factors , although important are not insurmountable , as is evident from comparison of trends in bangladesh and pakistan . ecps are safe , in fact safer than pregnancies , especially those that are unintended and where women do not have access to safe services . however , more than half of the participants thought that ecps can harm future pregnancies . this harm might have been considered either due to the perceived teratogenic effects of the pills or delaying of future conceptions . in either case it will be difficult for the lhss to promote ecps if they are not convinced themselves . the non abortive mechanism of action and absence of serious side effects may be stressed during the introduction phase of ecps . the former , in general , face a lack of facilities like schools and hospitals . this area of residence seems to play some role in the knowledge and attitudes of the lhss . it was found to be associated with the question regarding the abortive nature of ecps and the belief that ecps can lead to evil practices in the society . a significant number of lhss of rural settings believed that ecps were abortive drugs and that they may lead to evil practices in society . however , the cumulative score of attitudes was only related to the knowledge of the participants . this seems to be a significant find , assuring that good quality trainings and targeting the identified gaps of knowledge , may also improve the attitudes of the workforce . ecps may have the potential to significantly reduce the morbidity and mortality associated with unsafe abortions which are a major cause of maternal deaths in developing countries . in pakistan , the national program of family planning bridges the gap between the community and health care system . these attitudes have shown to be related to their knowledge of the subject . for smooth introduction of ecps in its arsenal of the family planning methods , national program of family planning may consider addressing the gaps in knowledge and misplaced beliefs .

they have traditionally been considered benign , but recent evidence indicates that frequent apbs are a strong predictor of atrial fibrillation development and may be associated with increased risk of cardiovascular death in general population . in addition , frequent apbs may cause cardiomyopathy . her heart was structurally normal heart and there was no evidence of any sustained tachyarrhythmias . to our knowledge , this is the first report on successful ablation of frequent apbs in the non - coronary aortic cusp . a 59-year - old female with a long history of highly symptomatic frequent apbs presented to our hospital for catheter ablation . p wave was negative in inferior leads ii , iii , avf , positive in avr and v1v3 , indifferent in lead i , avl and v4v6 ( fig . 1 ) . apb burden in repeated holter recordings was 2040% ( 29000 apbs in the last holter recording ) . neither atrial fibrillation nor any other sustained supraventricular tachyarrhythmia was detected . clinical examination and thyroid function were normal . diagnostic catheters were placed in the coronary sinus and right ventricular apex vie right femoral vein . mapping and ablation was performed retrogradely via right femoral artery with a 3.5 mm tip open irrigated ablation catheter ( biosense webster navistar rmt , diamond bar , ca , usa ) using remote magnetic navigation ( epoch , stereotaxis inc . , st louis , mo , usa ) and 3d electroanatomical mapping system ( carto3 , biosense webster ) . mapping was started from the right atrium because of the p wave morphology and coronary sinus activation sequence . fast anatomical activation mapping demonstrated that the earliest activation in the right atrium was in the vicinity of the his bundle ( fig . 2 ) . no ablation attempt was made although local activation at this site preceded the p wave because of high risk of damage to the atrioventricular conduction system . meticulous mapping at the aortic root demonstrated earliest local activation of the clinical apb in the non - coronary sinus of valsalva anterior and superior to the his bundle . at this site no his potential was detected , and the local electrogram was earlier than in the para - hisian area in right atrium and preceded the p wave by about 50 ms ( fig . 3 ) . radiofrequency energy delivery at the non - coronary aortic cusp resulted in immediate cessation of the apbs . no junctional beats or pr prolongation were observed during ablation ( 120 s with maximum power of 35 w ) . total duration of the procedure was 110 min and fluoroscopy exposure 2 min 35 s ( 3.0 gy / cm2 ) , respectively . at discharge a routine holter recording was scheduled at 3 months and the patient was advised to contact her physician for ecg monitoring if any arrhythmia symptoms recurred . during the follow - up of 12 months the patient reported no recurrence of symptoms and there were no clinically relevant apbs in the holter recording . atrial premature beats are present in 1020% of the general population . in most cases they are benign , but frequent apbs have been associated with development of atrial fibrillation and cardiomyopathy as well as with increased risk of cardiovascular mortality and stroke , , . in our case no sustained atrial tachyarrhythmias were documented , and left ventricular function was normal despite long history of frequent apbs . nevertheless , it is possible that the high apb burden could have caused atrial fibrillation and/or apb - induced cardiomyopathy if not treated . the majority of apbs arise from within and around the pulmonary veins ( pv ) . other sites of origin include left atrial posterior wall , ligament of marshall , coronary sinus , superior and inferior vena cava , crista terminals and tricuspid annulus . catheter ablation has become the treatment of choice in patients with various paroxysmal supraventricular tachycardias and ventricular premature beats , but it is used quite rarely to eliminate isolated apbs . the frequently multiple sites of origin and capricious manifestation have made it difficult to ablate extrapulmonary abps . however , in patients with frequent monomorphic apbs the focus can usually be identified by careful activation mapping and treated by ablation . to the best of our knowledge this is the first report on successful ablation of frequent apbs in the non - coronary aortic cusp . based on our experience the main benefit of using magnetic navigation in cases like this is that mapping with a softer and more flexible magnetic catheter is less likely to cause perforation and to provoke catheter - induced extrasystoles than mapping with a manually steered ablation catheter . in addition , the magnetic technology has proven to reduce personnel and patient radiation exposure . it offers important safety benefit by reducing the risk of perforation but also makes creation of transmural lesions difficult in some areas ( e.g. , cavo - tricuspid isthmus ) . the p wave morphology and right atrial mapping demonstrated that the apbs originated from a site close to the his bundle . we and others have previously shown that sustained atrial tachycardia in this area can often be effectively and safely ablated within the non - coronary aortic cusp which is anatomically in close proximity to the interatrial septum and his bundle , . in the current case , the local atrial activation at the non - coronary aortic cusp was about 20 ms earlier than that recorded at the his region in the right atrium . at the ablation site no his potential was visible , and the catheter was stable and far from the coronary artery ostia . this case demonstrated that in addition to focal atrial tachyarrhythmias and ventricular premature beats aortic root can be a source for frequent apbs . due to the close anatomic relationship between the atrioventricular node and the non - coronary aortic cusp mapping of the aortic root and non - coronary cusp

the incidences of neuropsychiatric manifestations , like affective disorder or depression , are increasingly reported in general psychiatric and neurological practices , due to high - stress lifestyle . there are many contributing psychological , neuropsychiatric , and medical factors that should be investigated . the antiphospholipid syndrome ( aps ) , also known as lupus anticoagulant syndrome or anticardiolipin antibody syndrome , is a rare form of autoimmune coagulopathy.1,2 aps is characterized and diagnosed by recurrent vascular thrombosis or pregnancy - related morbidity , in the presence of circulating antiphospholipid ( apl ) antibodies . it is usually observed in young adults with , the most common neurologic manifestation being transient ischemic attack ; other manifestations include stroke , seizure , or acute encephalopathy.1,2 it can be easily overlooked if the patient presents with progressive neuropsychiatric disorders , such as depression or dementia . herein , we report two young women with aps who presented with similar neuropsychiatric disorders but different radiological manifestations . a 35-year - old female experienced progressive mental decline and depression for more than 2 years . initially she was treated at a psychiatric outpatient department ( opd ) , for 1 year . the depressive symptoms improved , but cognitive impairment did not show improvement ; hence , she was referred to the neurological opd . initial laboratory analysis revealed elevated erythrocyte sedimentation rate ( esr ) ( 312 mm / h ) and positive antinuclear antibody ( ana ) test ( 1:80 ) . dementia was suspected , based on the clinical presentation , and she was admitted for further examination . her history revealed four episodes of fetal abortion and diagnosis of aps 3 years previously , by medical record ; she had discontinued treatment for aps for 2 years . laboratory findings indicated anemia ( hemoglobin : 11.3 g / dl ) , thrombocytopenia ( platelet count : 76000/l ) , and abnormal coagulation function ( partial thromboplastin time [ ptt ] : 73.4/29.3 s ; prothrombin time [ pt ] : 11.2/10.8 s ; international normalized ratio [ inr ] : 1.16 ) . her autoimmune profiles were positive for anticardiolipin antibody ( acl ) , lupus anticoagulant antibody ( lac ) , ana , rheumatoid arthritis factor ( ra ) , apl immunoglobulin ( ig)g and igm , anti - ro , and showed decreased complement protein ( c)3 and c4 levels ( table 1 ) . the findings of brain magnetic resonance imaging ( mri ) were multiple old infarcts with encephalomalacia in bilateral cerebral hemispheres and the left cerebellar hemisphere ( figure 1 ) . her cognitive ability screening instrument ( casi ) score was 19 ( cutoff value is 85 , below is abnormal result).3 the casi - estimated mini - mental state examination ( mmse - ce ) score was 9 ( cutoff value is 25 , below is abnormal result ) . assessment with the neuropsychiatric inventory ( npi ) indicated disinhibited behavior . according to the report of neuropsychological testing warfarin was prescribed to control coagulopathy , along with a disease - modifying antirheumatic drug ( hydroxychloroquine ) . however , we lost this patient to follow - up for unknown reason . a 22-year - old unmarried female developed progressively depressive mood over a period of 1 month . gradually , her verbal output decreased , with incoherent speech , and she developed mild left upper limb weakness . intermittent involuntary movements of her four limbs were observed for several days before her referral to the neurological opd . neurological examination revealed abulia and slight decrease in the muscle power of her left upper limb . immediately brain computed topography ( ct ) was performed ; the results revealed a hyperdense , gyriform lesion along the cortices of the right temporoparietal lobe , with perifocal edema ( figure 2a ) . following this , she was transferred to a medical center hospital . at the emergency department , the laboratory blood test revealed anemia ( hemoglobin : 11.3 g / dl ) , thrombocytopenia ( platelet count : 75000/l ) , and normal coagulation function ( ptt : 30.6/28.6 s ; pt : 10.9/10.7 s ; inr : 1.04 ) . the brain ct findings were discussed with a neurosurgeon , and arteriovenous malformation ( avm ) with hemorrhage was suspected ( figure 2 ) . the brain mri finding was subacute to chronic infarction with hemorrhagic transformation in the right temporoparietal lobe ; the differential diagnosis was encephalitis with petechial hemorrhage ( figure 3 ) . further laboratory findings indicated an abnormal autoimmune function ( positive acl , lac , and anti-2 glycoprotein - i antibody , and elevated c3 level ) ( table 1 ) , and abnormal thyroid function . based on the neuropsychological testing , borderline dementia with depression and delusion were diagnosed ( table 2 ) . an eeg examination showed normal background activity with an intermittent theta wave over the right hemisphere . the final diagnosis was established after consulting the rheumatologist ; the aps was complicated by cerebral hemorrhagic infarction accompanied by borderline dementia , depression , suspected seizure , and concomitant hyperthyroidism . the follow - up brain ct 17 days later revealed encephalomalacia in the right temporoparietal lobe , with partial resolution of hemorrhagic transformation . because of the bleeding tendency underlying the recent hemorrhagic infarction , we prescribed acetylsalicylic acid instead of warfarin for temporary stroke prevention . a 35-year - old female experienced progressive mental decline and depression for more than 2 years . initially she was treated at a psychiatric outpatient department ( opd ) , for 1 year . the depressive symptoms improved , but cognitive impairment did not show improvement ; hence , she was referred to the neurological opd . initial laboratory analysis revealed elevated erythrocyte sedimentation rate ( esr ) ( 312 mm / h ) and positive antinuclear antibody ( ana ) test ( 1:80 ) . dementia was suspected , based on the clinical presentation , and she was admitted for further examination . her history revealed four episodes of fetal abortion and diagnosis of aps 3 years previously , by medical record ; she had discontinued treatment for aps for 2 years . laboratory findings indicated anemia ( hemoglobin : 11.3 g / dl ) , thrombocytopenia ( platelet count : 76000/l ) , and abnormal coagulation function ( partial thromboplastin time [ ptt ] : 73.4/29.3 s ; prothrombin time [ pt ] : 11.2/10.8 s ; international normalized ratio [ inr ] : 1.16 ) . her autoimmune profiles were positive for anticardiolipin antibody ( acl ) , lupus anticoagulant antibody ( lac ) , ana , rheumatoid arthritis factor ( ra ) , apl immunoglobulin ( ig)g and igm , anti - ro , and showed decreased complement protein ( c)3 and c4 levels ( table 1 ) . the findings of brain magnetic resonance imaging ( mri ) were multiple old infarcts with encephalomalacia in bilateral cerebral hemispheres and the left cerebellar hemisphere ( figure 1 ) . her cognitive ability screening instrument ( casi ) score was 19 ( cutoff value is 85 , below is abnormal result).3 the casi - estimated mini - mental state examination ( mmse - ce ) score was 9 ( cutoff value is 25 , below is abnormal result ) . assessment with the neuropsychiatric inventory ( npi ) indicated disinhibited behavior . according to the report of neuropsychological testing warfarin was prescribed to control coagulopathy , along with a disease - modifying antirheumatic drug ( hydroxychloroquine ) . a 22-year - old unmarried female developed progressively depressive mood over a period of 1 month . gradually , her verbal output decreased , with incoherent speech , and she developed mild left upper limb weakness . intermittent involuntary movements of her four limbs were observed for several days before her referral to the neurological opd . neurological examination revealed abulia and slight decrease in the muscle power of her left upper limb . immediately brain computed topography ( ct ) was performed ; the results revealed a hyperdense , gyriform lesion along the cortices of the right temporoparietal lobe , with perifocal edema ( figure 2a ) . following this , the laboratory blood test revealed anemia ( hemoglobin : 11.3 g / dl ) , thrombocytopenia ( platelet count : 75000/l ) , and normal coagulation function ( ptt : 30.6/28.6 s ; pt : 10.9/10.7 s ; inr : 1.04 ) . the brain ct findings were discussed with a neurosurgeon , and arteriovenous malformation ( avm ) with hemorrhage was suspected ( figure 2 ) . the brain mri finding was subacute to chronic infarction with hemorrhagic transformation in the right temporoparietal lobe ; the differential diagnosis was encephalitis with petechial hemorrhage ( figure 3 ) . further laboratory findings indicated an abnormal autoimmune function ( positive acl , lac , and anti-2 glycoprotein - i antibody , and elevated c3 level ) ( table 1 ) , and abnormal thyroid function . based on the neuropsychological testing , borderline dementia with depression and delusion were diagnosed ( table 2 ) . an eeg examination showed normal background activity with an intermittent theta wave over the right hemisphere . the final diagnosis was established after consulting the rheumatologist ; the aps was complicated by cerebral hemorrhagic infarction accompanied by borderline dementia , depression , suspected seizure , and concomitant hyperthyroidism . the follow - up brain ct 17 days later revealed encephalomalacia in the right temporoparietal lobe , with partial resolution of hemorrhagic transformation . because of the bleeding tendency underlying the recent hemorrhagic infarction , we prescribed acetylsalicylic acid instead of warfarin for temporary stroke prevention . in the two young patients with initial presentations of progressive depression and mild cognitive impairment , it is likely that an organic brain lesion could have been overlooked during diagnosis . notably , detailed review and investigation should be performed when patients present with obscure stress events , failed medication response , and persistent neuropsychiatric symptoms . the common differential diagnoses of young patients with neuropsychiatric disorders or dementia include structural disease , autoimmune encephalopathy , vasculopathies , psychiatric illness , etc . the reversible and treatable underlying disorders should be given priority.4 central nervous system ( cns ) involvement is a prominent feature of aps ; many acute neurological manifestations have been described in aps patients . however , there are limited reports on the psychiatric manifestations associated with aps , such as depression and mania.5 primary aps is considered a rare autoimmune disease and secondary aps is closely related to systemic lupus erythematosus ( sle ) , hematologic disorders , infection , and some medications . in the case of sle , there is an association between neuropsychiatric symptoms and specific antibodies;6,7 in particular , apl antibodies have been associated with stroke , vascular dementia , and epilepsy.810 in our first case , the patient did not completely satisfy the diagnostic criteria of probable sle , after consultation with the rheumatologist.11 however , sle might be diagnosed at a later stage in the clinical course , and hence , regular follow - up visits were strongly advised . in the second case , unlike the usual images of acute stroke , those of aps might mimic tumor mass , vascular malformation , and inflammatory lesion at the subacute to chronic stage . it is necessary to conduct careful investigations , with the cooperation of experts from various disciplines . another important inference from these two case studies is that aps might present as similar neuropsychiatric disorders ( ie , depression or dementia ) arising from different cerebrovascular lesions involving the contributory circuits and resulting in vascular depression and vascular dementia . early intervention will allow good prognosis . regarding the treatment of aps - related neuropsychiatric disorders , the symptomatic control of vascular complications and management of underlying autoimmune coagulopathy ( disease - modifying antirheumatic drugs and anticoagulation agents ) are suggested . for vascular depression , the antidepressant of selective serotonin reuptake inhibitor ( ssri ) type might be the considered choice because of its lesser influence on the cardiovascular system . on the other hand , the circulation - promoting agents and acetylcholinesterase inhibitors could be useful for vascular dementia , according to the patient s clinical status . additionally , many nonpharmacological interventions are available and are suggested as concomitant therapy for intensive care of neuropsychiatric disorders . because the incidences of progressive neuropsychiatric disorders in the young are increasing , we have underscored the clinical relevance of the differential diagnoses of systemic autoimmune diseases with cns involvement , such as aps . brain imaging is an essential tool to prevent any delay in the detection of structural lesions and facilitate the early intervention with good prognosis .

one major concern of endodontists is to prevent microleakage after root canal filling , in order to prevent bacterial invasion to the root canal space . that is because microorganisms play a significant role in pulpal and periapical diseases [ 1 , 2 ] . reported that in coronally unsealed endodontically treated teeth , saliva penetration was seen after 30 days regardless of the obturation technique ( lateral versus vertical condensation ) . trope et al . explained that endotoxins could penetrate through an obturated root canal in less than 21 days , if the coronal seal is not achieved . for the first time , bowman used gutta - percha as a root filling material and mentioned that one of the practical ways to limit microorganisms in the root canal system is to achieve a three dimensional root canal filling . this method can deprive the remaining microorganisms after cleaning and shaping of the canal system . the most popular material for root canal filling is the beta phase of gutta - percha . it is stated that guttapercha in alpha phase has special characteristics such as less shrinkage , lower viscosity and more adherence than the beta phase [ 57 ] . controversy exists regarding which phase of gutta - percha can provide a better apical seal . compared the radiographic quality of obturation with alpha and beta phases of gutta - percha ( thermafil versus lateral condensation method ) . they reported that the quality of the alpha phase of gutta - percha was significantly better . reported that more gutta - percha was seen in the lateral canals when alpha phase gutta - percha coated rigid carrier technique was used ; whereas more sealer was present when using cold lateral condensation and these differences were statistically significant . pommel and campes revealed that after one month , microbial leakage in thermafil method and vertical condensation technique was significantly less than that in lateral condensation and single cone methods . on the other hand , de deus et al . showed that there was no statistically significant difference in the sealing ability of lateral condensation , warm vertical condensation and thermafil methods in a period of 100 days . . showed that there was no statistically significant difference between thermafil and lateral condensation when evaluating dye microleakage after 24 hours , seven days and five months . compared the apical seal of lateral condensation , thermafil and one step technique by assessing microbial leakage . it should be noted that in all previous studies , the alpha phase of gutta - percha was used by the thermafil method [ 913 ] but the aim of this study was to compare the apical sealing ability of alpha and beta gutta - percha cones by using warm vertical condensation method . in this experimental study , 50 extracted human premolars with a single root canal were selected . the teeth , which had been previously treated endodontically , had more than one root canal , caries , calcification , fracture , internal or external root resorption , curvature and open apex were excluded from the study . access cavity was prepared in all samples . a # 15 k - file ( dentsply maillefer , ballaigues , switzerland ) was used to measure the canal 's diameter ( if # 15 k - file was loose , the tooth was excluded from the study ) . all samples were prepared with mtwo rotary files ( vdw , munich , germany ) up to # 35.04 . then , the canals were dried with paper points ( meta bio - med co. ltd . , the teeth were divided into three groups , according to the obturation technique used : 1- obturation with alpha phase gutta - percha ( vdw , munich , germany ) ( n=20).2- obturation with beta phase gutta - percha ( vdw , munich , germany ) ( n=20).3- control groups with no obturation . 1- obturation with alpha phase gutta - percha ( vdw , munich , germany ) ( n=20 ) . 2- obturation with beta phase gutta - percha ( vdw , munich , germany ) ( n=20 ) . a # 35.04 gutta - percha master cone and ah26 sealer were used by means of beefill pack ii ( vdw , munich , germany ) . after obturation , gutta - percha was cut with a # 30 plagger ( dentsply maillefer , ballaigues , switzerland ) and compressed . all samples were cut 10 mm coronal to the apex with a diamond disc ( d+z , bern , switzerland ) . the outer surfaces of the samples other than the apical 2 mm were covered with two layers of nail varnish for prevention of bacterial leakage from the accessory and lateral canals except for the apical foramen . samples were mounted and fixed in the plastic vial caps and sealed with cyanoacrylate glue ; then , they were sterilized with ethylene oxide gas for 24 hours . afterwards , mueller hinton broth ( merck , darmstadt , germany ) was added to the vials in such a way that the apical 2 mm of the roots was in contact with the medium . all obturated samples were exposed to the bacterial suspension of e. faecalis every three days coronally . samples in the control group were considered as negative controls ( n=2 ) and were not exposed to bacterial suspension ; the remaining control samples ( n=8 ) served as positive controls and were exposed to the bacterial suspension . the number of days required for the contamination of the entire root canals was recorded . the outer surfaces of the samples other than the apical 2 mm were covered with two layers of nail varnish for prevention of bacterial leakage from the accessory and lateral canals except for the apical foramen . samples were mounted and fixed in the plastic vial caps and sealed with cyanoacrylate glue ; then , they were sterilized with ethylene oxide gas for 24 hours . afterwards , mueller hinton broth ( merck , darmstadt , germany ) was added to the vials in such a way that the apical 2 mm of the roots was in contact with the medium . all obturated samples were exposed to the bacterial suspension of e. faecalis every three days coronally . samples in the control group were considered as negative controls ( n=2 ) and were not exposed to bacterial suspension ; the remaining control samples ( n=8 ) served as positive controls and were exposed to the bacterial suspension . the number of days required for the contamination of the entire root canals was recorded . distribution of bacterial leakage time ( by days ) in alpha and beta phase gutta - percha the negative control samples ( without bacteria and no canal filling ) showed no turbidity after 24 hours of incubation . the positive control samples ( with bacteria and no canal filling ) showed 100% turbidity after 24 hours . all samples in groups g1 and g2 were infected with e. faecalis after 30 days . the mean time of bacterial leakage in g1 and g2 was 12.059.85 and 10.57.8 days , respectively . the difference between the two groups was not statistically significant in this respect ( p=0.74 ) . the proper obturation of the root canal system is a key factor to achieve successful results in endodontic treatments . although beta phase guttapercha is the most commonly used obturation material , the alpha phase gutta - percha can flow better in the root canals and fill more lateral canals ; thus , it can provide a better apical seal . in this in vitro study , we used bacterial leakage model that is more similar to clinical situation than other microleakage evaluation methods . dye penetration is not a proper method to simulate the clinical setting . since e. faecalis is responsible for one - third of endodontic treatment failures , in the present study , we used e. faecalis suspension [ 1619 ] . used dye penetration method to assess the microleakage and they described that sealing ability of ah26 was better than that of tubliseal , sealapex and apexit . zhang et al . reported that alpha phase gutta - percha with warm vertical compaction technique moved significantly more into the lateral canals and depressions than the beta - phase gutta - percha . they showed that beta phase guttapercha had more shrinkage and led to higher level of microleakage . in this study , we did not observe any turbidity in muller hinton broth medium in the negative control group , which means nail varnish and cyanoacrylate glue prevent bacterial leakage from other parts of the root , except for the apical foramen . also , we observed complete turbidity in the positive control group . results of the current study showed that there was no statistically significant difference in apical bacterial leakage between alpha and beta phases of gutta - percha . in this in vitro study , we concluded that the type of gutta - percha crystallization had no impact on its apical sealing ability .

in this review we consider the problem of detection of deterministic gravitational - wave signals in the noise of a detector and the question of estimation of their parameters . the examples of deterministic signals are gravitational waves from rotating neutron stars , coalescing compact binaries , and supernova explosions . the case of detection of stochastic gravitational - wave signals in the noise of a detector is reviewed in . a very powerful method to detect a signal in noise that is optimal by several criteria consists of correlating the data with the template that is matched to the expected signal . this matched - filtering technique is a special case of the maximum likelihood detection method . in this review we describe the theoretical foundation of the method and we show how it can be applied to the case of a very general deterministic gravitational - wave signal buried in a stationary and gaussian noise . early gravitational - wave data analysis was concerned with the detection of bursts originating from supernova explosions . it involved analysis of the coincidences among the detectors . with the growing interest in laser interferometric gravitational - wave detectors that are broadband it was realized that sources other than supernovae can also be detectable and that they can provide a wealth of astrophysical information [ 122 , 77 ] . for example , the analytic form of the gravitational - wave signal produced during the inspiral phase of a compact binary coalescence is known in terms of a few parameters to a good approximation ( see , e.g. , and section 2.4 of ) . consequently one can detect such a signal by correlating the data with the predicted waveform ( often called the template ) and maximizing the correlation with respect to the parameters of the waveform . using this method one can pick up a weak signal from the noise by building a large signal - to - noise ratio over a wide bandwidth of the detector . this observation has led to rapid development of the theory of gravitational - wave data analysis . it became clear that the detectability of sources is determined by optimal signal - to - noise ratio , which is the power spectrum of the signal divided by the power spectrum of the noise integrated over the bandwidth of the detector . an important landmark was a workshop entitled gravitational wave data analysis held in dyffryn house and gardens , st . nicholas near cardiff , in july 1987 . the meeting acquainted physicists interested in analyzing gravitational - wave data with the basics of the statistical theory of signal detection and its application to detection of gravitational - wave sources . as a result of subsequent studies , the fisher information matrix was introduced to the theory of the analysis of gravitational - wave data [ 50 , 76 ] . the diagonal elements of the fisher matrix give lower bounds on the variances of the estimators of the parameters of the signal and can be used to assess the quality of astrophysical information that can be obtained from detections of gravitational - wave signals [ 41 , 75 , 25 ] . it was also realized that the application of matched - filtering to some sources , notably to continuous sources originating from neutron stars , will require extraordinary large computing resources . this gave a further stimulus to the development of optimal and efficient algorithms and data analysis methods . a very important development was the work by cutler et al . where it was realized that for the case of coalescing binaries matched filtering was sensitive to very small post - newtonian effects of the waveform . this leads to a much better verification of einstein s theory of relativity and provides a wealth of astrophysical information that would make a laser interferometric gravitational - wave detector a true astronomical observatory complementary to those utilizing the electromagnetic spectrum . as further development of the theory , methods were introduced to calculate the quality of suboptimal filters , to calculate the number of templates required to do a search using matched - filtering , to determine the accuracy of templates required , and to calculate the false alarm probability and thresholds . an important point is the reduction of the number of parameters that one needs to search for in order to detect a signal . namely estimators of a certain type of parameters , called extrinsic parameters , can be found in a closed analytic form and consequently eliminated from the search . thus , a computationally - intensive search need only be performed over a reduced set of intrinsic parameters [ 76 , 68 , 78 ] . techniques reviewed in this paper have been used in the data analysis of prototypes of gravitational - wave detectors [ 100 , 99 , 12 ] and in the data analysis of gravitational - wave detectors currently in operation [ 133 , 23 , 4 , 3 , 2 ] . one method is to measure changes induced by gravitational waves on the distances between freely - moving test masses using coherent trains of electromagnetic waves . the other method is to measure the deformation of large masses at their resonance frequencies induced by gravitational waves . the first idea is realized in laser interferometric detectors ( both earth - based [ 105 , 141 , 53 ] and space - borne [ 85 , 137 ] antennas ) and doppler tracking experiments , whereas the second idea is implemented in resonant mass detectors ( see , e.g. , ) . we start by describing the change of a photon s frequency caused by a passing gravitational wave and registered by particles ( representing different parts of a gravitational - wave detector ) freely falling in the field of the gravitational wave . the detailed derivation of the formulae we show here can be found in chapter 5 of ( see also [ 47 , 15 , 120 ] ) . an equivalent derivation of the response of test masses to gravitational waves in the local lorentz gauge ( without making use of the long - wavelength approximation ) is given in . we employ here the transverse traceless ( tt ) coordinate system ( more about the tt gauge can be found , e.g. , in section 35.4 of or in section 1.3 of ) . a spacetime metric describing a plane gravitational wave traveling in the + z direction of the tt coordinate system ( with coordinates x ct , x x , x y , x z ) , is described by the line element 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}{{\rm{s}}^2}= - { c^2}{\rm{d}}{t^2 } + \left({1 + { h _ + } \left({t - { z \over c } } \right ) } \right){\rm{d}}{x^2 } + \left({1 - { h _ + } \left({t - { z \over c } } \right ) } \right){\rm{d}}{y^2 } + 2{h _ \times}\left({t - { z \over c } } \right){\rm{d}}x{\rm{d}}y + { \rm{d}}{z^2},$$\end{document } where h+ and h are the two independent polarizations of the wave . we assume that the wave is weak , i.e. , for any instant of time t , 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert { h _ + } ( t)\vert \ll 1,\,\vert { h _ \times}(t)\vert \ll 1.$$\end{document } we will neglect all terms of order h or higher . the form of the line element ( 1 ) implies that the functions h+(t ) and h(t ) describe the wave - induced perturbation of the flat minkowski metric at the origin of the tt coordinate system ( where x = y = z = 0 ) . it is convenient to introduce the three - dimensional matrix of the spatial metric perturbation produced by the gravitational wave ( at the coordinate system s origin ) , 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{h}}(t ) : = \left({\begin{array}{*{20}c } { { h _ + } ( t ) } & { { h _ \times}(t ) } & 0 \\ { { h _ \times}(t ) } & { - { h _ + } ( t ) } & 0 \\ 0 & 0 & 0 \end{array } } \right).$$\end{document } let two particles freely fall in the field ( 1 ) of the gravitational wave , and let their spatial coordinates remain constant , so the particles world lines are described by equations 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t({\tau _ a } ) = { \tau _ a},\,x({\tau _ a } ) = { x_a},\,y({\tau _ a } ) = { y_a},\,z({\tau _ a } ) = { z_a},\,a = 1,2,$$\end{document } where ( xa , ya , za ) are spatial coordinates of the ath particle and a is its proper time . these two particles measure , in their proper reference frames , the frequency of the same photon traveling along a null geodesic x = x( ) , where is some affine parameter . the coordinate time , at which the photon s frequency is measured by the ath particle , is equal to ta ( a = 1 , 2 ) ; we assume that t2 > t1 . let us introduce the coordinate time duration t12 of the photon s trip and the euclidean coordinate distance l12 between the particles : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{12 } } : = { t_2 } - { t_1},{l_{12 } } : = \sqrt { { { ( { x_2 } - { x_1})}^2 } + { { ( { y_2 } - { y_1})}^2 } + { { ( { z_2 } - { z_1})}^2}.}$$\end{document } let us also introduce the 3-vector n of unit euclidean length directed along the line connecting the two particles . we arrange the components of this vector into the column 3 1 matrix n ( thus , we distinguish here the 3-vector n from its components being the elements of the matrix n ; the same 3-vector can be decomposed into components in different spatial coordinate systems ) : 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{n : = ( cos}}\,\alpha { \rm{,cos}}\beta { \rm{,cos}}\gamma)^{\rm{t } } = \left({\begin{array}{*{20}c } { \cos \alpha } \\ { \cos \beta } \\ { \cos \gamma } \\ if one neglects the spacetime curvature caused by the gravitational wave , then , , and are the angles between the path of the photon in the 3-space and the coordinate axis x , y , or z , respectively ( obviously , , , 0 ; ; and cos + cos + cos = 1 ) . let us denote the value of the frequency registered by the ath particle by a ( a = 1 , 2 ) and let us finally define the relative change of the photon s frequencies , 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{12 } } : = { { { \nu _ 2 } } \over { { \nu _ 1 } } } - 1.$$\end{document } then , it can be shown ( see chapter 5 of for details ) that the frequency ratio y12 can be written [ making use of the quantities introduced in eqs . ( 3 ) and ( 5)(6 ) ] as follows ( the dot means here matrix multiplication ) : 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{12 } } = { 1 \over { 2(1 - \cos \gamma)}}{{\rm{n}}^{\rm{t } } } \cdot \left({\rm{h}\left({{t_1 } - { { { z_1 } } \over c } } \right ) - { \rm{h}}\left({{t_1 } - { { { z_1 } } \over c } + ( 1 - \cos \gamma){{{l_{12 } } } \over c } } \right ) } \right ) \cdot { \rm{n + \mathcal{o}(}}{{\rm{h}}^2}).$$\end{document } it is convenient to introduce the unit 3-vector k directed from the origin of the coordinate system to the source of the gravitational wave . in the coordinate system adopted by us the wave is traveling in the + z direction . therefore , the components of the 3-vector k , arranged into the column matrix k , are 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{k } } = { ( 0,0 , - 1)^{\rm{t}}}.$$\end{document } the positions of the particles with respect to the origin of the coordinate system we describe by the 3-vectors xa ( a = 1 , 2 ) , the components of which we put into the column matrices xa : 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm x } _ a } = { ( { x_a},{y_a},{z_a})^{\rm{t}}},\,a = 1,2.$$\end{document } making use of eqs . ( 9)(10 ) we rewrite the basic formula ( 8) in the following form 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{12 } } = { { { { \rm{n}}^{\rm{t } } } \cdot \left({{\rm{h}}\left({{t_1 } + { { { { \rm{k}}^{\rm{t } } } \cdot { { \rm{x}}_1 } } \over c } } \right ) - { \rm{h}}\left({{t_1 } + { { { l_{12 } } } \over c } + { { { { \rm{k}}^{\rm{t } } } \cdot { { \rm{x}}_2 } } \over c } } \right ) } \right ) \cdot { \rm{n } } } \over { 2(1 + { { \rm{k}}^{\rm{t } } } \cdot { \rm{n } } ) } } + { \mathcal o}({h^2}).$$\end{document } to obtain the response for all currently working and planned detectors it is enough to consider a configuration of three particles shown in figure 1 . two particles model a doppler tracking experiment , where one particle is the earth and the other is a distant spacecraft . three particles model a ground - based laser interferometer , where the masses are suspended from seismically - isolated supports or a space - borne interferometer , where the three test masses are shielded in satellites driven by drag - free control systems . in figure 1 we have introduced the following notation : o denotes the origin of the tt coordinate system related to the passing gravitational wave , xa(a = 1 , 2 , 3 ) are 3-vectors joining o and the particles , na and la ( a = 1 , 2 , 3 ) are , respectively , 3-vectors of unit euclidean length along the lines joining the particles and the coordinate euclidean distances between the particles , where a is the label of the opposite particle . we still assume that the spatial coordinates of the particles do not change in time . figure 1schematic configuration of three freely - falling particles as a detector of gravitational waves . the particles are labelled 1 , 2 , and 3 , their positions with respect to the origin o of the coordinate system are given by 3-vectors xa ( a = 1 , 2 , 3 ) . the euclidean separations between the particles are denoted by la , where the index a corresponds to the opposite particle . the unit 3-vectors na point between pairs of particles , with the orientation indicated . the particles are labelled 1 , 2 , and 3 , their positions with respect to the origin o of the coordinate system are given by 3-vectors xa ( a = 1 , 2 , 3 ) . the euclidean separations between the particles are denoted by la , where the index a corresponds to the opposite particle . let us denote by 0 the frequency of the coherent beam used in the detector ( laser light in the case of an interferometer and radio waves in the case of doppler tracking ) . let the particle 1 emit the photon with frequency 0 at the moment t0 towards the particle 2 , which registers the photon with frequency at the moment \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t\prime } = { t_0 } + { l_3}/c + { \mathcal o(h)}$\end{document}. the photon is immediately transponded ( without change of frequency ) back to the particle 1 , which registers the photon with frequency at the moment \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t = { t_0 } + 2{l_3}/c + { \mathcal o(h)}$\end{document}. we express the relative changes of the photon s frequency y12 ( 0)/0 and y21 ( )/ as functions of the instant of time t. making use of eq . ( 11 ) we obtain 12a\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{12}}(t ) = { 1 \over { 2(1 - { { \rm{k}}^{\rm{t } } } \cdot { { \rm{n}}_3})}}{\rm{n}}_3^{\rm{t } } \cdot \left({{\rm{h}}\left({t - { { 2{l_3 } } \over c } + { { { { \rm{k}}^{\rm{t } } } \cdot { { \rm{x}}_1 } } \over c } } \right ) - { \rm{h}}\left({t - { { { l_3 } } \over c } + { { { { \rm{k}}^{\rm{t } } } \cdot { { \rm{x}}_2 } } \over c } } \right ) } \right ) \cdot { { \rm{n}}_3 } + \mathcal{o}({h^2}),$$\end{document } 12b\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{21}}(t ) = { 1 \over { 2(1 + { { \rm{k}}^{\rm{t}}}\cdot{{\rm{n}}_3})}}{\rm{n}}_3^{\rm{t}}\cdot\left({{\rm{h}}\left({t - { { { l_3 } } \over c } + { { { { \rm{k}}^{\rm{t}}}\cdot{{\rm{x}}_2 } } \over c } } \right ) - { \rm{h}}\left({t + { { { { \rm{k}}^{\rm{t}}}\cdot{{\rm{x}}_1 } } \over c } } \right ) } \right)\cdot{{\rm{n}}_3 } + { \mathcal o}({h^2}).$$\end{document } the total frequency shift y121 ( 0)/0 of the photon during its round trip can be computed from the one - way frequency shifts y12 and y21 given above : 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{121 } } = { \nu \over { { \nu _ 0 } } } - 1 = { \nu \over { { \nu { \prime}}}}{{{\nu { \prime } } } \over { { \nu _ 0 } } } - 1 = ( { y_{21 } } + 1)({y_{12 } } + 1 ) - 1 = { y_{12 } } + { y_{21 } } + { \mathcal o}({h^2}).$$\end{document } let l be the size of the detector and /(2 ) be the reduced wavelength of the gravitational wave impinging on the detector . in the long - wavelength approximation the condition l is fulfilled . time delays caused by the finite speed of the wave propagating across the detector are of order t l / c , but 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega \delta t \sim { l \over -{{\rlap{-}\lambda } } } \ll 1,$$\end{document } so time delays across the detector are much shorter than the period of the gravitational wave and can be neglected . it means that with a good accuracy the gravitational - wave field can be treated as being uniform ( but time - dependent ) in the space region that covers the entire detector . to detect gravitational waves with some dominant angular frequency one must collect data over time intervals longer ( sometimes much longer ) than the gravitational - wave period . ( 8) for the relative frequency shift , the typical value of the quantity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar t : = { t_1 } - { z_1}/c$\end{document } will be much larger than the retardation time t l12/c therefore , we can expand this equation with respect to t and keep terms only linear in t . after doing this one obtains ( see section 5.3 in for more details ) : 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{12 } } = - { { { l_{12 } } } \over { 2c}}{{\rm{n}}^{\rm{t}}}\cdot{\rm{\dot h}}({t_1 } - { { { z_1 } } \over c})\cdot{\rm{n } } + { \mathcal o}({h^2},\delta { t^2}),$$\end{document } where overdot denotes differentiation with respect to time . for the configuration of particles shown in figure 1 , the relative frequency shifts y12 and y21 given by eqs . ( 12 ) can be written , by virtue of the formula ( 15 ) , in the form 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{12}}(t ) = { y_{21}}(t ) = - { { { l_3 } } \over { 2c}}{\rm{n}}_3^{\mathrm{t } } \cdot { \rm{\dot h}}\left({t + { { { { \rm{k}}^{\rm{t } } } \cdot { { \rm{x}}_1 } } \over c } } \right ) \cdot { { \rm{n}}_3 } + \mathcal{o}({h^2},\delta { t^2}),$$\end{document } so that they are equal to each other up to terms \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o({h^2},\delta { t^2})}$\end{document}. the photon s total round - trip frequency shift y121 [ cf . eq . ( 13 ) ] is thus equal to 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${y_{121}}(t ) = - { { { l_3 } } \over { 2c}}{\rm{n}}_3^{\rm{t}}\cdot{\rm{\dot h}}\left({t + { { { { \rm{k}}^{\rm{t}}}\cdot{{\rm{x}}_1 } } \over c } } \right)\cdot{{\rm{n}}_3 } + { \mathcal o}({h^2},\delta { t^2}).$$\end{document } there are important cases where the long - wavelength approximation is not valid . these include satellite doppler tracking measurements and the space - borne lisa detector for gravitational - wave frequencies larger than a few mhz . real gravitational - wave detectors do not stay at rest with respect to the tt coordinate system related to the passing gravitational wave , because they also move in the gravitational field of the solar system bodies , as in the case of the lisa spacecraft , or are fixed to the surface of the earth , as in the case of earth - based laser interferometers or resonant bar detectors . let us choose the origin o of the tt coordinate system employed in section 2.1 to coincide with the solar system barycenter ( ssb ) . the motion of the detector with respect to the ssb will modulate the gravitational - wave signal registered by the detector . one can show that as far as the velocities of the particles ( modeling the detector s parts ) with respect to the ssb are non - relativistic , which is the case for all existing or planned detectors , eqs . ( 12 ) can still be used , provided the 3-vectors xa and na ( a = 1 , 2 , 3 ) will be interpreted as made of the time - dependent components describing the motion of the particles with respect to the ssb . it is often convenient to introduce the proper reference frame of the detector with coordinates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({{\hat x}^a})$\end{document}. because the motion of this frame with respect to the ssb is non - relativistic , we can assume that the transformation between the ssb - related coordinates ( x ) and the detector s proper reference frame coordinates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({{\hat x}^a})$\end{document } has the form 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat t = t,\quad\quad{\hat x^i}(t,{x^k } ) = \hat x_{\hat o}^i(t ) + o_j^i(t){x^j},$$\end{document } where the functions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat x}^i}_{\hat o}(t)$\end{document } describe the motion of the origin of the proper reference frame with respect to the ssb , and the functions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{o}}_j^i(t)$\end{document } account for the different orientations of the spatial axes of the two reference frames . ( 12 ) in the detector s coordinates rather than in the tt coordinates . for instance , the matrix of the wave - induced spatial metric perturbation in the detector s coordinates is related to the matrix h of the spatial metric perturbation produced by the wave in the tt coordinate system through equation 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{\hat h}}(t ) = { ( { \rm{o}}{(t)^{- 1}})^{\rm{t } } } \cdot { \rm{h}}(t ) \cdot { \rm{o}}{(t)^{- 1}},$$\end{document } where the matrix o has elements \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{o}}_j^i$\end{document}. if the transformation matrix o is orthogonal , then o = ot , and eq . ( 19 ) simplifies to 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{\hat h}}(t ) = { \rm{o}}(t ) \cdot { \rm{h}}(t ) \cdot { \rm{o}}{(t)^{\rm{t}}}.$$\end{document } see [ 31 , 54 , 68 , 78 ] for more details . for a ground - based laser - interferometric detector , the long - wavelength approximation can be employed ( however , see [ 27 , 115 , 114 ] for a discussion of importance of high - frequency corrections , which modify the interferometer response function computed within the long - wavelength approximation ) . in the case of an interferometer in a standard michelson and equal - arm configuration ( such configurations can be represented by figure 1 with the particle 1 corresponding to the corner station of the interferometer and with l2 = l3 = l ) , the observed relative frequency shift (t)/0 is equal to the difference of the round - trip frequency shifts in the two detector s arms : 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu ( t ) } \over { { \nu _ 0 } } } = { y_{131}}(t ) - { y_{121}}(t).$$\end{document } let ( xd , yd , zd ) be the components ( with respect to the tt coordinate system ) of the 3-vector rd connecting the origin of the tt coordinate system with the corner station of the interferometer . then x1 = ( xd , yd , zd ) , k ( 17 ) , the relative frequency shift ( 21 ) can be written as 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu ( t ) } \over { { \nu _ 0 } } } = { l \over c}\left({{\rm{n}}_2^t \cdot { \rm{\dot h}}\left({t - { { { z_d } } \over c } } \right ) \cdot { { \rm{n}}_2 } - { \rm{n}}_3^{\rm{t } } \cdot { \rm{\dot h}}\left({t - { { { z_d } } \over c } } \right ) \cdot { { \rm{n}}_3 } } \right).$$\end{document } the difference (t ) of the phase fluctuations measured , say , by a photo detector , is related to the corresponding relative frequency fluctuations (t ) by 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu ( t ) } \over { { \nu _ 0 } } } = { 1 \over { 2\pi { \nu _ 0}}}{{{\rm{d}}\delta \phi ( t ) } \over { { \rm{d}}t}}.$$\end{document } one can integrate eq . ( 22 ) to write the phase change (t ) as 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \phi ( t ) = 4\pi { \nu _ 0}lh(t),$$\end{document } where the dimensionless function h , 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t):={1 \over 2}\left({{\rm{n}}_2^{\rm{t } } \cdot { \rm{h}}\left({t - { { { z_{\rm{d } } } } \over c } } \right ) \cdot { { \rm{n}}_2 } - { \rm{n}}_3^{\rm{t } } \cdot { \rm{h}}\left({t - { { { z_{\rm{d } } } } \over c } } \right ) \cdot { { \rm{n}}_3 } } \right),$$\end{document } is the response function of the interferometric detector to a plane gravitational wave in the long - wavelength approximation . ( 22)(23 ) one should assume that the quantities n2 , n3 , and zd [ entering eq . ( 22 ) ] do not depend on time t. but the formulae ( 24)(25 ) can also be used in the case when those quantities are time dependent , provided the velocities a of the detector s parts with respect to the ssb are non - relativistic . the error we make in such cases is on the order of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o(hv)}$\end{document } , where v is a typical value of the velocities a . thus , the response function of the earth - based interferometric detector equals 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t ) = { 1 \over 2}\left({{{\rm{n}}_2}{{(t)}^{\rm{t}}}\cdot{\rm{h}}\left({t - { { { z_{\rm{d}}}(t ) } \over c } } \right)\cdot{{\rm{n}}_2}(t ) - { { \rm{n}}_3}{{(t)}^{\rm{t}}}\cdot{\rm{h}}\left({t - { { { z_{\rm{d}}}(t ) } \over c } } \right)\cdot{{\rm{n}}_3}(t ) } \right),$$\end{document } where all quantities here are computed in the ssb - related tt coordinate system . the same response function can be written in terms of a detector s proper - reference - frame quantities as follows 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t ) = { 1 \over 2}\left({{\rm{\hat n}}_2^{\rm{t}}\cdot{\rm{\hat h}}\left({t - { { { z_{\rm{d}}}(t ) } \over c } } \right)\cdot{{{\rm{\hat n}}}_2 } - { \rm{\hat n}}_3^{\rm{t}}\cdot{\rm{\hat h}}\left({t - { { { z_{\rm{d}}}(t ) } \over c } } \right)\cdot{{{\rm{\hat n}}}_3 } } \right),$$\end{document } where the matrices and h are related to each other by means of formula ( 20 ) . in eq . ( 27 ) the proper - reference - frame components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{{\rm{\hat n}}}_2}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{{\rm{\hat n}}}_3}$\end{document } of the unit vectors directed along the interferometer arms can be treated as constant ( i.e. , time independent ) quantities . from eqs . ( 26 ) and ( 3 ) it follows that the response function h is a linear combination of the two wave polarizations h+ and h , so it can be written as 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t ) = { f _ + } ( t){h _ + } \left({t - { { { z_{\rm{d}}}(t ) } \over c } } \right ) + { f _ \times}(t){h _ \times}\left({t - { { { z_{\rm{d}}}(t ) } \over c } } \right).$$\end{document } the functions f+ and f are the interferometric beam - pattern functions . they depend on the location of the detector on earth , the position of the gravitational - wave source in the sky , and the polarization angle of the wave ( this angle describes the orientation , with respect to the detector , of the axes relative to which the plus and cross polarizations of the wave are defined , see , e.g. , figure 9.2 in ) . derivation of the explicit formulae for the interferometric beam patterns f+ and f can be found , e.g. , in appendix c of . in the long - wavelength approximation , the response function of the interferometric detector can be derived directly from the equation of geodesic deviation . then the response is defined as the relative difference between the wave - induced changes of the proper lengths of the two arms , i.e. , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h(t ) : = ( \delta { { \hat l}_2}(t ) - \delta { { \hat l}_3}(t))/{{\hat l}_0}$\end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat l}_0 } + \delta { { \hat l}_2}(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat l}_0 } + \delta { { \hat l}_3}(t)$\end{document } are the instantaneous values of the proper lengths of the interferometer s arms and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat l}_0}$\end{document } is the unperturbed proper length of these arms . in the case of an earth - based resonant - bar detector the long - wavelength approximation is very accurate and the dimensionless detector s response function can be defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h(t ) : = \delta \hat l(t)/{{\hat l}_0}$\end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \hat l(t)$\end{document } is the wave - induced change of the proper length \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat l}_0}$\end{document } of the bar . the response function h can be computed in terms of the detector s proper - reference - frame quantities from the formula ( see , e.g. , section 9.5.2 in ) 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t ) = { { \rm{\hat n}}^t } \cdot { \rm{\hat h}}\left({t - { { { z_{\rm{d}}}(t ) } \over c } } \right ) \cdot { \rm{\hat n}},$$\end{document } where the column matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{\hat n}}}$\end{document } consists of the components ( computed in the proper reference frame of the detector ) of the unit vector n directed along the symmetry axis of the bar . the response function ( 29 ) can be written as a linear combination of the wave polarizations h+ and h , i.e. , the formula ( 28 ) is also valid for the resonant - bar response function but with some bar - pattern functions f+ and f different from the interferometric beam - pattern functions . derivation of the explicit form of the bar patterns can be found , e.g. , in appendix c of . in many cases of interest the response function of the detector to a plane gravitational wave can be written as a linear combination of n waveforms hk(t ; ) which all depend on the same set of parameters = ( 1 , , m ) ] with constant amplitudes ak(k = 1 , , n ) , 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t;\theta ) = \sum\limits_{k = 1}^n { { a_k}{h_k}(t;\xi)},$$\end{document } where the vector collects all the signal s parameters . it is convenient to introduce column matrices 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm a } : = \left({\begin{array}{*{20}c } { { a_1 } } \\ \vdots \\ { { a_n } } \\ \end{array } } \right),\ , { \rm h}(t;\xi ) : = \left({\begin{array}{*{20}c } { { h_1}(t;\xi ) } \\ \vdots \\ { { h_n}(t;\xi ) } \\ \end{array } } \right).$$\end{document } then one can briefly write = ( a , ) and the response ( 30 ) can be written as 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t;\theta ) = { { \rm{a}}^{\rm{t } } } \cdot { \rm{h}}(t;\xi).$$\end{document } the functions hk ( k = 1 , , n ) are independent of the parameters a. the parameters a are called extrinsic ( or amplitude ) parameters whereas the parameters are called intrinsic . ( 32 ) with n = 4 is a model of the response of the space - based detector lisa to gravitational waves from a binary system . also for n = 4 the same equation models the response of a ground - based detector to a continuous source of gravitational waves like a rotating neutron star . for ground - based detectors the long - wavelength approximation can be applied and within this approximation the functions hk ( k = 1 , , 4 ) are given by 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { h_1}(t;\xi ) = u(t;\xi)\cos \phi ( t;\xi ) , } \\ { { h_2}(t;\xi ) = v(t;\xi)\cos \phi ( t;\xi ) , } \\ { { h_3}(t;\xi ) = u(t;\xi)\sin \phi ( t;\xi ) , } \\ { { h_4}(t;\xi ) = v(t;\xi)\sin \phi ( t;\xi ) , } \\ \end{array}$$\end{document } where (t ; ) is the phase modulation of the signal and u(t ; ) and v(t ; ) are slowly varying amplitude modulations . the gravitational - wave signal from spinning neutron stars may consist of several components of the form ( 32 ) . for short observation times over which the amplitude modulation functions are nearly constant , the response of the ground - based detector can further be approximated by 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_j}(\delta ) = { c_{0j}}{p_j}(r ) + { c_{1j}}{p_j}({r{\prime}}),\,j = 0,1,$$\end{document } where a0 and 0 are constant amplitude and initial phase , respectively , and g(t ; ) is a slowly varying function of time . ( 34 ) is a good model for the response of a detector to the gravitational wave from a coalescing compact binary system [ 135 , 30 ] . we would like to stress that not all deterministic gravitational - wave signals may be cast into the general form ( 32 ) . the gravitational - wave signal will be buried in the noise of the detector and the data from the detector will be a random ( or stochastic ) process . consequently , the problem of extracting the signal from the noise is a statistical one . the basic idea behind signal detection is that the presence of the signal changes the statistical characteristics of the data x , in particular its probability distribution . when the signal is absent the data have probability density function ( pdf ) p0(x ) , and when the signal is present the pdf is p1(x ) . a thorough introduction to probability theory and mathematical statistics can be found , e.g. , in [ 51 , 130 , 131 , 101 ] . a full exposition of statistical theory of signal detection that is only outlined here can be found in the monographs [ 147 , 74 , 143 , 139 , 87 , 58 , 107 ] . a general introduction to stochastic processes is given in and advanced treatment of the subject can be found in [ 84 , 146 ] . a concise introduction to the statistical theory of signal detection and time series analysis the problem of detecting the signal in noise can be posed as a statistical hypothesis testing problem . the null hypothesis h0 is that the signal is absent from the data and the alternative hypothesis h1 is that the signal is present . a hypothesis test ( or decision rule ) is a partition of the observation set into two subsets , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } and its complement \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r\prime}$\end{document}. if data are in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } we accept the null hypothesis , otherwise we reject it . a type i error is choosing hypothesis h1 when h0 is true and a type ii error is choosing h0 when h1 is true . in signal detection theory the probability of a type i error is called the false alarm probability , whereas the probability of a type ii error is called the false dismissal probability . 1 ( false dismissal probability ) is the probability of detection of the signal . in hypothesis testing theory , the probability of a type i error is called the significance of the test , whereas 1 ( probability of type ii error ) is called the power of the test . the subject is covered in detail in many books on statistics , for example , see [ 72 , 51 , 80 , 83 ] . in the bayesian approach we assign costs to our decisions ; in particular we introduce positive numbers cij , i , j = 0 , 1 , where cij is the cost incurred by choosing hypothesis hi when hypothesis hj is true . we define the conditional risk r of a decision rule for each hypothesis as 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_j}(\delta ) = { c_{0j}}{p_j}(\mathcal{r } ) + { c_{1j}}{p_j}({\mathcal{r}{\prime } } ) , \quad\quad j = 0,1,$$\end{document } where pj is the probability distribution of the data when hypothesis hj is true . next , we assign probabilities 0 and 1 = 1 0 to the occurrences of hypotheses h0 and h1 , respectively . we define the bayes risk as the overall average cost incurred by the decision rule : 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r(\delta ) = { \pi _ 0}{r_0}(\delta ) + { \pi _ 1}{r_1}(\delta).$$\end{document } finally we define the bayes rule as the rule that minimizes the bayes risk r( ) . very often in practice we do not have control over or access to the mechanism generating the state of nature and we are not able to assign priors to various hypotheses . in such a case one criterion is to seek a decision rule that minimizes , over all , the maximum of the conditional risks , r0( ) and r1( ) . a decision rule that fulfills that criterion the imposition of a specific cost structure on the decisions made is not possible or desirable . the neyman - pearson approach involves a trade - off between the two types of errors that one can make in choosing a particular hypothesis . the neyman - pearson design criterion is to maximize the power of the test ( probability of detection ) subject to a chosen significance of the test ( false alarm probability ) . it is remarkable that all three very different approaches bayesian , minimax , and neyman - pearson lead to the same test called the likelihood ratio test . the likelihood ratio is the ratio of the pdf when the signal is present to the pdf when it is absent : 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda ( x ) : = { { { p_1}(x ) } \over { { p_0}(x)}}.$$\end{document } we accept the hypothesis h1 if > k , where k is the threshold that is calculated from the costs cij , priors i , or the significance of the test depending on which approach is being used . let h be the gravitational - wave signal we are looking for and let n be the detector s noise . for convenience we assume that the signal h is a continuous function of time t and that the noise n is a continuous random process . results for the discrete - in - time data that we have in practice can then be obtained by a suitable sampling of the continuous - in - time expressions . assuming that the noise is additive the dat x can be written as 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x(t ) = n(t ) + h(t).$$\end{document } the autocorrelation function of the noise n is defined as 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${k_n}(t , t{\prime } ) : = { \rm e}[n(t)n({t{\prime}})],$$\end{document } where e denotes the expectation value . let us further assume that the detector s noise n is a zero - mean and gaussian random process . it can then be shown that the logarithm of the likelihood function is given by the following cameron - martin formula 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\log \lambda [ x ] = \int\nolimits_0^{{t_o } } { q(t)x(t){\rm{d}}t - { 1 \over 2}\int\nolimits_0^{{t_o } } { q(t)h(t){\rm{d}}t,}}$$\end{document } where 0 ; to is the time interval over which the data was collected and the function q is the solution of the integral equation 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t ) = \int\nolimits_0^{{t_o } } { { k_n}(t,{t\prime})q({t\prime}){\rm{d}}t.}$$\end{document } for stationary noise , its autocorrelation function ( 39 ) depends on times t and t only through the difference t t. it implies that there exists then an even function n of one variable such that 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm e}[n(t)n({t{\prime } } ) ] = { \kappa _ n}(t - { t{\prime}}).$$\end{document } spectral properties of stationary noise are described by its one - sided spectral density , defined for non - negative frequencies f 0 through relation 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_n}(f ) = 2\int\nolimits_{- \infty}^\infty { { \kappa _ n}(t){e^{2\pi i ft}}{\rm{d}}t.}$$\end{document } for negative frequencies f < 0 , by definition , sn(f ) = 0 . the spectral density sn can also be determined by correlations between the fourier components of the detector s noise 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{e}}[\tilde n(f){\tilde n^{\ast}}({f{\prime } } ) ] = { 1 \over 2}{s_n}(\vert f\vert)\delta ( f - { f{\prime}}),\ , - \infty < f,{f{\prime } } < \infty.$$\end{document } for the case of stationary noise with one - sided spectral density sn , it is convenient to define the scalar product ( xy ) between any two waveforms x and y , 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(x\vert y ) : = 4\re \int\nolimits_0^\infty { { { \tilde x(f){{\tilde y}^ { \ast}}(f ) } \over { { s_n}(f)}}{\rm{d}}f,}$$\end{document } where denotes the real part of a complex expression , the tilde denotes the fourier transform , and the asterisk is complex conjugation . making use of this scalar product , the log likelihood function ( 40 ) can be written as 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\log \lambda [ x ] = ( x\vert h ) - { 1 \over 2}(h\vert h).$$\end{document } from the expression ( 46 ) we see immediately that the likelihood ratio test consists of correlating the data x with the signal h that is present in the noise and comparing the correlation to a threshold . an important quantity is the optimal signal - to - noise ratio defined by 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho : = \sqrt { ( h\vert h).}$$\end{document } by means of eq . ( 45 ) it can be written as 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rho ^2 } = 4\int\nolimits_0^\infty { { { \vert \tilde h(f){\vert ^2 } } \over { { s_n}(f)}}{\rm{d}}f.}$$\end{document } we see in the following that determines the probability of detection of the signal . the higher the signal - to - noise ratio the higher the probability of detection . an interesting property of the matched filter is that it maximizes the signal - to - noise ratio over all linear filters . very often we know the waveform of the signal that we are searching for in the data in terms of a finite number of unknown parameters . an estimator of a parameter is a function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \theta ( x)$\end{document } that assigns to each data set the best guess of the true value of . note that because \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \theta ( x)$\end{document } depends on the random data it is a random variable . ideally we would like our estimator to be ( i ) unbiased , i.e. , its expectation value to be equal to the true value of the parameter , and ( ii ) of minimum variance . such estimators are rare and in general difficult to find . we assign a cost function c( , ) of estimating the true value of as . we then associate with an estimator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \theta}$\end{document } a conditional risk or cost averaged over all realizations of data x for each value of the parameter : 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_\theta}(\hat \theta ) = { { \rm{e}}_\theta}[c(\hat \theta , \theta ) ] = \int\nolimits_x { c(\hat \theta ( x),\theta)p(x,\theta ) } { \rm{d}}x,$$\end{document } where x is the set of observations and p(x , ) is the joint probability distribution of data x and parameter . we further assume that there is a certain a priori probability distribution ( ) of the parameter . we then define the bayes estimator as the estimator that minimizes the average risk defined as 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r(\hat \theta ) = { \rm{e}}[{r_\theta}(\hat \theta ) ] = \int\nolimits_x { \int\nolimits_\theta { c(\hat \theta ( x),\theta ) } p(x,\theta ) } \pi ( \theta){\rm{d}}\theta { \rm{d}}x,$$\end{document } where e is the expectation value with respect to an a priori distribution , and is the set of observations of the parameter . it is not difficult to show that for a commonly used cost function 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c({\theta { \prime}},\theta ) = { ( { \theta { \prime}},\theta)^2},$$\end{document } the bayesian estimator is the conditional mean of the parameter given data x , i.e. , 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat \theta ( x ) = e[\theta \vert x ] = \int\nolimits_\theta { \theta p(\theta \vert x)d\theta,}$$\end{document } where p(x ) is the conditional probability density of parameter given the data x. suppose that in a given estimation problem we are not able to assign a particular cost function c( , ) . then a natural choice is a uniform cost function equal to 0 over a certain interval i of the parameter . from bayes theorem we have 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(\theta \vert x ) = { { p(x,\theta)\pi ( \theta ) } \over { p(x)}},$$\end{document } where p(x ) is the probability distribution of data x. then , from eq . ( 50 ) one can deduce that for each data point x the bayes estimate is any value of that maximizes the conditional probability p(x ) . the density p(x ) is also called the a posteriori probability density of parameter and the estimator that maximizes p(x ) is called the maximum a posteriori ( map ) estimator . it is denoted by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat \theta}_{{\rm{map}}}}$\end{document}. we find that the map estimators are solutions of the following equation 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\partial \log p(x,\theta ) } \over { \partial \theta } } = - { { \partial \log \pi ( \theta ) } \over { \partial \theta}},$$\end{document } which is called the map equation . often we do not know the a priori probability density of a given parameter and we simply assign to it a uniform probability . in such a case maximization of the a posteriori probability is equivalent to maximization of the probability density p(x , ) treated as a function of . we call the function l( , x ) p(x , ) the likelihood function and the value of the parameter that maximizes l( , x ) the maximum likelihood ( ml ) estimator . instead of the function l we can use the function ( , x ) = l( , x)/p(x ) ( assuming that p(x ) > 0 ) . then the ml estimators are obtained by solving the equation 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\partial \log \lambda ( \theta , x ) } \over { \partial \theta } } = 0,$$\end{document } which is called the ml equation . there is a useful lower bound on variances of the parameter estimators called the cramr - rao bound , which is expressed in terms of the fisher information matrix ( ) for the signal h(t ; ) , which depends on k parameters collected into the vector = ( 1 , , k ) , the components of the matrix ( ) are defined as 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma { ( \theta)_{ij } } : = { \rm{e}}\left [ { { { \partial \log \lambda [ x;\theta ] } \over { \partial { \theta _ i}}}{{\partial \log \lambda [ x;\theta ] } \over { \partial { \theta _ j } } } } \right ] = - { \rm{e}}\left [ { { { { \partial ^2}\log \lambda [ x;\theta ] } \over { \partial { \theta _ i}\partial { \theta _ j } } } } \right],\,i , j = 1 , \ldots , k.$$\end{document } the cramr - rao bound states that for unbiased estimators the covariance matrix c( ) of the estimators fulfills the inequality 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{c}}(\theta ) \geq\gamma { ( \theta)^{- 1}}.$$\end{document } ( the inequality a b for matrices means that the matrix a b is nonnegative definite . ) a very important property of the ml estimators is that asymptotically ( i.e. , for a signal - to - noise ratio tending to infinity ) they are ( i ) unbiased , and ( ii ) they have a gaussian distribution with covariance matrix equal to the inverse of the fisher information matrix . in the case of gaussian noise the components of the fisher matrix are given by 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma { ( \theta)_{ij } } = \left({\left . { { { \partial h(t;\theta ) } \over { \partial { \theta _ i } } } } \right\vert { { \partial h(t;\theta ) } \over { \partial { \theta _ j } } } } \right),\,i , j = 1 , \ldots , k,$$\end{document } where the scalar product ( ) is defined in eq . ( 45 ) . in this section , we study the detection of a deterministic gravitational - wave signal h(t ; ) of the general form given by eq . ( 32 ) and the estimation of its parameters using the maximum - likelihood ( ml ) principle . we assume that the noise n(t ) in the detector is a zero - mean , gaussian , and stationary random process . the data x in the detector , in the case when the gravitational - wave signal h(t ; ) is present , is x(t ; ) = n(t ) + h(t ; ) . the parameters = ( a , ) of the signal ( 32 ) split into extrinsic ( or amplitude ) parameters a and intrinsic ones . for the gravitational - wave signal h(t ; a , ) of the form given in eq . ( 32 ) the log likelihood function ( 46 ) can be written as 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\log \lambda [ x;{\rm{a}},\xi ] = { { \rm{a}}^{\rm{t } } } \cdot { \rm{n}}[x;\xi ] - { 1 \over 2}{{\rm{a}}^{\rm{t } } } \cdot { \rm{m(}}\xi ) \cdot { \rm{a,}}$$\end{document } where the components of the column n 1 matrix n and the square n n matrix m are given by 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n_k}[x;\xi ] : = ( x\vert { h_k}(t;\xi)),\,{m_{kl}}(\xi ) : = ( { h_k}(t;\xi)\vert { h_l}(t;\xi)),\,k , l = 1 , \ldots , n.$$\end{document } the ml equations for the extrinsic parameters a , log[x ; a , ]/a = 0 , can be solved explicitly to show that the ml estimators of the parameters a are given by 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{\hat a}}[x;\xi ] = { \rm{m}}{(\xi)^{- 1 } } \cdot { \rm{n}}[x;\xi ] .$$\end{document } replacing the extrinsic parameters a in eq . ( 59 ) by their ml estimators , we obtain the reduced log likelihood function , 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal f}[x;\xi ] : = \log \lambda [ x;{\rm{\hat a}}[x;\xi ] , \xi ] = { 1 \over 2}{\rm n}{[x;\xi ] ^{\rm{t } } } \cdot { \rm{m}}{(\xi)^{- 1 } } \cdot { \rm{n}}[x;\xi ] , $ $ \end{document } that we call the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic depends nonlinearly on the intrinsic parameters of the signal , it does not depend on the extrinsic parameters a. the procedure to detect the gravitational - wave signal of the form ( 32 ) and estimate its parameters consists of two parts . the first part is to find the ( local ) maxima of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic ( 62 ) in the intrinsic parameters space . the ml estimators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \xi}$\end{document } of the intrinsic parameters are those values of for which the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic attains a maximum . the second part is to calculate the estimators of the extrinsic parameters a from the analytic formula ( 61 ) , where the matrix m and the correlations n are calculated for the parameters replaced by their ml estimators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \xi}$\end{document } obtained from the first part of the analysis . see section 4.6 for a discussion of the algorithms to find the ( local ) maxima of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic can also be used in the case when the intrinsic parameters are known . an example of such an analysis called a targeted search is the search for a gravitational - wave signal from a known pulsar . in this case assuming that gravitational - wave emission follows the radio timing , the phase of the signal is known from pulsar observations and the only unknown parameters of the signal are the amplitude ( or extrinsic ) parameters a [ see eq . ( 30 ) ] . to detect the signal one calculates the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic for the known values of the intrinsic parameters and compares it to a threshold . when a statistically - significant signal is detected , one then estimates the amplitude parameters from the analytic formulae ( 61 ) . in it was shown that the maximum - likelihood \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic can be interpreted as a bayes factor with a simple , but unphysical , amplitude prior ( and an additional unphysical sky - position weighting ) . using a more physical prior based on an isotropic probability distribution for the unknown spin - axis orientation of emitting systems , a new detection statistic ( called the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } monte carlo simulations for signals with random ( isotropic ) spin - axis orientations show that the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal b}$\end{document}-statistic is more powerful ( in terms of its expected detection probability ) than the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic . a modified version of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic that can be more powerful than the original one has been studied in . the detectability of the signal h(t ; ) is determined by the signal - to - noise ratio . in general it depends on all the signal s parameters and can be computed from [ see eq . ( 47 ) ] 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho ( \theta ) = \sqrt { ( h(t;\theta)\vert h(t;\theta)}).$$\end{document } the signal - to - noise ratio for the signal ( 32 ) can be written as 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho ( { \rm{a}},\xi ) = \sqrt { { { \rm{a}}^{\rm{t } } } \cdot { \rm{m}}(\xi ) \cdot { \rm{a}}},$$\end{document } where the components of the matrix m( ) are defined in eq . the accuracy of estimation of the signal s parameters is determined by fisher information matrix . the components of in the case of the gaussian noise can be computed from eq . ( 58 ) . for the signal given in eq . ( 32 ) the signal s parameters ( collected into the vector ) split into extrinsic and intrinsic parameters : = ( a , ) , where a = ( a1 , , an ) and = ( 1 , , m ) . therefore , we use calligraphic lettering to denote the intrinsic parameter indices : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\xi _ { \mathcal a}},{\mathcal a } = 1 , \ldots , m$\end{document}. the matrix has dimension ( n + m ) ( n + m ) and it can be written in terms of four block matrices for the two sets of the parameters a and , 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma ( { \rm a},\xi ) = \left({\begin{array}{*{20}c } { { \gamma _ { { \rm{aa}}}}(\xi ) } & { { \gamma _ { { \rm a}\xi}}({\rm a},\xi ) } \\ { { \gamma _ { { \rm a}\xi}}{{({\rm a},\xi)}^{\rm{t } } } } & { { \gamma _ { \xi \xi}}({\rm a},\xi ) } \\ \end{array } } \right),$$\end{document } where aa is an n n matrix with components ( h/aih/aj ) ( i , j = 1 , , n ) , a is an n m matrix with components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\partial h/\partial { a_i}\backslash \partial h/\partial { \xi _ \mathcal a})(i = 1 , \ldots , n,\mathcal a = 1 , \ldots , m)$\end{document } , and finally is m m matrix with components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\partial h/\partial { \xi _ \mathcal a}\backslash \partial h/\partial { \xi _ \mathcal b})(\mathcal a,\mathcal b = 1 , \ldots , m)$\end{document}. we introduce two families of the auxiliary n n square matrices \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{f}}_{(\mathcal a)}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{s}}_{({\mathcal a}{\mathcal b})}}({\mathcal a},{\mathcal b } = 1 , \ldots , m)$\end{document } , which depend on the intrinsic parameters only ( the indexes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal a},{\mathcal b}$\end{document } within parentheses mean that they serve here as the matrix labels ) . the components of the matrices \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{f}}_{(\mathcal a)}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{s}}_{({\mathcal a}{\mathcal b})}}$\end{document } are defined as follows : 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f_{{{(\mathcal{a})}^{ij}}}}(\xi ) : = \left({{h_i}(t;\xi)\left\vert { { { \partial { h_j}(t;\xi ) } \over { \partial { \xi _ \mathcal{a } } } } } \right . } \right),\,i , j = 1 , \ldots , n , \,\mathcal{a } = 1 , \ldots , m.$$\end{document } 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{{({\mathcal a}{\mathcal b})}^{ij}}}}(\xi ) : = \left({{{\partial { h_i}(t;\xi ) } \over { \partial { \xi _ { \mathcal a}}}}\left\vert { { { \partial { h_j}(t;\xi ) } \over { \partial { \xi _ { \mathcal b } } } } } \right . } \right),\,i , j = 1 , \ldots , n,\,{\mathcal a},{\mathcal b } = 1 , \ldots , m.$$\end{document } making use of the definitions ( 60 ) and ( 66)(67 ) one can write the more explicit form of the matrices aa , a , and , 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { aa}}(\xi ) = { \rm{m}}(\xi),$$\end{document } 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { a\xi}}({\rm{a}},\xi ) = ( { { \rm{f}}_{(1)}}(\xi ) \cdot { \rm{a } } \cdots { { \rm{f}}_{(m)}}(\xi ) \cdot { \rm{a}}),$$\end{document } 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { \xi \xi}}({\rm{a}},\xi ) = \left({\begin{array}{*{20}c } { { { \rm{a}}^{\rm{t } } } \cdot { { \rm{s}}_{(11)}}(\xi ) \cdot { \rm{a } } \cdots { { \rm{a}}^{\rm{t } } } \cdot { { \rm{s}}_{(1m)}}(\xi ) \cdot { \rm{a } } } \\ { \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots } \\ { { { \rm{a}}^{\rm{t } } } \cdot { { \rm{s}}_{(m1)}}(\xi ) \cdot { \rm{a } } \cdots { { \rm{a}}^{\rm{t } } } \cdot { { \rm{s}}_{(mm)}}(\xi ) \cdot { \rm{a } } } \\ \end{array } } \right).$$\end{document } the notation introduced above means that the matrix a can be thought of as a 1 m row matrix made of n 1 column matrices \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{f}}_{(\mathcal a)}}$\end{document}. thus , the general formula for the component of the matrix a is 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${({\gamma _ { { \rm{a}}\xi}})_{i\mathcal{a } } } = ( { { \rm{f}}_{(\mathcal{a ) } } \cdot { \rm{a}})_i } = \sum\limits_{j = 1}^n { { f_{(\mathcal{a})ij}}{a_j } , \quad\quad \mathcal{a } = 1 , \ldots , m , \quad i = 1 , \ldots , n.}$$\end{document } the general component of the matrix is given by 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${({\gamma _ { \xi \xi}})_{{\mathcal a}{\mathcal b } } } = { { \rm a}^{\rm t } } \cdot { s_{(\mathcal{a}\mathcal{b } ) } } \cdot { \rm a } = \sum\limits_{i = 1}^n { \sum\limits_{j = 1}^n { { s_{({\mathcal a}{\mathcal b})ij}}{a_i}{a_j},\,{\mathcal a},\mathcal{b } = 1 , \ldots , m.}}$$\end{document } the covariance matrix c , which approximates the expected covariances of the ml estimators of the parameters , is defined as applying the standard formula for the inverse of a block matrix to eq . ( 65 ) , one gets 73\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm c}({\rm{a}},\xi ) = \left({\begin{array}{*{20}c } { { { \rm c}_{{\rm{aa}}}}({\rm{a}},\xi ) } & { { { \rm c}_{{\rm{a}}\xi}}({\rm{a}},\xi ) } \\ { { { \rm c}_{{\rm{a}}\xi}}{{({\rm{a}},\xi)}^t } } & { { { \rm c}_{\xi \xi}}({\rm{a}},\xi ) } \\ \end{array } } \right),$$\end{document } where the matrices caa , ca , and c can be expressed in terms of the matrices aa = m , a , and as follows : 74\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{c}}_{{\rm{aa}}}}({\rm{a}},\xi ) = { \rm{m(}}\xi)^{- 1 } + { \rm{m(}}\xi)^{- 1 } \cdot { \gamma _ { { \rm{a}}\xi}}({\rm{a,}}\xi ) \cdot \bar \gamma { ( { \rm{a,}}\xi)^{- 1 } } \cdot { \gamma _ { { \rm{a}}\xi}}{({\rm{a,}}\xi)^{\rm{t } } } \cdot { \rm{m(}}\xi)^{- 1},$$\end{document } 75\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{c}}_{{\rm{a}}\xi}}({\rm{a}},\xi ) = - { \rm{m}}{(\xi)^{- 1}}\cdot{\gamma _ { { \rm{a}}\xi}}({\rm{a}},\xi)\cdot\bar \gamma { ( { \rm{a}},\xi)^{- 1}},$$\end{document } 76\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{c}}_{\xi \xi}}({\rm{a}},\xi ) = \bar \gamma { ( { \rm{a}},\xi)^{- 1}}.$$\end{document } in eqs . ( 74)(76 ) we have introduced the m m matrix : 77\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\bar \gamma ( { \rm{a,}}\xi ) : = { \gamma _ { \xi \xi}}({\rm{a,}}\xi ) - { \gamma _ { { \rm{a}}\xi}}{({\rm{a}},\xi)^{\rm{t } } } \cdot { \rm{m(}}\xi)^{- 1 } \cdot { \gamma _ { { \rm{a}}\xi}}({\rm{a,}}\xi).$$\end{document } we call the matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document } ( which is the schur complement of the matrix m ) the projected fisher matrix ( onto the space of intrinsic parameters ) . because the matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document } is the inverse of the intrinsic - parameter submatrix c of the covariance matrix c , it expresses the information available about the intrinsic parameters that takes into account the correlations with the extrinsic parameters . the matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document } is still a function of the putative extrinsic parameters . we next define the normalized projected fisher matrix ( which is the m m square matrix ) 78\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bar \gamma _ n}({\rm{a}},\xi ) : = { { \bar \gamma ( { \rm{a,}}\xi ) } \over { \rho { { ( { \rm{a,}}\xi)}^2}}},$$\end{document } where is the signal - to - noise ratio . making use of the definition ( 77 ) and eqs . ( 71)(72 ) we can show that the components of this matrix can be written in the form 79\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$({\bar \gamma _ n}{({\rm{a}},\xi)_{\mathcal{a}\mathcal{b } } } ) = { { { { \rm{a}}^{\rm{t } } } \cdot { { \rm{a}}_{(\mathcal{a}\mathcal{b})}}(\xi ) \cdot { \rm{a } } } \over { { { \rm{a}}^{\rm{t } } } \cdot { \rm{m(}}\xi ) \cdot { \rm{a}}}},\mathcal{a},\mathcal{b } = 1 , \ldots , m,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{a}}_{({\mathcal a}{\mathcal b})}}$\end{document } is the n n matrix defined as 80\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{a}}_{(\mathcal{a}\mathcal{b})}}(\xi ) : = { s_{(\mathcal{a}\mathcal{b})}}(\xi ) - { { \rm{f}}_{(\mathcal{a})}}{(\xi)^{\rm{t } } } \cdot { \rm{m(}}\xi)^{- 1 } \cdot { { \rm{f}}_{(b)}}(\xi),\,\mathcal{a},\mathcal{b } = 1 , \ldots , m.$$\end{document } from the rayleigh principle it follows that the minimum value of the component \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${({{\bar \gamma}_n}({\rm{a}},\xi))_{{\mathcal a}{\mathcal b}}}$\end{document } is given by the smallest eigenvalue of the matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{m}}^{- 1 } } \cdot { { \rm{a}}_{({\mathcal a}{\mathcal b})}}$\end{document}. similarly , the maximum value of the component \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${({{\bar \gamma}_n}({\rm{a}},\xi))_{{\mathcal a}{\mathcal b}}}$\end{document } is given by the largest eigenvalue of that matrix . because the trace of a matrix is equal to the sum of its eigenvalues , the m m square matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } with components 81\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${(\tilde \gamma ( \xi))_{\mathcal{a}\mathcal{b } } } : = { 1 \over n}{\rm{tr(m(}}\xi)^{- 1 } \cdot { { \rm{a}}_{(\mathcal{a}\mathcal{b})}}(\xi)),\,\mathcal{a},\mathcal{b } = 1 , \ldots , m.$$\end{document } expresses the information available about the intrinsic parameters , averaged over the possible values of the extrinsic parameters . we shall call \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } the reduced fisher matrix . we shall see that the reduced fisher matrix plays a key role in the signal processing theory that we present here . it is used in the calculation of the threshold for statistically significant detection and in the formula for the number of templates needed to do a given search . for the case of the signal 82\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t;{a_0},{\phi _ 0},\xi ) = { a_0}g(t;\xi)\cos ( \phi ( t;\xi ) - { \phi _ 0}),$$\end{document } the normalized projected fisher matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar \gamma}_n}$\end{document } is independent of the extrinsic parameters a0 and 0 , and it is equal to the reduced matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } . the components of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } are given by 83\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde \gamma _ { \mathcal{a}\mathcal{b } } } = { ( { \gamma _ 0})_{\mathcal{a}\mathcal{b } } } - { { { { ( { \gamma _ 0})}_{{\phi _ 0}\mathcal{a}}}{{({\gamma _ 0})}_{{\phi _ 0}\mathcal{b } } } } \over { { { ( { \gamma _ 0})}_{{\phi _ 0}{\phi _ 0}}}}},$$\end{document } where 0 is the fisher matrix for the signal g(t ; ) cos ( (t ; ) 0 ) . we first present the false alarm and detection probabilities when the intrinsic parameters of the signal are known . in this case the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic is a quadratic form of the random variables that are correlations of the data . as we assume that the noise in the data is gaussian and the correlations are linear functions of the data , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } is a quadratic form of the gaussian random variables . consequently the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic has a distribution related to the distribution . one can show ( see section iii b in ) that for the signal given by eq . ( 30 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$2{\mathcal f}$\end{document } has a distribution with n degrees of freedom when the signal is absent and noncentral distribution with n degrees of freedom and non - centrality parameter equal to the square of the signal - to - noise ratio when the signal is present . as a result the pdfs p0 and p1 of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic , when the intrinsic parameters are known and when respectively the signal is absent or present in the data , are given by 84\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_0}(\mathcal{f } ) = { { { \mathcal{f}^{n/2 - 1 } } } \over { ( n/2 - 1)!}}\exp ( - \mathcal{f}),$$\end{document } 85\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_1}(\rho,\mathcal{f } ) = { { { { ( 2\mathcal{f})}^{(n/2 - 1)/2 } } } \over { { \rho ^{n/2 - 1}}}}{i_{n/2 - 1}}\left({\rho \sqrt { 2\mathcal{f } } } \right)\exp \left({- \mathcal{f } - { 1 \over 2}{\rho ^2 } } \right),$$\end{document } where n is the number of degrees of freedom of distribution and in/21 is the modified bessel function of the first kind and order n/2 1 . the false alarm probability pf is the probability that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } exceeds a certain threshold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal f}_0}$\end{document } when there is no signal . in our case we have 86\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{\rm{f}}}({\mathcal{f}_0 } ) : = \int\nolimits_{{\mathcal{f}_0}}^\infty { { p_0}(\mathcal{f})d\mathcal{f } = \exp ( - { \mathcal{f}_0})\sum\limits_{k = 0}^{n/2 - 1 } { { { { \mathcal{f}_0}^k } \over { k!}}.}}$$\end{document } the probability of detection pd is the probability that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } exceeds the threshold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal f}_0}$\end{document } when a signal is present and the signal - to - noise ratio is equal to : 87\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{\rm{d}}}(\rho , { \mathcal{f}_0 } ) : = \int\nolimits_{{\mathcal{f}_0}}^\infty { p_1}(\rho , \mathcal{f}){\rm{d\mathcal{f}}}.$$\end{document } the integral in the above formula can be expressed in terms of the generalized marcum q - function [ 132 , 58 ] , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p_{\rm{d}}}(\rho , { { \mathcal f}_0 } ) = q(\rho , \sqrt { 2{{\mathcal f}_0}})$\end{document}. we see that when the noise in the detector is gaussian and the intrinsic parameters are known , the probability of detection of the signal depends on a single quantity : the optimal signal - to - noise ratio . next we return to the case in which the intrinsic parameters are not known . then the statistic \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}[x;\xi ] $ \end{document } given by eq . ( 62 ) is a certain multiparameter random process called the random field ( see monographs [ 5 , 6 ] for a comprehensive discussion of random fields ) . if the vector has one component the random field is simply a random process . for random fields we define the autocovariance function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } just in the same way as we define such a function for a random process : 88\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{c}(\xi , { \xi { \prime } } ) : = { { \rm{e}}_0}[\mathcal{f}[x;\xi ] \mathcal{f}[x;{\xi { \prime } } ] ] - { { \rm{e}}_0}[\mathcal{f}[x;\xi ] ] { { \rm{e}}_0}[\mathcal{f}[x;{\xi { \prime}}]],$$\end{document } where and are two values of the intrinsic parameter set , and e0 is the expectation value when the signal is absent . one can show that for the signal ( 30 ) the autocovariance function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } is given by 89\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal c}(\xi , { \xi \prime } ) = { 1 \over 2}{\rm{tr}}\left({{\rm{q(}}\xi , { \xi { \prime } } ) \cdot { \rm{m(}}{\xi { \prime}})^{- 1 } \cdot { \rm{q(}}\xi,{\xi { \prime}})^{\rm{t } } \cdot { \rm{m(}}\xi)^{- 1 } } \right),$$\end{document } where q is an n n matrix with components 90\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{q(}}\xi , { \xi { \prime}})_{ij } : = ( { h_i}(t;\xi)\vert { h_j}(t;{\xi { \prime}})),\,i , j = 1 , \ldots n.$$\end{document } obviously q( , ) = m( ) , therefore \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}(\xi , \xi ) = n/2$\end{document}. one can estimate the false alarm probability in the following way . the autocovariance function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } tends to zero as the displacement = increases ( it is maximal for = 0 ) . thus we can divide the parameter space into elementary cells such that in each cell the autocovariance function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } is appreciably different from zero . the realizations of the random field within a cell will be correlated ( dependent ) , whereas realizations of the random field within each cell and outside of the cell are almost uncorrelated ( independent ) . thus , the number of cells covering the parameter space gives an estimate of the number of independent realizations of the random field . we choose the elementary cell with its origin at the point to be a compact region with boundary defined by the requirement that the autocovariance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}(\xi , { \xi \prime})$\end{document } between the origin and any point at the cell s boundary equals half of its maximum value , i.e. , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}(\xi , \xi)/2$\end{document}. thus , the elementary cell is defined by the inequality 91\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{c}(\xi , { \xi { \prime } } ) \leq { 1 \over 2}\mathcal{c}(\xi , \xi ) = { n \over 4},$$\end{document } with at the cell s center and on the cell s boundary . to estimate the number of cells we perform the taylor expansion of the autocovariance function up to the second - order terms : 92\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{c}(\xi , \xi \prime ) \cong \frac{n}{2 } + \sum\limits_{\mathcal{a } = 1}^m { { { \left . { \frac{{\partial \mathcal{c}(\xi , \xi \prime ) } } { { \partial { \xi _ \mathcal{a}}\prime } } } \right|}_{\xi \prime = \xi } } } \delta { \xi _ \mathcal{a } } + \frac{1}{2}\sum\limits_{\mathcal{a},\mathcal{b } = 1}^m { { { \left . { \frac{{{\partial ^2}\mathcal{c}(\xi , \xi \prime ) } } { { \partial { { \xi ' } _ \mathcal{a}}\partial { { \xi ' } _ \mathcal{b } } } } } \right|}_{\xi \prime = \xi } } } \delta { \xi _ \mathcal{a}}\delta { \xi _ \mathcal{b}}.$$\end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } attains its maximum value when = 0 , we have 93\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left . { { { \partial { \mathcal c}(\xi , { \xi \prime } ) } \over { \partial \xi _ a\prime } } } \right|_{{\xi \prime } = \xi } } = 0,\quad{\mathcal a } = 1 , \ldots , m.$$\end{document } let us introduce the symmetric matrix g with components 94\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mathcal{a}\mathcal{b}}}(\xi { \frac{1}{{2\mathcal{c}(\xi , \xi ) } } \frac{{{\partial ^2}\mathcal{c}(\xi , \xi \prime ) } } { { \partial { { \xi ' } _ \mathcal{a}}\partial { { \xi ' } _ \mathcal{b } } } } } \right|_{\xi \prime = \xi } } , \quad \mathcal{a},\mathcal{b } = 1 , \ldots , m.$$\end{document } then , the inequality ( 91 ) for the elementary cell can approximately be written as 95\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sum\limits_{\mathcal{a},\mathcal{b } = 1}^m { { g_{\mathcal{a}\mathcal{b}}}(\xi)\delta { \xi _ \mathcal{a}}\delta { \xi _ \mathcal{b } } \leq{1 \over 2}.}$$\end{document } it is interesting to find a relation between the matrix g and the fisher matrix . one can show ( see , appendix b ) that the matrix g is precisely equal to the reduced fisher matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } given by eq . if the components of the matrix g are constant ( i.e. , they are independent of the values of the intrinsic parameters of the signal ) , the above equation defines a hyperellipsoid in m - dimensional ( m is the number of the intrinsic parameters ) euclidean space . the m - dimensional euclidean volume vcell of the elementary cell given by eq . ( 95 ) equals 96\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${v_{{\rm{cell } } } } = { { { { ( \pi/2)}^{m/2 } } } \over { \gamma ( m/2 + 1)\sqrt { \det \,{\rm{g}}}}},$$\end{document } where denotes the gamma function . we estimate the number ncells of elementary cells by dividing the total euclidean volume v of the m - dimensional intrinsic parameter space by the volume vcell of one elementary cell , i.e. , we have 97\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n_{{\rm{cells } } } } = { v \over { { v_{{\rm{cell}}}}}}.$$\end{document } the components of the matrix g are constant for the signal h(t ; a0 , 0 , ) = a0 cos ( (t ; ) 0 ) , provided the phase (t ; ) is a linear function of the intrinsic parameters . to estimate the number of cells in the case when the components of the matrix g are not constant , i.e. , when they depend on the values of the intrinsic parameters , one replaces eq . ( 97 ) by 98\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n_{{\rm{cells } } } } = { { \gamma ( m/2 + 1 ) } \over { { { ( \pi/2)}^{m/2}}}}\int\nolimits_v { \sqrt { \det { \rm{g(}}\xi ) } { \rm{d}}v.}$$\end{document } this formula can be thought of as interpreting the matrix g as the metric on the parameter space . this interpretation appeared for the first time in the context of gravitational - wave data analysis in the work by owen , where an analogous integral formula was proposed for the number of templates needed to perform a search for gravitational - wave signals from coalescing binaries . the concept of number of cells was introduced in and it is a generalization of the idea of an effective number of samples introduced in for the case of a coalescing binary signal . we approximate the pdf of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic in each cell by the pdf \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p_0}({\mathcal f})$\end{document } of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic when the parameters are known [ it is given by eq . the values of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic in each cell can be considered as independent random variables . the probability that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } does not exceed the threshold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal f}_0}$\end{document } in a given cell is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1 - { p_f}({{\mathcal f}_0})$\end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p_f}({{\mathcal f}_0})$\end{document } is given by eq . consequently the probability that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } does not exceed the threshold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal f}_0}$\end{document } in all the ncells cells is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${[1 - { p_f}({{\mathcal f}_0})]^{{n_{{\rm{cells}}}}}}$\end{document}. thus , the probability \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_f^t$\end{document } that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } exceeds \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal f}_0}$\end{document } in one or more cells is given by 99\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\rm{f}}^t({\mathcal{f}_0 } ) = 1 - { [ 1 - { p_{\rm{f}}}({\mathcal{f}_0})]^{{n_{{\rm{cells}}}}}}.$$\end{document } by definition , this is the false alarm probability when the phase parameters are unknown . the number of false alarms nf is given by 100\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n_{\rm{f } } } = { n_{{\rm{cells}}}}p_{\rm{f}}^t({\mathcal{f}_0}).$$\end{document } a different approach to the calculation of the number of false alarms using the euler characteristic of level crossings of a random field is described in . it was shown ( see ) that for any finite \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal f}_0}$\end{document } and ncells , eq . also in a tighter upper bound for the false alarm probability was derived by modifying a formula obtained by mohanty . the formula amounts essentially to introducing a suitable coefficient multiplying the number ncells of cells . when the signal is present in the data a precise calculation of the pdf of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic is very difficult because the presence of the signal makes the data s random process non - stationary . as a first approximation we can estimate the probability of detection of the signal when the intrinsic parameters are unknown by the probability of detection when these parameters are known [ it is given by eq . this approximation assumes that when the signal is present the true values of the intrinsic parameters fall within the cell where the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic has a maximum . this approximation will be the better the higher the signal - to - noise ratio is . to search for gravitational - wave signals we evaluate the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic on a grid in parameter space . the grid has to be sufficiently fine such that the loss of signals is minimized . in order to estimate the number of points of the grid , or in other words the number of templates that we need to search for a signal , the natural quantity to study is the expectation value of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic when the signal is present . thus , we assume that the data x contains the gravitational - wave signal h(t ; ) defined in eq . ( 32 ) , so x(t ; ) = h(t ; ) + n(t ) . the parameters = ( a , ) of the signal consist of extrinsic parameters a and intrinsic parameters . the data x will be correlated with the filters hi(t ; ) ( i = 1 , , n ) parameterized by the values of the intrinsic parameters . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic can thus be written in the form [ see eq . ( 62 ) ] 101\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{f}[x(t;{\rm{a,}}\xi { \rm{);}}{\xi { \prime } } ] = { 1 \over 2}{\rm{n}}{[x(t;{\rm{a}},\xi);{\xi { \prime}}]^{\rm{t } } } \cdot { \rm{m(}}{\xi { \prime}})^{- 1}\cdot{\rm{n}}[x(t;{\rm{a}},\xi);{\xi { \prime}}],$$\end{document } where the matrices m and n are defined in eqs . the expectation value of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic ( 101 ) is 102\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{e}}[\mathcal{f}[x(t;{\rm{a}},\xi);{\xi \prime } ] ] = { 1 \over 2}(n + { { \rm{a}}^{\rm{t } } } \cdot { \rm{q}}(\xi , { \xi { \prime } } ) \cdot { \rm{m}}{({\xi { \prime}})^{- 1 } } \cdot { \rm{q}}{(\xi , { \xi { \prime}})^{\rm{t } } } \cdot { \rm{a}}),$$\end{document } where the matrix q is defined in eq . let us rewrite the expectation value ( 102 ) in the following form , 103\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{e}}[{\mathcal f}[x(t;{\rm{a}},\xi);{\xi \prime } ] ] = { 1 \over 2}(n + \rho { ( { \rm{a}},\xi)^2}{\mathcal{c}_{\rm{n}}}({\rm{a}},\xi , { \xi { \prime}})),$$\end{document } where is the signal - to - noise ratio and where we have introduced the normalized correlation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal c}_{\rm{n}}}$\end{document } , 104\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal c}_{\rm{n}}}({\rm{a}},\xi , { \xi \prime } ) : = { { { { \rm{a}}^{\rm{t } } } \cdot { \rm{q}}(\xi , { \xi \prime})\cdot{\rm{m}}{{({\xi \prime})}^{- 1}}\cdot{\rm{q}}{{(\xi , { \xi \prime})}^{\rm{t}}}\cdot{\rm{a } } } \over { { { \rm{a}}^{\rm{t}}}\cdot{\rm{m}}(\xi)\cdot{\rm{a}}}}.$$\end{document } from the rayleigh principle it follows that the minimum value of the normalized correlation function is equal to the smallest eigenvalue of the matrix m( ) q( , ) m( ) q( , ) , whereas the maximum value is given by its largest eigenvalue . we define the reduced correlation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } as 105\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{c}(\xi , { \xi { \prime } } ) : = { 1 \over 2}{\rm{tr}}({\rm{m}}{(\xi)^{- 1 } } \cdot { \rm{q}}(\xi , { \xi { \prime } } ) \cdot { \rm{m}}{({\xi { \prime}})^{- 1 } } \cdot { \rm{q}}{(\xi , { \xi { \prime}})^{\rm{t}}}).$$\end{document } as the trace of a matrix equals the sum of its eigenvalues , the reduced correlation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } is equal to the average of the eigenvalues of the normalized correlation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal c}_{\rm{n}}}$\end{document}. in this sense we can think of the reduced correlation function as an the advantage of the reduced correlation function is that it depends only on the intrinsic parameters , and thus is suitable for studying the number of grid points on which the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic needs to be evaluated . we also note that the normalized correlation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } precisely coincides with the autocovariance function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic given by eq . ( 89 ) . as in the calculation of the number of cells in order to estimate the number of templates we perform a taylor expansion of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } up to second order terms around the true values of the parameters , and we obtain an equation analogous to eq . ( 95 ) , 106\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sum\limits_{\mathcal{a},\mathcal{b } = 1}^m { { g_{\mathcal{a}\mathcal{b}}}\delta { \xi _ \mathcal{a}}\delta { \xi _ \mathcal{b } } = 1 - { c_0},}$$\end{document } where g is given by eq . ( 94 ) . by arguments identical to those in deriving the formula for the number of cells we arrive at the following formula for the number of templates : 107\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n_t } = { 1 \over { { { ( 1 - { c_0})}^{m/2}}}}{{\gamma ( m/2 + 1 ) } \over { { \pi ^{m/2}}}}\int\nolimits_v { \sqrt { \det g(\xi ) } { \rm d}v.}$$\end{document } when c0 = 1/2 the above formula coincides with the formula for the number ncells of cells , eq . we would like to place the templates sufficiently closely so that the loss of signals is minimized . the formula ( 107 ) for the number of templates assumes that the templates are placed in the centers of hyperspheres and that the hyperspheres fill the parameter space without holes . in order to have a tiling of the parameter space without holes we can place the templates in the centers of hypercubes , which are inscribed in the hyperspheres . then the formula for the number of templates reads 108\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n_t } = { 1 \over { { { ( 1 - { c_0})}^{m/2}}}}{{{m^{m/2 } } } \over { { 2^m}}}\int\nolimits_v { \sqrt { \det { \rm{g}}(\xi ) } { \rm{d}}v.}$$\end{document } for the case of the signal given by eq . ( 34 ) our formula for the number of templates is equivalent to the original formula derived by owen . owen has also introduced a geometric approach to the problem of template placement involving the identification of the fisher matrix with a metric on the parameter space . an early study of the template placement for the case of coalescing binaries can be found in [ 121 , 45 , 26 ] . applications of the geometric approach of owen to the case of spinning neutron stars and supernova bursts are given in [ 33 , 16 ] . the problem of how to cover the parameter space with the smallest possible number of templates , such that no point in the parameter space lies further away from a grid point than a certain distance , is known in mathematical literature as the covering problem . this was first studied in the context of gravitational - wave data analysis by prix [ ] . the maximum distance of any point to the next grid point is called the covering radius r. an important class of coverings are lattice coverings . we define a lattice in m - dimensional euclidean space to be the set of points including 0 such that if u and v are lattice points , then also u + v and u v are lattice points . the basic building block of a lattice is called the fundamental region . a lattice covering is a covering of by spheres of covering radius r , where the centers of the spheres form a lattice . the most important quantity of a covering is its thickness defined as 109\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta : = { { { \rm{volume}}\,{\rm{of}}\,{\rm{one}}\,m-{\rm{dimensional}}\,{\rm{sphere } } } \over { { \rm{volume}}\,{\rm{of}}\,{\rm{the}}\,{\rm{fundamental}}\,{\rm{region}}}}.$$\end{document } in the case of a two - dimensional euclidean space the best covering is the hexagonal covering and its thickness 1.21 . for dimensions higher than 2 the best covering these are a*m lattices , which have thicknesses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\theta _ { a_m^{\ast}}}$\end{document } equal to 110\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\theta _ { a_m^{\ast } } } = { v_m}\sqrt { m + 1 } { \left({{{m(m + 2 ) } \over { 12(m + 1 ) } } } \right)^{m/2}},$$\end{document } where vm is the volume of the m - dimensional sphere of unit radius . the advantage of an a*m lattice over the hypercubic lattice grows exponentially with the number of dimensions . for the case of gravitational - wave signals from spinning neutron its thickness is around 1.84 , which is much better than the cubic grid with a thickness of approximately 2.72 . it is worse than the best 3-dimensional lattice covering , which has a thickness of around 1.46 . in a grid was constructed in the 4-dimensional parameter space spanned by frequency , frequency derivative , and sky position of the source , for the case of an almost monochromatic gravitational - wave signal originating from a spinning neutron star . the grid was then constrained so that the nodes of the grid coincide with fourier frequencies . this allowed the use of a fast fourier transform ( fft ) to evaluate the maximum - likelihood \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic efficiently ( see section 4.6.2 ) . efficient 2-dimensional banks of templates suitable for directed searches ( in which one assumes that the position of the gravitational - wave source in the sky is known , but one does not assume that the wave s frequency and its derivative are a priori known ) were constructed in . all grids found in enable usage of the fft algorithm in the computation of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic ; they have thicknesses 0.116% larger than the thickness of the optimal 2-dimensional hexagonal covering . in the construction of grids the dependence on the choice of the position of the observational interval with respect to the origin of time axis was employed . also the usage of the fft algorithms with nonstandard frequency resolutions achieved by zero padding or folding the data was discussed . the above template placement constructions are based on a fisher matrix with constant coefficients , i.e. , they assume that the parameter manifold is flat . this problem is to use stochastic template banks where a grid in the parameter space is randomly generated by some algorithm [ 89 , 57 , 86 , 119 ] . to extract gravitational - wave signals from the detector s noise one very often uses filters that are not optimal . we may have to choose an approximate , suboptimal filter because we do not know the exact form of the signal ( this is almost always the case in practice ) or in order to reduce the computational cost and to simplify the analysis . in the case of the signal of the form given in eq . ( 32 ) the most natural and simplest way to proceed is to use as detection statistic the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } statistic where the filters hk(t ; ) ( k = 1 , , n ) are the approximate ones instead of the optimal ones hk(t ; ) ( k = 1 , , n ) matched to the signal . in general the functions hk(t ; ) will be different from the functions hk(t ; ) used in optimal filtering , and also the set of parameters will be different from the set of parameters in optimal filters . we call this procedure the suboptimal filtering and we denote the suboptimal statistic by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal f}_{\rm{s}}}$\end{document}. it is defined as [ see eq . ( 62 ) ] 111\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal{f}_{\rm{s}}}[x;\zeta ] : = { 1 \over 2}{{\rm{n}}_{\rm{s}}}{[x;\zeta ] ^{\rm t } } \cdot { { \rm{m}}_{\rm{s}}}{(\zeta)^{- 1 } } \cdot { { \rm{n}}_{\rm{s}}}[x;\zeta ] , $ $ \end{document } where the data - dependent n 1 column matrix ns and the square n n matrix ms have components [ see eq . ( 60 ) ] 112\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n_{{\text{s}}\;i}}[x;\zeta ] : = ( x(t)|{h'_i}(t;\zeta ) ) , \quad { m_{{\text{s}}ij}}(\zeta ): = ( { h'_i}(t;\zeta ) |{h'_j}(t;\zeta ) ) , \quad i , j = 1 , \ldots , n.$$\end{document } we need a measure of how well a given suboptimal filter performs . to find such a measure we calculate the expectation value of the suboptimal statistic \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal f}_{\rm{s}}}$\end{document } in the case where the data contains the gravitational - wave signal , i.e. , when x(t ; a , ) = n(t ) + h(t ; a , ) . we get 113\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{e}}[{{\mathcal{f}}_s}[x(t;{\rm{a}},\xi);\zeta ] ] = { 1 \over 2}(n + { { \rm{a}}^{\rm t } } \cdot { \rm{q } } _ { \rm{s}}(\xi , \zeta ) \cdot { { \rm{m}}_{\rm{s}}}{(\zeta)^{- 1 } } \cdot { \rm q}_{\rm{s}}{(\xi , \zeta)^{\rm{t } } } \cdot \rm{a}),$$\end{document } where we have introduced the matrix qs with components 114\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_{{\text{s}}ij}}(\xi , \zeta ): = ( { h_i}(t;\xi ) |{h'_j}(t;\zeta ) ) , \quad i , j = 1 , \ldots , n.$$\end{document } let us rewrite the expectation value ( 113 ) in the following form , 115\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{e}}[{{\mathcal f}_{\rm{s}}}[x(t;{\rm{a}},\xi);\zeta ] ] = { 1 \over 2}\left({n + \rho { { ( { \rm{a}},\xi)}^2}{{{{\rm{a}}^t } \cdot { { \rm{q}}_{\rm{s}}}(\xi , \zeta ) \cdot { { \rm{m}}_{\rm{s}}}{{(\zeta)}^{- 1 } } \cdot { { \rm{q}}_{\rm{s}}}{{(\xi , \zeta)}^t } \cdot { \rm{a } } } \over { { { \rm{a}}^{\rm{t } } } \cdot m(\xi ) \cdot { \rm{a } } } } } \right),$$\end{document } where is the optimal signal - to - noise ratio [ given in eq . this expectation value reaches its maximum equal to ( n + )/2 when the filter is perfectly matched to the signal . therefore , a natural measure of the performance of a suboptimal filter is the quantity ff defined by 116\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\text{ff}(\xi ): = \mathop { \max } \limits_{(a,\varsigma ) } \sqrt { \frac{{{\text{a}^\text{t } } \cdot { \text{q}_\text{s}}(\xi , \varsigma ) \cdot { \text{m}_\text{s}}{{(\varsigma ) } ^ { - 1 } } \cdot { \text{q}_\text{s}}{{(\xi , \varsigma ) } ^\text{t } } \cdot \text{a}}}{{{\text{a}^\text{t } } \cdot \text{m}(\xi ) \cdot \text{a } } } } .$$\end{document } we call the quantity ff the generalized fitting factor . from the rayleigh principle , it follows that the generalized fitting factor is the maximum of the largest eigenvalue of the matrix m( ) qs( , ) ms( ) qs( , ) over the intrinsic parameters of the signal . in the case of a gravitational - wave signal given by 117\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s(t;{a_0},\xi ) = { a_0}h(t;\xi),$$\end{document } the generalized fitting factor defined above reduces to the fitting factor introduced by apostolatos : 118\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{ff}(\xi ) = \max\limits_\zeta { { ( h(t;\xi)\vert { h{\prime}}(t;\zeta ) ) } \over { \sqrt { ( h(t;\xi)\vert h(t;\xi ) ) } \sqrt { ( { h{\prime}}(t;\zeta)\vert { h{\prime}}(t;\zeta))}}}.$$\end{document } the fitting factor is the ratio of the maximal signal - to - noise ratio that can be achieved with suboptimal filtering to the signal - to - noise ratio obtained when we use a perfectly matched , optimal filter . ( 117 ) , ff is independent of the value of the amplitude a0 . for the case of a signal of the form 119\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s(t;{a_0},{\phi _ 0},\xi ) = { a_0}\cos ( \phi ( t;\xi ) + { \phi _ 0}),$$\end{document } where 0 is a constant phase , the maximum over 0 in eq . ( 118 ) can be obtained analytically . moreover , assuming that over the bandwidth of the signal the spectral density of the noise is constant and that over the observation time cos(t ; ) oscillates rapidly , the fitting factor is approximately given by 120\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{ff}}(\xi ) \cong \max\limits_\zeta { \left [ { { { \left({\int\nolimits_0^{{t_0 } } { \cos ( \phi ( t;\xi ) - { \phi { \prime}}(t;\zeta))dt } } \right)}^2 } + { { \left({\int\nolimits_0^{{t_0 } } { \sin ( \phi ( t;\xi ) - { \phi { \prime}}(t;\zeta))dt } } \right)}^2 } } \right]^{1/2}}.$$\end{document } in designing suboptimal filters one faces the issue of how small a fitting factor one can accept . 97 . assuming that the amplitude of the signal and consequently the signal - to - noise ratio decreases inversely proportionally to the distance from the source this corresponds to 10% loss of the signals that would be detected by a matched filter . proposals for good suboptimal ( search ) templates for the case of coalescing binaries are given in [ 35 , 134 ] and for the case - spinning neutron stars in [ 65 , 18 ] . in order to detect signals we search for threshold crossings of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic over the intrinsic parameter space . once we have a threshold crossing we need to find the precise location of the maximum of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } in order to estimate accurately the parameters of the signal . the first step is a coarse search where we evaluate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } on a coarse grid in parameter space and locate threshold crossings . the second step , called a fine search , is a refinement around the region of parameter space where the maximum identified by the coarse search is located . one is to refine the grid around the threshold crossing found by the coarse search [ 94 , 92 , 134 , 127 ] , and the other is to use an optimization routine to find the maximum of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } [ 65 , 78 ] . as initial values to the optimization routine one useful method is the nelder - mead algorithm , which does not require computation of the derivatives of the function being maximized . usually the grid in parameter space is very large and it is important to calculate the optimum statistic as efficiently as possible . in special cases the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic given by eq . for example , in the case of coalescing binaries \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } can be expressed in terms of convolutions that depend on the difference between the time - of - arrival ( toa ) of the signal and the toa parameter of the filter . such convolutions can be efficiently computed using ffts . for continuous sources , like gravitational waves from rotating neutron stars observed by ground - based detectors or gravitational waves form stellar mass binaries observed by space - borne detectors , the detection statistic \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } involves integrals of the general form 121\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_0^{{t_0 } } { x(t)m(t;\omega , \tilde \xi ) } \exp ( i\omega { \phi _ { \bmod}}(t;\tilde \xi))\exp ( i\omega t){\rm{d}}t,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \xi}$\end{document } are the intrinsic parameters excluding the frequency parameter , m is the amplitude modulation function , and mod the phase modulation function . the amplitude modulation function is slowly varying compared to the exponential terms in the integral ( 121 ) . we see that the integral ( 121 ) can be interpreted as a fourier transform ( and computed efficiently with an fft ) , if mod = 0 and if m does not depend on the frequency . in the long - wavelength approximation the amplitude function m does not depend on the frequency . in this case , eq . ( 121 ) can be converted to a fourier transform by introducing a new time variable tb , 122\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{\rm{b}}}(t;\tilde \xi ) : = t + { \phi _ { \bmod}}(t;\tilde \xi).$$\end{document } thus , in order to compute the integral ( 121 ) , for each set of the intrinsic parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \xi}$\end{document } we multiply the data by the amplitude modulation function m , resample according to eq . ( 122 ) , and perform the fft . in the case of lisa detector data when the amplitude modulation m depends on frequency we can divide the data into several band - passed data sets , choosing the bandwidth for each set to be sufficiently small so that the change of m exp(imod ) is small over the band . in the integral ( 121 ) we can then use as the value of the frequency in the amplitude and phase modulation function the maximum frequency of the band of the signal ( see for details ) . fisher matrix has been extensively used to assess the accuracy of estimation of astrophysically - interesting parameters of different gravitational - wave signals . for ground - based interferometric detectors , the first calculations of the fisher matrix concerned gravitational - wave signals from inspiralling compact binaries ( made of neutron stars or black holes ) in the leading - order quadrupole approximation [ 50 , 76 , 63 ] and from quasi - normal modes of kerr black hole . cutler and flanagan initiated the study of the implications of the higher - order post - newtonian ( pn ) phasing formula as applied to the parameter estimation of inspiralling binary signals . they used the 1.5pn phasing formula to investigate the problem of parameter estimation , both for spinning and non - spinning binaries , and examined the effect of the spin - orbit coupling on the estimation of parameters . the effect of the 2pn phasing formula was analyzed independently by poisson and will and krlak , kokkotas and schfer . in both cases the focus was to understand the leading - order spin - spin coupling term appearing at the 2pn level when the spins were aligned perpendicularly to the orbital plane . compared to , also included a priori information about the magnitude of the spin parameters , which then leads to a reduction in the rms errors in the estimation of mass parameters . the case of a 3.5pn phasing formula was studied in detail by arun et al . . more recently the fisher matrix was employed to assess the errors in estimating the parameters of nonspinning black - hole binaries using the complete inspiral - merger - ring - down waveforms . various authors have investigated the accuracy with which the lisa detector can determine binary parameters including spin effects . cutler determined lisa s angular resolution and evaluated the errors of the binary masses and distance considering spins aligned or anti - aligned with the orbital angular momentum . hughes investigated the accuracy with which the redshift can be estimated ( if the cosmological parameters are derived independently ) , and considered the black - hole ring - down phase in addition to the inspiralling signal . seto included the effect of finite armlength ( going beyond the long wavelength approximation ) and found that the accuracy of the distance determination and angular resolution improve . this happens because the response of the instrument when the armlength is finite depends strongly on the location of the source , which is tightly correlated with the distance and the direction of the orbital angular momentum . vec - chio provided the first estimate of parameters for precessing binaries when only one of the two supermassive black holes carries spin . he showed that modulational effects decorrelate the binary parameters to some extent , resulting in a better estimation of the parameters compared to the case when spins are aligned or antialigned with orbital angular momentum . hughes and menou studied a class of binaries , which they called golden binaries , for which the inspiral and ring - down phases could be observed with good enough precision to carry out valuable tests of strong - field gravity . berti , buonanno and will have shown that inclusion of non - precessing spin - orbit and spin - spin terms in the gravitational - wave phasing generally reduces the accuracy with which the parameters of the binary can be estimated . this is not surprising , since the parameters are highly correlated , and adding parameters effectively dilutes the available information . extensive study of accuracy of parameter estimation for continuous gravitational - wave signals from spinning neutron stars was performed in . in seto used the fisher matrix to study the possibility of determining distances to rapidly rotating isolated neutron stars by measuring the curvature of the wave fronts . in order to test the performance of the maximization method of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic it is useful to perform monte carlo simulations of the parameter estimation and compare the simulated variances of the estimators with the variances calculated from the fisher matrix . such simulations were performed for various gravitational - wave signals [ 73 , 26 , 65 , 36 ] . in these simulations one observes that , above a certain signal - to - noise ratio , called the threshold signal - to - noise ratio , the results of the monte carlo simulations agree very well with the calculations of the rms errors from the inverse of the fisher matrix . however , below the threshold signal - to - noise ratio they differ by a large factor . exist more refined theoretical bounds on the rms errors that explain this effect , and they were studied in the context of the gravitational - wave signals from coalescing binaries . use of the fisher matrix in the assessment of accuracy of the parameter estimation has been critically examined in , where a criterion has been established for the signal - to - noise ratio above which the inverse of the fisher matrix approximates well covariances of the parameter estimators . in [ 148 , 142 ] the errors of ml estimators of parameters of gravitational - wave signals from nonspinning black - hole binaries were calculated analytically using a power expansion of the bias and the covariance matrix in inverse powers of the signal - to - noise ratio . the first - order term in this covariance matrix expansion is the inverse of the fisher information matrix . the use of higher - order derivatives of the likelihood function in these expansions makes the errors prediction sensitive to the secondary lobes of the pdf of the ml estimators . conditions for the validity of the cramr - rao lower bound are discussed in as well , and some new features in regions of the parameter space so far not explored are predicted ( e.g. , that the bias can become the most important contributor to the parameters errors for high - mass systems with masses 200 m and above ) . there exists a simple model that explains the deviations from the covariance matrix and reproduces well the results of the monte carlo simulations ( see also ) . the model makes use of the concept of the elementary cell of the parameter space that we introduced in section 4.3.2 . the calculation given below is a generalization of the calculation of the rms error for the case of a monochromatic signal given by rife and boorstyn . when the values of parameters of the template that correspond to the maximum of the functional \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } fall within the cell in the parameter space where the signal is present , the rms error is satisfactorily approximated by the inverse of the fisher matrix . however , sometimes , as a result of noise , the global maximum is in the cell where there is no signal . in the simplest case we can assume that the probability density of the values of the outliers is uniform over the search interval of a parameter , and then the rms error is given by 123\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sigma _ { { \rm{out}}}^2 = { { { \delta ^2 } } \over { 12}},$$\end{document } where is the length of the search interval for a given parameter . the probability that an outlier occurs will be higher the lower the signal - to - noise ratio is . then the total variance f the estimator of a parameter is the weighted sum of the two errors 124\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\sigma ^2 } = \sigma _ { { \rm{out}}}^2q + \sigma _ { { \rm{cr}}}^2(1 - q),$$\end{document } where cr is the rms errors calculated from the covariance matrix for a given parameter . one can show that the probability q can be approximated by the following formula : 125\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q = 1 - \int\nolimits_0^\infty { { p_1}(\rho , \mathcal{f}){{\left({\int\nolimits_0^\mathcal{f } { { p_0}(y){\rm{d}}y } } \right)}^{{n_{{\rm{cells } } } } - 1}}{\rm{d}}\mathcal{f},}$$\end{document } where p0 and p1 are the pdfs of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic ( for known intrinsic parameters ) when the signal is absent or present in data , respectively [ they are given by eqs . ( 84 ) and ( 85 ) ] , and where ncells is the number of cells in the intrinsic parameter space . ( 125 ) is in good but not perfect agreement with the rms errors obtained from the monte carlo simulations ( see ) . there are clearly other reasons for deviations from the cramr - rao bound as well . one important effect ( see ) is that the functional \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } has many local subsidiary maxima close to the global one . thus , for a low signal - to - noise ratio the noise may promote the subsidiary maximum to a global one . detection of a signal is signified by a large value of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic that is unlikely to arise from the noise - only distribution . if instead the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } is consistent with pure noise with high probability we can place an upper limit on the strength of the signal . one way of doing this is to take the loudest event obtained in the search and solve the equation 126\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{\rm{d}}}({\rho _ { { \rm{ul}}}},{{\mathcal f}_{\rm{l } } } ) = \beta$$\end{document } for signal - to - noise ratio ul , where pd is the detection probability given by eq . ( 87 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal f}_{\rm{l}}}$\end{document } is the value of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic corresponding to the loudest event , and is a chosen confidence [ 23 , 2 ] . then ul is the desired upper limit with confidence . when gravitational - wave data do not conform to a gaussian probability density assumed in eq . ( 87 ) , a more accurate upper limit can be obtained by injecting the signals into the detector s data and thereby estimating the probability of detection pd . for example a network of bar detectors can observe a gravitational - wave burst from the same supernova explosion , or a network of laser interferometers can detect the inspiral of the same compact binary system . the space - borne lisa detector can be considered as a network of three detectors that can make three independent measurements of the same gravitational - wave signal . simultaneous observations are also possible among different types of detectors . for example , a search for supernova bursts can be performed simultaneously by resonant and interferometric detectors . let us consider a network consisting of n gravitational - wave detectors and let us denote by xi the data collected by the ith detector ( i = 1 , , we assume that noises in all detectors are additive , so the data xi is a sum of the noise ni in the ith detector and eventually a gravitational - wave signal hi registered by the ith detector , 127\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x_i}(t ) = { n_i}(t ) + { h_i}(t),\,i = 1 , \ldots , n.$$\end{document } it is convenient to collect all the data streams , all the noises , and all the gravitational - wave signals into column n 1 matrices denoted respectively by x , n , and h , 128\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{x}}(t ) : = \left({\begin{array}{*{20}c } { { x_1}(t ) } \\ \vdots \\ { { x_n}(t ) } \\ \end{array } } \right),{\rm{n}}(t ) : = \left({\begin{array}{*{20}c } { { n_1}(t ) } \\ \vdots \\ { { n_n}(t ) } \\ \end{array } } \right),\,{\rm{h}}(t ) : = \left({\begin{array}{*{20}c } { { h_1}(t ) } \\ \vdots \\ { { h_n}(t ) } \\ \end{array } } \right),$$\end{document } then eqs . ( 127 ) can shortly be written as 129\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{x}}(t ) = { \rm{n}}(t ) + { \rm{h}}(t).$$\end{document } if additionally all detectors noises are stationary , gaussian , and continuous - in - time random processes with zero means , the network log likelihood function is given by 130\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\log \lambda [ { \rm{x } } ] = ( { \rm{x } } + { \rm{h } } ) - { 1 \over 2}({\rm{h } } + { \rm{h}}),$$\end{document } where the scalar product ( ) is defined by 131\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$({\rm{x}}\vert { \rm{y } } ) : = 4\re \int\nolimits_0^\infty { { \rm{\tilde x}}{{(f)}^{\rm{t } } } \cdot { { \rm{s}}_n}{{(f)}^{- 1 } } \cdot { \rm{\tilde y } } } { ( f)^{\ast}}{\rm{d}}f.$$\end{document } here sn is the one - sided cross spectral density matrix of the noises of the detector network , which is defined by ( here e denotes the expectation value ) 132\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{e}}[{\rm{\tilde n}}(f ) \cdot { { \rm{\tilde n}}^{\ast}}{({f{\prime}})^{\rm{t } } } ] = { 1 \over 2}\delta ( f - { f{\prime}}){{\rm{s}}_n}(\vert f\vert).$$\end{document } the analysis is greatly simplified if the cross spectrum matrix sn is diagonal . this is the case when the detectors of the network are in widely separated locations , like , for example , the two ligo detectors . however , this assumption is not always satisfied . an important case is the lisa detector where the noises of the three independent responses are correlated . nevertheless for the case of lisa one can find a set of three combinations for which the noises are uncorrelated [ 108 , 112 ] . when the cross spectrum matrix is diagonal the network log likelihood function is just the sum of the log likelihood functions for each detector . derivation of the likelihood function for an arbitrary network of detectors can be found in . applications of optimal filtering for observations of gravitational - wave signals from coalescing binaries by networks of ground - based detectors are given in [ 63 , 41 , 62 , 103 ] , and for the case of stellar - mass binaries observed by lisa space - borne detector in [ 78 , 118 ] . the single - detector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic for nearly monochromatic gravitational waves from spinning neutron stars was generalized to the case of a network of detectors ( also with time - varying noise curves ) in ( in this work the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}-statistic was also generalized from the usual single - source case to the case of a collection of known sources ) . the reduced fisher matrix [ defined in eq . ( 81 ) ] for the case of a network of interferometers observing spinning neutron stars has been derived and studied in . network searches for gravitational - wave burst signals of unknown shape are often based on maximization of the network likelihood function over each sample of the unknown polarization waveforms h+ and h and over sky positions of the source [ 52 , 95 ] . a least - squares - fit solution for the estimation of the sky location of the source and the polarization waveforms by a network of three detectors for the case of a broadband burst there is also another important method for analyzing the data from a network of detectors the search for coincidences of events among detectors . this analysis is particularly important when we search for supernova bursts , the waveforms of which are not very well known . such signals can be easily mimicked by the non - gaussian behavior of the detector noise . the idea is to filter the data optimally in each of the detectors and obtain candidate events . then one compares parameters of candidate events , like , for example , times of arrivals of the bursts , among the detectors in the network . this method is widely used in the search for supernovae by networks of bar detectors . a new geometric coincident algorithm of combining the data from a network of detectors was proposed in . this algorithm employs the covariances between signal s parameters in such a way that it associates with each candidate event an ellipsoidal region in parameter space defined by the covariance matrix . events from different detectors are deemed to be in coincidence if their ellipsoids have a nonzero overlap . the coincidence and the coherent strategies of multidetector detection of gravitational - wave signals from inspiralling compact binaries have been compared in [ 96 , 97 , 32 ] . considered detectors in pairs located in the same site and pairs of detectors at geographically separated sites . the case of three detectors ( like the network of two ligo detectors and the virgo detector ) has been considered in detail in , where it was demonstrated that the hierarchical coherent pipeline on gaussian data has a better performance than the pipeline with just the coincident stage . a general framework for studying the effectiveness of networks of interferometric gravitational - wave detectors has been proposed in . using this framework it was shown that adding a fourth detector to the existing network of ligo / virgo detectors can dramatically increase , by a factor of 2 to 4 , the detected event rate by allowing coherent data analysis to reduce the spurious instrumental coincident background . eqs . ( 61 ) and ( 62 ) provide maximum likelihood estimators only when the noise in which the signal is buried is gaussian . one is the central limit theorem , which states that the mean of any set of variables with any distribution having a finite mean and variance tends to the normal distribution . the other comes from the information theory and says that the probability distribution of a random variable with a given mean and variance , which has the maximum entropy ( minimum information ) is the gaussian distribution . nevertheless , analysis of the data from gravitational - wave detectors shows that the noise in the detector may be non - gaussian ( see , e.g. , figure 6 in ) . the noise in the detector may also be a non - linear and a non - stationary random process . the maximum likelihood method does not require that the noise in the detector be gaussian or stationary . however , in order to derive the optimum statistic and calculate the fisher matrix we need to know the statistical properties of the data . the probability distribution of the data may be complicated , and the derivation of the optimum statistic , the calculation of the fisher matrix components and the false alarm probabilities may be impractical . however , there is one important result that we have already mentioned . the matched - filter , which is optimal for the gaussian case is also a linear filter that gives maximum signal - to - noise ratio no matter what the distribution of the data . monte carlo simulations performed by finn for the case of a network of detectors indicate that the performance of matched - filtering ( i.e. , the maximum likelihood method for gaussian noise ) is satisfactory for the case of non - gaussian and stationary noise . allen et al . [ 10 , 11 ] derived an optimal ( in the neyman - pearson sense , for weak signals ) signal processing strategy , when the detector noise is non - gaussian and exhibits tail terms . this strategy is robust , meaning that it is close to optimal for gaussian noise but far less sensitive than conventional methods to the excess large events that form the tail of the distribution . this strategy is based on a locally optimal test that amounts to comparing a first non - zero derivative 133\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda _ n}[x ] = { \left . { { { { d^n}\lambda [ x\vert \epsilon ] } \over { d\epsilon^n } } } \right\vert _ { \epsilon = 0}}$$\end{document } of the likelihood ratio with respect to the amplitude of the signal with a threshold instead of the likelihood ratio itself . the non - stationarity in the case of gaussian and uncorrelated noise can be easily incorporated into matched filtering ( see appendix c of ) . let us assume that a noise sample nl in the data has a gaussian pdf with a variance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sigma _ l^2$\end{document } and zero mean ( l = 1 , , n , where n is the number of data points ) . we also assume that the noise samples are uncorrelated , then the autocorrelation function k(l , l ) of the noise is given by [ see eq . ( 39 ) ] 134\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k(l,{l{\prime } } ) = \sigma _ l^2{\delta _ { l{l{\prime}}}},$$\end{document } where ll is the kronecker delta function . in the case of a known signal hl and additive noise the optimal filter ql is the solution of the following equation [ which is a discrete version of the integral eq . ( 41 ) ] : 135\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_l } = \sum\limits_{{l{\prime } } = 1}^n { k(l,{l{\prime}})ql{\prime}.}$$\end{document } thus , we have the following equation for the filter ql : 136\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_l } = { { { h_l } } \over { \sigma _ l^2}},$$\end{document } and the following expression for the log likelihood ratio : 137\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ln \lambda [ x ] = \sum\limits_{l = 1}^n { { { { h_l}{x_l } } \over { \sigma _ l^2 } } - { 1 \over 2 } } \sum\limits_{l = 1}^n { { { h_l^2 } \over { \sigma _ l^2}}.}$$\end{document } thus , we see that for non - stationary , uncorrelated gaussian noise the optimal processing is identical to matched filtering for a known signal in stationary gaussian noise , except that we divide both the data xl and the signal hl by time - varying standard deviation of the noise . this may be thought of as a special case of whitening the data and then correlating it using a whitened filter . in the remaining part of this section we review some statistical tests and methods to detect non - gaussianity , non - stationarity , and non - linearity in the data . a classical test for a sequence of data to be gaussian is the kolmogorov - smirnov test . it calculates the maximum distance between the cumulative distribution of the data and that of a normal distribution , and assesses the significance of the distance . a similar test is the lillifors test , but it adjusts for the fact that the parameters of the normal distribution are estimated from the data rather than specified in advance . another test is the jarque - bera test , which determines whether sample skewness and kurtosis are unusually different from their gaussian values . this test assumes that the data has an underlying gaussian probability distribution but it is corrupted by some disturbances . outliers are removed one by one and the test is iterated until no outliers are detected . grubbs test is a test of the null hypothesis : h0 : there are no outliers in the data set xk.against the alternate hypothesis : h1 : there is at least one outlier in the data set xk . the grubbs test statistic is the largest absolute deviation from the sample mean in units of the sample standard deviation , so it is defined as 138\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = { { { { \max}_k}\vert { x_k } - \mu \vert } \over \sigma},$$\end{document } where and denote the sample mean and the sample standard deviation , respectively . the hypothesis of no outliers is rejected if 139\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g > { { n - 1 } \over { \sqrt n}}\sqrt { { { t_{\alpha/2(2n),n - 2}^2 } \over { n - 2 + t_{\alpha/(2n),n - 2}^2}},}$$\end{document } where t/(2n),n2 denotes the critical value of the t - distribution with n 2 degrees of freedom and a significance level of /(2n ) . h0 : there are no outliers in the data set xk . against the alternate hypothesis : h1 : there is at least one outlier in the data set xk . grubbs test has been used to identify outliers in the search of virgo data for gravitational - wave signals from the vela pulsar . a test to discriminate spurious events due to non - stationarity and non - gaussianity of the data from genuine gravitational - wave signals has been developed by allen . this test , called the time - frequency discriminator , is applicable to the case of broadband signals , such as those coming from compact coalescing binaries . let now xk and ul be two discrete - in - time random processes ( < k , l < ) and let ul be independent and identically distributed ( i.i.d . ) we call the process xk linear if it can be represented by 140\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x_k } = \sum\limits_{l = 0}^n { { a_l}{u_{k - l}},}$$\end{document } where al are constant coefficients . if ul is gaussian ( non - gaussian ) , we say that xl is linear gaussian ( non - gaussian ) . in order to test for linearity and gaussianity we examine the third - order cumulants of the data . the third - order cumulant ckl of a zero mean stationary process is defined by 141\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${c_{kl } } : = { \rm{e}}[{x_m}{x_{m + k}}{x_{m + l}}].$$\end{document } the bispectrum s2(f1 , f2 ) is the two - dimensional fourier transform of ckl . the bicoherence is defined as 142\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b({f_1},{f_2 } ) : = { { { s_2}({f_1},{f_2 } ) } \over { s({f_1 } + { f_2})s({f_1})s({f_2})}},$$\end{document } where s(f ) is the spectral density of the process xk . if the process is gaussian , then its bispectrum and consequently its bicoherence is zero . one can easily show that if the process is linear then its bicoherence is constant . thus , if the bispectrum is not zero , then the process is non - gaussian ; if the bicoherence is not constant then the process is also non - linear . consequently we have the following hypothesis testing problems : h1 : the bispectrum of xk is nonzero.h0 : the bispectrum of xk is zero . if hypothesis 1 holds , we can test for linearity , that is , we have a second hypothesis testing problem : 3.h1 : the bicoherence of xk is not constant.4.h1 : the bicoherence of xk is a constant . if hypothesis 4 holds , the process is linear . h1 : the bicoherence of xk is a constant . using the above tests we can detect non - gaussianity and , if the process is non - gaussian , non - linearity of the process . one can divide the data into short segments and for each segment calculate the mean , standard deviation and estimate the spectrum . one can then investigate the variation of these quantities from one segment of the data to the other . another quantity to examine is the autocorrelation function of the data . for a stationary process the autocorrelation function should decay to zero . a test to detect certain non - stationarities used for analysis of econometric time series is the dickey - fuller test . it models the data by an autoregressive process and it tests whether values of the parameters of the process deviate from those allowed by a stationary model . a robust test for detecting non - stationarity in data from gravitational - wave detectors has been developed by mohanty . the test involves applying student s t - test to fourier coefficients of segments of the data . it identifies places in the data stream where the characteristic statistics of the data change .

the purpose of this study was to assess predictability , efficacy , safety and stability in patients who received a toric implantable collamer lens to correct moderate to high myopic astigmatism . forty - three eyes of 23 patients underwent implantation of a toric implantable collamer lens ( staar surgical inc ) for astigmatism correction . 98 3.49 diopters ( d ) ( range : 0 to 13 d ) , and mean cylinder was 2.62 0.97 d ( range : 1.00 to 5.00 d ) . main outcomes measures evaluated during a 12-month follow - up included uncorrected visual acuity ( ucva ) , refraction , best - corrected visual acuity ( bcva ) , vault , and adverse events . at 12 months the mean snellen decimal ucva was 0.87 0.27 and mean bcva was 0.94 0.21 , with an efficacy index of 1.05 . more than 60% of the eyes gained 1 line of bcva ( 17 eyes , safety index of 1.14 ) . the treatment was highly predictable for spherical equivalent ( r = 0.99 ) and astigmatic components : j0 ( r = 0.99 ) and j45 ( r = 0.90 ) . the mean spherical equivalent dropped from 7.29 3.4 d to 0.17 0.40 d at 12 months . of the attempted spherical equivalent , 76.7% of the eyes were within 0.50 d and 97.7% eyes were within 1.00 d , respectively . the results of the present study support the safety , efficacy , and predictability of toric implantable collamer lens implantation to treat moderate to high myopic astigmatism . clinical results of posterior phakic intraocular lenses ( piols ) have confirmed this procedure as a promising treatment option for patients who can not undergo keratorefractive procedures.1 the main advantages are the correction of higher levels of myopia , hyperopia , and astigmatism and the reversibility of the procedure.212 presently , one of the most successful piol , known as implantable collamer lens ( icl ) , is the visian iclv4 manufactured by staar surgical ( monrovia , ca ; figure 1 ) . the visian icl is the only posterior piol currently under revision for approval by the united states food and drug administration ( fda ) for the treatment of astigmatism.10 several studies have been published on an anterior chamber iris - fixated toric piol1317 demonstrating a significant reduction in spherical and astigmatic refractive errors . however concerns still exist regarding potentially induced progressive endothelial cell loss.18 the recently published outcomes of the toric icl812 showed good efficacy and predictability . additionally , in cases in which keratorefractive or other alternative refractive procedures were not a good or feasible option , toric icl implantation showed promising results.1922 the purpose of this study was to assess safety , efficacy , predictability , and stability in patients who undergo toric icl implantation to correct moderate to high myopic astigmatism with follow - up visits up to 12 months . we prospectively studied 43 eyes of 23 patients , 17 ( 73.9% ) females and 6 ( 26.1% ) males , who underwent implantation of a toric icl to correct myopic astigmatism at the medipolis eye centre ( antwerp , belgium ) . mean patient age was 34.41 9.42 years ( range : 22 to 49 years ) . the inclusion criteria were best - corrected distance visual acuity ( bcva ) of 20/40 or better , stable refraction and clear central cornea . the exclusion criteria included age < 22 years , anterior chamber depth < 2.8 mm from endothelium , endothelial cell density < 2000 cell / mm , cataract , history of glaucoma or retinal detachment , macular degeneration or retinopathy , neuro - ophthalmic diseases , and history of ocular inflammation . before the ticl implantation , patients had a complete ophthalmologic examination , including refraction , keratometry , corneal topography , endothelial cell count , pachymetry , slit - lamp microscopy , goldmann applanation tonometry , and binocular indirect ophthalmoscopy . toric icl is a piol manufactured with an haptic design identical to the spherical icl2 in terms of size , thickness , and shape , with central convex / concave optical zone , and cylinder as necessary to address each patient s astigmatic condition.8 toric icl power calculation was performed using the software provided by the manufacturer . toric icl is manufactured to minimize rotation and required the surgeon to rotate the toric icl no more than 22.5 from the horizontal meridian . each toric icl comes with a guide demonstrating the amount and direction of rotation from the horizontal axis required of the toric icl to exactly align the toric icl cylinder axis to the patients required cylinder correction . all surgeries in this study were performed by one experienced surgeon ( elm ) using topical and intracameral anesthesia . on the day of surgery , 0.5% tropicamide and 5% phenylephrine hcl fifteen minutes before surgery a drop of preservative - free oxybuprocaine hcl 0.4% was instilled , followed by two to four drops of povidone iodine 5% . povidone iodine 10% ( isobetadine ; viatris , bad homburg , germany ) was applied to the eyelids , the patient was draped and a lid speculum inserted . four drops of preservative - free oxybuprocaine hcl 0.4% were used to further anaesthetize each eye . three minutes before the start of surgery povidone iodine 5% was administered to the ocular surface . the lens was removed from the sealed glass container and loaded inside the cartridge under the surgical microscope . the lens was loaded making sure that both longitudinal edges of the haptic were symmetrically tucked under the edge of the cartridge with the lens vaulted anteriorly . the coaxial forceps designed by zaldivar for icl loading ( asico , westmont , il ) was used to pull the lens through the cartridge tunnel . inspection of the lens inside the tunnel was done to exclude twisting of the lens which helped making the injection inside the anterior chamber symmetrical , smooth , and reproducible . a preinked surgical marker was used to mark the target axis for toric icl implantation using the previous dots marked at the slit lamp as reference for 0180. this provided the surgeon with extended lines over the cornea and pupil to facilitate alignment . a 1.0-mm side - port was made at 10 oclock in the left eye and at 5 oclock in the right eye followed by injection of intracameral preservative - free lidocaine 1% followed by viscoelastic ( methylcellulose ) with caution to not over - fill the anterior chamber . a 3-step temporal clear corneal incision : first a 600-m groove is made ; tilting the tip of the knife a little bit upward and the size of the incision was 2.6 mm ; this size allows smooth injection and has been reported to have negligible effect on postoperative astigmatism . firmly grasping the eye with the forceps , the tip of the cartridge was placed at the edge of the incision and the lens was slowly injected . the surgeon paid attention to the distal footplate to unfold in the anterior chamber before the trailing footplate was injected out of the cartridge . this prevents the lens from unfolding upside down in the anterior chamber . once the lens unfolded in the anterior chamber the marks on the distal and proximal footplates were checked for proper orientation . using the side - port and with vukich s or batlle s manipulator , the distal haptics were tucked under the iris . all manipulations were as peripheral as possible with no instruments touching the optic or crossing the pupillary zone . the proximal footplates were then tucked under the iris through the main incision . using the line marks on the cornea we checked for toric icl alignment . if rotation was needed we gently used the manipulator over the peripheral haptic and aligned the lens . flush of viscoelastic was done by injection of saline through the side - port and pushing down on the main incision . at the completion of the procedure we injected intracameral preservative - free antibiotics ( vancomycin 1 mg / ml ) , checked sealing of the incision and administered one drop of dorzolamide 20% and timolol 5% . in the recovery room a tablet of acetazolamide 250 mg was administered orally to reduce further the intraocular pressure . two hours after surgery the vault and alignment of the toric icl was checked at the slit - lamp and the intraocular pressure was measured . the tenets of the declaration of helsinki were followed in this study . informed consent stating the potential risks and benefits of the lens implantation was obtained from all patients prior to surgery . follow - up visits were done at week 1 , and months 1 , 3 , 6 , and 12 postoperatively . uncorrected visual acuity ( ucva ) , bcva , slit - lamp examination , refraction , central separation between the icl and the crystalline lens ( vault ) with slit lamp examination , and tonometry were evaluated . vault value was assessed by comparing the separation between the lens and the posterior surface of the ticl to the corneal thickness using an optical section during routine slit - lamp examination . estimation was registered in 0.25 steps ( ie , 0.25 corneal thickness [ ct ] , 0.5 ct , 0.75 ct , 1 ct , 1.25 ct , etc ) . for averaging , visual acuities were converted to logmar and then the means and standard deviations were back calculated to snellen acuity . assessment of toric icl outcomes was based on a comparison of preoperative with postoperative values of bcva and ucva for efficacy and safety and the achieved versus expected refractive outcomes postoperatively for predictability . efficacy index ( ratio of postoperative ucva over preoperative bcva ) and safety index ( ratio of postoperative over preoperative bcva ) were also calculated . spherocylindrical refractive results were converted into vector expressed by 3-dioptric powers : m , j0 , and j45 ; with m being equal to the spherical equivalent of the given refractive error , and j0 and j45 the two jackson cross cylinders equivalent to the conventional cylinder . manifest refractions in conventional script notation ( s [ sphere ] , c [ cylinder ] , [ axis ] ) were converted to power vector coordinates by the following formulas : m = s+c/2 ; j0=(c/2)*cos(2 ) and j45=(c/2)*sin(2)23 . data analysis was performed using spss for windows ( version 16.01 ; spss inc , chicago , il ) . hotelling t test of multivariate analysis was used to explore statistically significant differences for refractive values and visual acuity outcomes . no complications occurred during the surgical procedures . no eye needed explantation and only one eye needed repositioning due to off - axis alignment 10 months after implantation . decentration of the toric icl optic was not observed , and no case of pupillary block was detected . at 12 months postsurgery , the mean spherical error was 0.06 0.34 d ( ranging from 1.00 to 1.00 d ) and the mean cylinder value was 0.20 0.43 d ( ranging from 0 to 1.50 d ) . in relation to predictability , figure 2 shows the distribution of manifest refractive cylinder before toric icl implantation compared with the 12-month visit outcomes . at the 12-month follow - up visit , 43 ( 100% ) of eyes with the toric icl had 2.0 d of cylinder , compared with 17 ( 39.5% ) preoperatively , with 39 ( 90.7% ) eyes having a refractive cylinder 1.0 d. furthermore , no eyes ( 0.0% ) preoperatively had refractive cylinder of 0.50 d , whereas 36 eyes ( 83.7% ) had 0.5 d and 35 ( 81.4% ) had 0.25 d of refractive cylinder at 12 months . the deviation of the achieved from the expected m refraction was calculated ( see figure 3 ) . at 12 months , the mean m was 0.17 0.40 d with 33 ( 76.7% ) of the eyes within 0.50 d and 42 ( 97.7% ) were within 1.00 d of the desired m refraction ( r = 0.99 ) . for the astigmatic components , 42 eyes ( 97.7% ) and 36 ( 83.7% ) of eyes were within 0.50 d for j0 ( r = 0.99 ) and j45 ( r = 0.90 ) , respectively . the improvement in m refractive error the mean m dropped significantly from 7.29 3.4 d to 0.17 0.40 d at 12 months ( p < 0.05 ) . figure 5 shows the astigmatic components of the power vector as represented by the two - dimensional vector plot ( j0 , j45 ) where the tight distribution of points around zero is evident 12 months after surgery compared to preoperative data . a multivariate hotelling t test confirmed that the mean power vector after surgery was not significantly different from a vector of zero length ( p = 0.3256 ) . twelve months after surgery none of the examined eyes had lost > 2 lines of bcva ; one eye lost 2 lines , two ( 4.7% ) eyes lost 1 line , 10 eyes ( 23.3% ) did not change after surgery , while 13 eyes ( 30.2% ) gained 1 line , 14 ( 32.6% ) eyes gained 2 lines , and three eyes gained > 2 lines of bcva . all eyes showed a bcva of 20/63 or better with more than 93.0% ( 40 eyes ) of eyes with bcva of 20/32 or better and also an increase in the amount of eyes with bcva of 20/20 or better , from 30.2% ( 13 eyes ) at baseline to 69.8% ( 30 eyes ) after 12 months . in relation to the efficacy , figure 8 shows the changes in ucva and bcva . before surgery the mean snellen decimal ucva was 0.09 0.07 ( range : 0.05 to 0.2 ) while bcva was 0.83 0.18 ( range : 0.5 to 1.2 ) . at 12 months , the mean ucva was 0.87 0.27 ( range 0.2 to 1.2 ) and the mean bcva was 0.94 0.21 ( range 0.5 to 1.2 ) . eyes had 20/20 or better ucva , compared with 13 ( 30.2% ) bcva at baseline . furthermore , 31 ( 72% ) eyes had 12-month postoperative ucva equal to or better than preoperative bcva and 12 ( 28% ) eyes having 12-month postoperative ucva lower than preoperative bcva . during the entire follow - up period anterior subcapsular cataract development was not observed over the entire follow - up period and mean iop was 14.4 3.3 mmhg ( ranging from 8 to 20 mmhg ) . mean vault was 0.94 0.20 ct and changes during the follow - up period were very small without statistical significance during the follow - up period ( p > 0.05 ) . the present prospective study of 43 eyes of 23 patients analyzed the correction of myopic astigmatism with toric icl implantation . the aim of the study was to determine in our series whether this technique is a safe , predictable , and effective alternative for correcting myopia and astigmatism using piols . our outcomes revealed that toric icl implantation is highly predictable , stable and safe up to a period of 12 months postsurgery . the majority of eyes maintained or improved their bcva , which resulted in a high safety index ( 1.14 ) . conversely more than 60% of eyes showed a gain in bcva ( figure 6 ) . in addition , over 80% of eyes had bcva of 20/25 or better at 12 months and the percent of eyes with bcva of 20/20 or better increased from 30% preoperatively to 70% after toric icl implantation ( see figure 7 for a full description of cumulative visual acuity pre- and postsurgery ) . in order to compare with previous studies reporting data of the toric icl , the us fda toric icl clinical study8 showed that the toric icl implantation is a predictable and effective method for treating moderate to high astigmatism . in that study 76.4% of patients gained 1 line of bcva postoperatively and 76.5% of cases had postoperative ucva better than or equal to preoperative bcva , with 1.6% of eyes lost 2 lines of bcva after 12 months . it was also reported an improvement in mean manifest m value from 9.36 d preoperatively to 0.05 d postoperatively , with 76.9% of eyes within 0.50 d and 97.3% within 1.00 d as well a reduction of 73.6% in astigmatism with a mean manifest refractive cylinder reduction from 1.93 d to 0.51 d at 12 months . chang and lau9 analyzed the toric icl on 44 asian eyes at 6 months showing comparable outcomes to the fda toric icl study.8 recently , alfonso et al10 analyzed 55 eyes implanted with the toric icl at 12 months and reported high correlations between attempted and achieved refractive changes for sphere and cylinder components ( r 0.97 ) with 100% of eyes within 1.00 d , and more than 90% of eyes within 0.50 d. a similar study of these authors11 reported the analysis of the toric icl in highly astigmatic eyes ( > 4 d of cylinder ) showing also good outcomes with 93.3% of eyes having less than 1.00 d of cylinder . the mean spherical equivalent was 0.31 0.42 d ( range 1.00 to 0.75 d ) , with more than 70% of eyes within 0.50 d of the target . for the astigmatic components , 93.3% of eyes were within 1.00 d of j0 and all eyes were within 1.00 d of j45 . in addition , it is interesting to consider that toric icl implantation has also been used in cases where keratorefractive or alternative corrective refractive procedures were not a feasible option . for example , kamiya et al19 reported a marked improvement in ucva and bcva in two patients with nonprogressive keratoconus , showing a reduction in the manifest refraction from 10.00 6.00 100 d and 8.00 2.75 100 d to + 0.50 1.00 90 d and 0.25 1.25 100 d after surgery . mertens et al20 reported a bilateral implantation of a custom - designed toric icl in a patient with preoperative subjective refractive cylinder of 5.25 6 in the right eye and 5 176 in the left eye , changing to 0.5 77 and 0.5 115 after toric icl implantation , respectively . ucva improved to 20/20 in the right eye and 20/16 in the left eye , and bcva improved to 20/16 in the right eye and 20/10 in the left eye . in a series of 15 eyes with spherical icl and toric icl implantation after penetrating keratoplasty , alfonso et al21 reported an improvement in bcva , with 80% of eyes having bcva of 20/40 or better at 24 months postoperatively . about 46% of eyes gained 1 lines of bcva , 52% of all eyes did not change their bcva , and no patients lost 1 lines of bcva at follow - up . furthermore , 66.6% of eyes were within 0.50 d in m and astigmatic components showing values nearly 100% within 1.00 d in all eyes . good outcomes of these previous studies should be taken into account to consider toric icl implantation . in relation to adverse effects , the main concern related to icl implantation are anterior subcapsular cataract and increased iop,2428 presumably as a result of mechanical contact of the icl with the anterior lens capsule , or pigment dispersion and pupillary block due to angle closure , respectively . in our study , no chronic increased postoperative iop was found and no eye developed anterior subcapsular cataract over the entire follow - up period . furthermore , mean central separation between the toric icl and the crystalline lens ( vault ) during entire follow - up of this study was good and stable . longer follow - up is needed to properly analyze long - term effects of the toric icl on the ocular structures . to summarize , the results of the present study analyzing the toric icl for myopic astigmatism correction showed that this procedure is safe , stable , and predictable during the 12 months period of follow - up .

just as any other means of communication , snss allow its users to meet others , both familiar and non - familiar ones . it helps them to identify people available in the site with whom they can maintain relationships . most snss provide facilities for private messaging as well as for sending public comments and posts . many snss also have such features as photo sharing , video sharing , build - in blogging and instant messaging . snss differ widely on the basis of the basic objectives for which they are designed . there are an online business networking sites ( ryze.com ) , sites for arranging meetings for communities with like - minded interests ( meetup.com , evite.com ) and sites providing blogging service ( livejournal.com ) , but the most popular ones are those dedicated for initiation and maintenance of online friendships ( frendster.com , orkut.com , myspace.com and facebook.com ) . with increased use of snss people can initiate new contact along with the existing ones and these relationships that are formed over snss can extent to offline interactions also and hence that the social network of the person increases and correspondingly social capital also increases . it is also possible that with increased time spend on snss , people start spending lesser and lesser time for other social activities and community participation so that their social circle will get limited to these virtual social interactions . its usages are also associated with feeling better to handle loneliness and depression ; increase in self - esteem / social support , increase in time spent with online friends and increased general well - being . individuals with social anxiety show greater comfort and self - disclosure when socializing on - line compared with face - to - face interactions . heavy internet users experience a higher level of psychological distress and use internet significantly to meet their social needs . introverts are found to develop a compulsive pattern of internet use , in surfing or downloading . it ( surfing ) does not result in any long - term gratification this may lead to depression and social anxious feelings . there is low treatment seeking behavior and poor follow - up 's for psychological problems due to stigma , myths , availability of the facility or post treatment support group . epidemiological studies across different nations shows that only about one third of people with a mental disorder consult the mental health service provider , others seek help from non - specialized professionals . even in the high - income countries , a substantial number of patients with mental health problems do nt seek treatment from the mental health sector . in india , the present work explore the potentials of snss as an adjunctive treatment modality for initiating treatment contact as well as for managing psychological problems . the primary aim of this study is to explore the snss as an adjunctive treatment modality for psychological problems . the secondary aim of this study is to investigate the usages of snss in the subject with depression and anxiety spectrum disorder . a survey method was used to 28 participants which comprised of 18 males and 10 females participants from clinical population meeting inclusion criteria ( people who use any snss for at least 15 min or more per sessions ; between 18 and 35 years ; any of the depressive or anxiety spectrum disorder ; having an icd-10 diagnosis : f32 , f33 , f34.1 , f40 f48 ; working knowledge in english ) from out - patients and in - settings of national institute of mental health and neuroscience , bangalore , karnataka , india . tools : interview schedule developed by the team to covers the basic demographic details and other details concerning sns use as the sns that he likes the most ; the age of registering with snss ; the friends and family members attitude about his / her sns use and the impact of sns use on his / her personal or professional life , their opinion about the positive and negative aspects of use of snss . the extent to which they discussed their personal or illness related issues to sns friends . details of the illness were obtained from patient directly as well as from the psychiatry file . facebook intensity scale is a measure of facebook usage , which includes two self - reported assessments of facebook behavior , designed to measure the extent to which the participant was actively engaged in facebook activities : the number of facebook friends and the amount of time spent on facebook on a typical day . this measure also includes a series of likert - scale attitudinal questions designed to tap the extent to which the participant was emotionally connected to facebook and the extent to which facebook was integrated into their daily activities . the present work has nimhans institute ethics committee , bangalore , karnataka , india approval . the primary aim of this study is to explore the snss as an adjunctive treatment modality for psychological problems . the secondary aim of this study is to investigate the usages of snss in the subject with depression and anxiety spectrum disorder . a survey method was used to 28 participants which comprised of 18 males and 10 females participants from clinical population meeting inclusion criteria ( people who use any snss for at least 15 min or more per sessions ; between 18 and 35 years ; any of the depressive or anxiety spectrum disorder ; having an icd-10 diagnosis : f32 , f33 , f34.1 , f40 f48 ; working knowledge in english ) from out - patients and in - settings of national institute of mental health and neuroscience , bangalore , karnataka , india . tools : interview schedule developed by the team to covers the basic demographic details and other details concerning sns use as the sns that he likes the most ; the age of registering with snss ; the friends and family members attitude about his / her sns use and the impact of sns use on his / her personal or professional life , their opinion about the positive and negative aspects of use of snss . the extent to which they discussed their personal or illness related issues to sns friends . details of the illness were obtained from patient directly as well as from the psychiatry file . facebook intensity scale is a measure of facebook usage , which includes two self - reported assessments of facebook behavior , designed to measure the extent to which the participant was actively engaged in facebook activities : the number of facebook friends and the amount of time spent on facebook on a typical day . this measure also includes a series of likert - scale attitudinal questions designed to tap the extent to which the participant was emotionally connected to facebook and the extent to which facebook was integrated into their daily activities . the present work has nimhans institute ethics committee , bangalore , karnataka , india approval . the inpatients and out - patients with the diagnosis of anxiety spectrum disorder or depressive illnesses were approached . prior consent was obtained from the treating team for inclusion of these participants in the study as well as the informed consent was obtained from the participants . descriptive statistics such as mean , standard deviation , percentages and frequencies were used to analyze demographic data and pattern of use of snss . the mean age of registering with snss was 21 years 0.46.7% of family members were unconcerned about their usages . sample includes subjects 10 obsessive compulsive ; 8 mild depression ; 5 social anxiety a ; 5 with adjustment problems . nearly 5% of them had the problematic usage of facebook in term of losing control , having desire to be online and repeated engagement despite its negative effects on their interpersonal life . a significant positive correlation exists between number of friends and emotional connectedness to the snss ( r = 0.32 , p < 0.01 ) and between number of hours spent on snss and emotional connectedness to snss ( r = 0.36 , p < 0.01 ) [ table 1 ] . correlation of emotional connectedness to snss with number of friends and time spent nearly , 2% misrepresented themselves to opposite sex as well as in relation to working style , leisure time activity , social gathering , financial status and physical appearance [ table 2 ] . percentage distribution of content of communication over snss table 2 shows the content of communication that occur over snss . analysis of others usages of snss also revealed the following areas : communicating with friends was the most frequently identified ( 50% ) positive aspect of snss . snss provided an opportunity for reconciliations ( 24% ) or to make up the fights and to escape form the problems ( 24% ) . compared with males , females used snss more frequently to manage real - life problems ( p = 0.04 ) . usages of snss for emotional support was identified as an advantage in form of to talking to already known friends as well as it also enhances the accessibility toward existing friends at times of distress for them . it had membership of fifty two people and they used to provide comments / suggestions / references to available information related to psychological problems as well as for cheering up . it helped them to know others people with similar illness , nature of illness and biopsychosocial nature and to initiate treatment . participants perceived the online group helpful in terms of getting information about the disorder , getting expert opinion about the illness as well as the management strategies . others usages included feeling better with available online support , increased awareness about the illness , sharing of emotional difficulties , used relaxation techniques or maintaining contact with the treating doctor . usages of snss for emotional support was identified as an advantage in form of to talking to already known friends as well as it also enhances the accessibility toward existing friends at times of distress for them . it had membership of fifty two people and they used to provide comments / suggestions / references to available information related to psychological problems as well as for cheering up . it was used to provide reassurance and comment . it helped them to know others people with similar illness , nature of illness and biopsychosocial nature and to initiate treatment . participants perceived the online group helpful in terms of getting information about the disorder , getting expert opinion about the illness as well as the management strategies . others usages included feeling better with available online support , increased awareness about the illness , sharing of emotional difficulties , used relaxation techniques or maintaining contact with the treating doctor . snss ( facebook ) are used as a way to seek emotional support on the basis of the frequent updates of emotional content that users put in their profile ; reconciliations , escape form the problems or to manage the loneliness ; provide emotional connectedness [ table 1 ] ; getting information about illness and its treatment and interaction with experts . 2% misrepresented themselves to the opposite sex as well as in relation to working style , leisure time activity , social gathering , financial status and physical appearance ; 52.30% discuss their psychological problems in general term and 14% discuss in complete details and others use it for discussion of neutral topics [ table 2 ] . facebook were used for social interaction , passing time , entertainment , companionship and communication . expressing one 's current emotional state dominated use of facebook 's status - update tool . larger networks and larger estimated audiences were associated with higher levels of life satisfaction and perceived social support on facebook . loneliness as a predictor of increased use of the internet as well as snss has been well documented in the literature . the present work reflect the usages of facebook for building support system as well as for improving one 's psychological well - being . the work has implications in terms of initiating treatment contact , its role in getting support to manage the psychological problems , assessing the efficacy of e support group to develop psychological support and evolving snss as a adjunctive treatment modality of psychological problems .

secondary metabolites have long served as inspirational structural and functional scaffolds for the development of new - in - class pharmaceuticals [ 1 - 2 ] . a longstanding era of secondary metabolite discovery followed the discovery of penicillin in the 1920s , and by the 1990s , approximately 80% of commercial drugs were natural products or natural product derivatives [ 3 - 4 ] . this percentage has decreased over the last few decades due to the expansion of combinatorial synthetic methods and an increase in the rediscovery rates of natural products through traditional discovery campaigns . however , with the continued expansion of microbial genome and metagenome sequence information , a resurgence in interdisciplinary academic and industrial natural product discovery campaigns is well underway [ 5 - 11 ] . several major challenges exist for the discovery of new microbial natural product - derived drug leads , such as : 1 ) our inability to culture the majority of microbes from environmental samples ( e.g. , the great plate count anomaly ) [ 12 - 17 ] ; 2 ) our general lack of robust tools to broadly activate bioactive small molecule production from diverse silent pathways in the microbes ( or heterologous expression hosts ) that we can readily cultivate in the lab [ 18 - 21 ] ; and 3 ) our inefficiencies in quickly identifying and dereplicating unknown metabolites from expressed pathways with often unpredictable structural and functional properties . as a result , there is a continued general need for the development of interdisciplinary approaches to link orphan biosynthetic gene clusters to the bioactive small molecules they produce . multipartite animal - microbe symbioses have provided rich sources of novel bacterial small molecules that naturally function in animal environments , enhancing their pharmacological potential [ 23 - 28 ] . by understanding the ecological niche , new small molecules can be stimulated , discovered , and investigated with overarching ecological and functional contexts . indeed , since the turn of the century , we have come to appreciate humans and all other animals as being our resident microbes , the human microbiota , is one such source that has emerged as a prominent player in regulating both human health and disease , and thus its metabolite coding capacity , the human microbiome , is a rich reservoir of potential clinically - relevant small molecules [ 30 - 32 ] . the microbiota affects the host through various mechanisms including the exchange of nutrients , regulation of the immune system , protection from pathogens , and metabolism of indigestible compounds [ 26 , 33 - 34 ] . because of the importance of this symbiotic relationship , dysregulation of the microbiota communities ( dysbiosis ) has been correlated with the onset of serious health issues , including obesity , diabetes , inflammatory bowel diseases , and cancers [ 35 - 40 ] . small molecule metabolites regularly mediate host - microbe interactions . and despite the ecological importance of microbial natural products , we know very little about the structures and functional roles of these compounds and how they affect human health . ( meta)genomics - guided approaches have started to shed light on the extent of this question . sequencing of the human microbiota [ 41 - 42 ] revealed that human - associated bacteria encode for a wide diversity of biosynthetic gene clusters , with over 3,000 biosynthetic gene clusters being widely distributed among the sequenced microbiota of healthy individuals . much of the chemical diversity encompassed by the small molecule products of these gene clusters is found in bacteria that are associated with the oral and gut cavities . the majority of these compounds have not yet been characterized . one of the more heavily studied biosynthetic gene clusters from the microbiome is the colibactin pathway . the colibactin gene cluster is a ~55 kb biosynthetic gene cluster that produces a family of polyketide - nonribosomal peptide hybrid molecules . this gene cluster is found among the enterobacteriaceae , including escherichia coli , citrobacter koseri , klebsiella pneumoniae , and enterobacter aerogenes . additionally , the gene cluster has been discovered in the microbiota of infected coral and of honeybees exhibiting an intestinal scab phenotype [ 46 - 47 ] . bacteria expressing the pathway induce dna double strand breaks and cause genomic instability of mammalian cells [ 48 - 49 ] . the presence of this gene cluster is epidemiologically associated with long - term persistence in the host . under inflammatory conditions , such as in inflammatory bowel disease ( ibd ) , enterobacteriaceae members containing this gene cluster proliferate . as a result of the cytotoxicity exhibited by the small molecules from this pathway , the colibactin pathway has been directly linked to colorectal tumorogenesis in colitis mouse models [ 38 - 39 , 52 ] . however , other strains containing the colibactin cluster , such as e. coli nissle 1917 , paradoxically have also been demonstrated to exhibit probiotic effects for patients with ulcerative colitis . gaining mechanistic insights for these functional disconnects mechanistic understanding of the phenotypes exhibited by this pathway have been hindered by the lack of colibactin structural information . fortunately , structural and small molecule functional data are starting to emerge , providing new vantage points to experimentally elucidate the mechanistic underpinnings for the various colibactin pathway functions [ 54 - 61 ] . in this mini - review , we focus on the colibactin pathway as a central thematic example of linking biosynthetic gene clusters to the small molecules they produce and draw connections to other pathways invoking related biosynthetic logic . we highlight pathway - targeted molecular networking as one approach to more finely map expressed secondary metabolic pathways within complex metabolomes to aid in secondary metabolite identification and characterization . lastly , we discuss a few of the disconnects between secondary metabolite structure and biosynthetic predictions as illustrative examples for the continued need of enzymological characterizations of orphan biosynthetic gene clusters . the structure first , then hunt for its responsible gene cluster paradigm , is transitioning to sequence first , then hunt for the many possible products encoded in the ( meta)genomic information . genes - to - molecules discovery approaches inherently reduce rediscovery rates of known metabolites , as novel gene clusters , i.e. , novel briefly , natural products of the polyketide and the nonribosomal peptide families , for example , are often biosynthesized according to a biosynthetic code [ 64 - 66 ] . many of these biosynthetic systems follow a co - linearity rule , in which the organization of domains dictates the order of biosynthetic operations in the pathway . because of this logic and the wealth of mechanistic enzymology knowledge in this area , it is possible to predict with some accuracy the possible core structure(s ) of assembly line - derived polyketides and nonribosomal peptides [ 64 - 66 ] . new bioinformatic programs such as antismash [ 67 - 70 ] and clusterfinder have integrated a variety of existing bioinformatic algorithms and have largely automated the process of finding novel secondary metabolite gene clusters and predicting possible core structures . however , many nonlinear and iterative pathways among other confounding factors , such as hypothetical proteins serving as novel biocatalysts , necessitate a continued need for biosynthetic code breaking . indeed , about half of the genes in the human microbiome are listed as hypotheticals and are completely unknown . additionally , many putative secondary metabolite pathways are emerging in genome databases that contain proteins with no significant similarity to previously reported pathways and are not detected in algorithms that rely on currently known pathways as inputs , raising genome - guided opportunities for the discovery of new small molecule classes . when comparing experimental conditions , a versus b , metabolomics groups often make functional claims based on the responses of molecules that can be mapped to established external and internal databases . without question , many functional insights have been gained from these approaches , but what about all of the product ions that are not found in any current database ? in secondary metabolite discovery operations , mapping small molecules to known databases most often fails , as novel metabolites by definition have not yet been characterized . foundational approaches are emerging that are beginning to address this key bottleneck in microbial secondary metabolism . specifically , molecular networking techniques enhance diverse investigations of secondary metabolite discovery , regulation , and their functional roles by interconnecting structurally related molecules in silico . this unbiased approach scores tandem ms ( ms ) spectra based on small molecule fragmentation similarities . the molecules are then represented in a molecular network as interconnected nodes based on fragmentation relationships . using this method , an individual node , or ( mof ) groups with similar mofs , forming structurally related clusters , or molecular families . molecular networking has found many recent uses in investigating metabolic responses from individual microorganisms to complex cell - to - cell interactions . for example , coupling nanospray desorption electrospray ionization ( nanodesi ) ms with molecular networking , the metabolic status of living microbial colonies has been characterized . by growing the colony next to a competing species , the metabolic response of the microbial colony can be assessed . from the ms / ms network , small molecules that are stimulated by the challenge can be grouped into general families of metabolites . consequently , if a novel molecule is not yet in a database but falls within a known molecular family in the network , structural information can more readily be proposed . these foundational methods accelerate natural product dereplication approaches , facilitate the study of intraspecies , interspecies , and even interkingdom interactions [ 75 - 76 ] , and significantly aid in the decoding of orphan biosynthetic gene clusters and the cryptic small molecules they produce . when collecting high - resolution tandem ms data in untargeted fragmentation modes , the subsequent molecular networks generated from this data are enriched in the more abundant molecules primary metabolites , media components , and abundant secondary metabolites and often lack less abundant molecules encoded by the pathway of interest . to address this roadblock , we conducted a modified pathway - targeted molecular networking strategy for the colibactin pathway that ties into the existing untargeted networking frameworks and resources . in our modified strategy , we first collect high - resolution untargeted metabolomics data ( ms ) in isogenic wildtype and mutant strains to identify parent ions dependent on the presence of the secondary metabolic pathway , which can be cleanly deleted or inserted into a heterologous host without significant effects to cell growth . then , we run a tandem ms experiment focusing on the fragmentation of only those unique pathway - dependent features for molecular networking . multiple tandem ms datasets can be pooled if needed to enhance overall pathway - targeted network coverage . to illustrate the output of this approach , fig . ( 1 ) compares an untargeted and a pathway - targeted molecular network from e. coli heterologously expressing an example secondary metabolic pathway . we recently applied this approach to the bacterial colibactin pathway found in select strains of e. coli and elsewhere [ 56 - 57 , 62 ] . the result was a focused network map of the colibactin pathway that contained critically important , lower abundance ions that were missed in untargeted fragmentation modes . because pathway intermediates and products inherently share structural similarities , the networks from freshly prepared organic extracts greatly facilitated the structural predictions of colibactin pathway - dependent molecules in the network , some of which were unstable and decomposed during chemical processing , relative to a handful of stable pathway - dependent reference molecules extensively characterized by nmr and/or synthesis . we have found this approach to be more generally applicable in conducting detailed systems - level biosynthetic analyses of secondary metabolic pathways , such as determining genetic mutation consequences at the metabolic network level ( pinpointing bottlenecks in secondary metabolism ) and assessing nrps amino acid substrate specificities at the pathway level using a combination of system - wide isotopic labeling studies and pathway - targeted molecular networking . while pathway - targeted molecular networking has the potential to aid in natural product discovery efforts , there are a few limitations that must be considered . pathway - targeted comparative metabolomics requires the ability to acquire spectra from both a producing and non - producing strain ( comparison of a functionally expressed pathway versus a strain lacking a functional secondary metabolic pathway ) . silent under laboratory growth conditions and that the producing host is genetically tractable , or that the pathway can be transferred and functionally expressed in a heterologous host . despite these limitations , pathway - targeted molecular networking is a useful tool in metabolite discovery efforts and can be coupled to emerging synthetic biology techniques for pathway activation . hydrolytic maturation of secondary metabolites and the incorporation of unexpected amino acids and how pathway - targeted molecular networking has and continues to aid in the elucidation of diverse structures from this important pathway . more and more examples have emerged where sequencing of a biosynthetic gene cluster leads to a predicted biosynthetic code that does not match its expected product . one important mechanism underlying this disconnect is in the hydrolytic maturation of nonribosomal peptides and hybrids thereof , in which a larger precursor is cleaved to form smaller constituents . analogous proteolytic events occur during the maturation of many ribosomally synthesized and post - translationally modified peptides ( ripps ) from larger precursor peptides . an important nonribosomal peptide example is in the biosynthesis of the prototypical monocyclic -lactam antibiotic family , the nocardicins , which are broad spectrum antibacterials first described in the 1970s . when townsend and co - workers reported the biosynthetic gene cluster for nocardicin in 2004 through a structure - guided sequencing approach , they noted that the gene cluster encoded five modules for the predicted production of a pentapetide , but nocardicin a consisted of only a modified tripeptide sequence . the group proposed that either the pathway contained inactive modules , which was the favored mechanism at the time , or engaged in proteolytic processing to explain the discrepancy between the number of modules in the biosynthetic gene cluster and the number of amino acids in the final structure of the mature antibiotic . because a candidate protease was not found in the gene cluster , experimental support for the less favored proteolytic mechanism came later through a model protease cleavage assay and biochemical analysis of individual catalytic domains [ 83 - 84 ] . hydrolytic processing of polyketide synthase / nonri - bosomal peptide synthetase ( pks / nrps ) hybrid products was proposed as the primary route for zwittermicin biosynthesis . the structure of zwittermicin was first described in 1994 , but it was not until the gene cluster was identified in 2009 that it was proposed that zwittermicin is one of two major metabolites cleaved from a larger precursor molecule . this precursor , prezwittermicin a , was proposed to be capped with an n - terminal n - acyl - d - asparagine . a transmembrane peptidase , zmam , which is encoded in the gene cluster , was then proposed to cleave the n - acyl - d - asparagine during export from the cell , releasing mature zwittermicin a. however , the first strong complementary experimental evidence for this maturation mechanism was presented in two related pathways , small molecule structures of prexenocoumacins from the xenocoumacin antibiotic pathway and shortly thereafter an x - ray crystal structure of a peptidase from the colibactin genotoxin pathway like those found in the zwittermicin and xenocoumacin pathways . from the xenocoumacin pathway , deletion of this peptidase led to the characterization of a family of prexenocoumacins that are capped with an n - acyl - d - asparagine . the capped prexenocoumacins exhibited no detectable bioactivity . due to the differences in antibiotic activity between prexenocoumacins and xenocoumacins , the complementary x - ray structure and biochemical analysis of the related peptidase clbp in the colibactin pathway , showed that the transmembrane peptidase was necessary for genotoxicity and was located on the inner membrane facing the periplasm , supporting the cleavage of precolibactins in the periplasm during export . the authors noted the key similarities between the colibactin pathway and the xenocoumacin / zwittermicin pathways , but the structures of precolibactins remained unknown . clbp - dependent n - acyl - d - asparagines , e.g. n - myristoyl - d - asn and clbp precursor analogs , from the colibactin pathway were identified later in accordance with the above biosynthetic logic [ 54 - 56 ] . the function of this n - terminal cap is likely to protect the producing strain from genotoxicity . however , the liberated n - myristoyl - d - asparagine has also been shown to have biological activities in vitro , including weak bacterial growth inhibitory effects against gram - positive bacteria and antagonistic activities against the serotonin-7 receptor and the dopamine-5 transporter . speculatively , these activities may contribute to bacteria - bacteria and/or host - bacteria interactions in the gut . an untargeted network of a single e. coli organic extract ( left ) contains over 1500 molecular features ( mofs ) . endogenous metabolites , media components , as well as metabolites of interest are included in this network . a targeted analysis fragmenting gene cluster - dependent features returns approximately 50 mofs ( right ) with higher pathway coverage . the production of each of these features is dependent on the presence of a gene cluster of interest . nodes are colored according to average ionization intensity , with white nodes being present at low abundance and black nodes being present at high abundance . a variety of other nrps / pks molecules are enzymatically hydrolyzed during maturation . didemnin b is a cyclic depsipeptide that has been investigated for use as an anticancer agent . didemnin x and y contain the core structure of the active didemnin b with an additional n - terminal -hydroxyl - polyglutamine cap . these derivatives were isolated with the didmenin gene cluster in hand , enabling a comparative gene cluster small molecule structural outcome correlation . zeamine is a pks / nrps hybrid molecule that was originally isolated from dickeya zeae . pre - zeamine was isolated from serratia plymuthica and contains a c - terminal pentapeptide that is proposed to be cleaved by a peptidase in the gene cluster . pyoverdine is a fluorescent siderophore produced by psuedomonas aeruginosa that is required for virulence [ 92 - 93 ] . periplasmic proteins are responsible for the maturation of the chromophore as well as cleavage of a myristoyl moiety [ 94 - 95 ] . in vitro reconstitution of the saframycin biosynthetic pathway revealed that it is synthesized with an n - terminal long - chain acyl group . amicoumacin , a compound from b. subtilis that is structurally related to xenocoumacin , also employs a hydrolytic maturation strategy in which an n - terminal acyl - asparagine or acyl - glutamine is removed upon activation [ 98 - 99 ] . as with xenocoumacin , the acylated compound is inactive , while the mature compound is a potent antibiotic . ( 2 ) summarizes currently reported nrps and nrps / pks - derived structures that undergo enzymatic hydrolytic maturation . for a dedicated review of nrps / pks products that mature via enzy- the active cleaved metabolite is shown in black and the leader structural features , which may also have biological activity , are shown in grey . a , the proposed thiazoline and thiazole order and its stereochemistry were predicted by bioinformatics , tandem ms , and isotopic labeling studies , and further experimental evidence is needed . matic hydrolysis , the reader is also directed to a recent review by bode and co - workers . hydrolytic maturation of secondary metabolites has been well established when extending beyond nrps / pks assembly lines . for example , cofactors used in primary metabolism can be incorporated into secondary metabolites and hydrolyzed into smaller structural units . in contrast to the examples discussed above , these compounds retain part of the building blocks incorporated during biosynthesis , rather than using hydrolyzable motifs encoded in thiotemplate assembly line biosynthesis . in thienamycin biosynthesis , notably , the phosphopantethiene arms between coa and pks / nrps carrier proteins are shared , and polar coa analogs produced inside of the cell typically remain sequestered in the intracellular environment . two hydrolases associated with the thienamycin biosynthetic gene cluster are responsible for the stepwise hydrolysis of coa to pantetheine . a third hydrolase processes the carbapenem - pantetheine adduct into thienamycin , which is exported for antimicrobial defense / signaling . in other selected examples , redox - relevant cofactors such as glutathione and mycothiol are also used as sulfur donors in the biosynthesis of secondary metabolites . gliotoxin , an epidithiodiketopiperazine produced by the fungus aspergillus fumigatus , is a nonribosomal peptide virulence factor [ 101 - 102 ] . deletion of individual genes in the gliotoxin cluster revealed that glutathione serves as an unusual sulfur donor . cleavage of the c - s bond and oxidation to the disulfide result in mature gliotoxin . an analogous mechanism has been observed in the biosynthesis of the saccharide antibiotic lincomycin a produced by gram - positive streptomyces . it is intriguing that the latter two examples exploit general cellular toxin detoxification strategies , the thiol nucleophiles glutathione and mycothiol , which typically neutralize electrophilic toxins , for channeling potentially toxic antibiotic intermediates . with genome sequence information now complementing structural characterization efforts , diverse maturation strategies are now emerging as more prominent routes for nonribosomal peptide processing . moving forward , we expect more related nonribosomal peptide maturation strategies to emerge . with further mechanistic characterizations and a better understanding of the processing enzymes , current bioinformatics approaches , which rely on homology to known systems or machine learning algorithms trained on known systems , still fail to identify putative atypical gene clusters . because microbial biosynthetic gene clusters represent chemical traits and are well known to transfer from one organism to another via horizontal gene transfer , genome synteny analyses examining the co - localization of genetic loci in phylogenetically - related organisms continue to provide a promising route for the discovery of atypical natural product enzymes and pathways [ 28 , 106 ] . the continued characterization of atypical secondary metabolic pathways , new biosynthetic enzymes , and new maturation strategies , and their subsequent integration into online bioinformatics programs , such as antismash , is needed . nrpss can sample from about 500 amino acid substrates , which provide a good variety of potential monomer building blocks for secondary metabolite structural diversification . the continued characterization of these building blocks and their associated biosynthetic enzymes will aid in both nonribosomal , and more increasingly , in ribosomal peptide / protein engineering . while the gene cluster for colibactin was described in 2006 , the recently proposed structure of precolibactin a , which accounts for a majority of the enzymatic domains in the biosynthetic pathway , contains an unexpected cyclopropane moiety . this moiety was similarly found in a smaller precolibactin shunt product , where the cyclopropane was proposed to be derived from the amino acid building block 1-aminocyclopropane-1-carboxylic acid ( acc ) [ 57 - 59 ] . in plants , acc is an intermediate in the production of ethylene , a signaling hormone . acc is biosyntheized from s - adenosylmethionine in a plp - dependent manner . in the proposed mechanism , this mechanism is paralleled in the synthesis of coronatine , albeit with a different leaving group . coronamic acid is a cyclopropyl containing intermediate that is produced through a cryptic halogenation event . a carrier protein - tethered isoleucine is chlorinated at the -carbon by an -ketogluterate non - heme fe - dependent oxygenase . deprotonation of the -carbon yields the enolate , which then displaces chloride , forming the cyclopropane moiety . other variations of this mechanism are found in the biosynthesis of , for example , kutzneride 2 and curacin a . none of the previously described mechanisms for the biosynthesis of cyclopropane substructures readily appear to be encoded in the colibactin gene cluster . isotope labeling studies indicate that the four carbons ( aminobutyryl ) are derived from methionine [ 57 - 58 ] , which was also previously observed in the biosynthesis of the cytotrienin cyclopropyl moiety . since acc is derived from methionine in bacteria , it is possible that free acc is loaded onto the carrier protein ; however , feeding studies with free deuterated acc showed no detectable incorporation of the free amino acid in bacterial cell cultures . one nrps module with an unusual architecture is involved in the production of the spirobicyclic structural feature [ 57 - 58 ] . in contrast to canonical nrps modules that contain a condensation domain , ( c ) , followed by an adenlyation domain , ( a ) , and then a thiolation domain , ( t ) , this particular protein has the architecture a - c - a - t . the second a - domain was speculatively proposed to potentially convert a carrier protein - tethered met into sam for cyclization . exactly how isotopically labeled met is processed to the acc - derived feature in colibactin remains a subject of current investigation . it is not without precedent , however , where nrps domains directly catalyze the formation of ring - strained units , such as in the biosynthesis of the -lactam nocardicin . there , the condensation domain catalyzes the condensation with the upstream intermediate as well as -lactam formation . more recent studies on clbh indicate that the a1 protein domain fragment activates serine in vitro as predicted by bioinformatics . free - standing carrier protein clbe accepts this substrate in vitro , which is further oxidized to -aminomalonate ( detected as its decarboxylation product ) by isolated dehydrogenases clbd and clbf . clbg and clbo , a discrete acyltransferase and pks module , respectively , which are currently unaccounted for in colibactin biosynthesis , might be available for incorporation of this rare extender unit . -aminomalonate extender units can also be found in the zwittermicin , guadinomine , and lumiquinone antibiotics . these genes are necessary for bacterial cells harboring the colibactin pathway to initiate mammalian cell dna damage , suggesting that functional ( pre)colibactin derivatives in the bacteria - host interaction may incorporate an -aminomalonate substrate . however , suggestions have been made for clbl s potential involvement in a second cleavage event based on gene deletion analysis . nucleophilic positions on guanine or adenine can attack the ring , particularly when the ring is positioned in conjugation with an --unsaturated carbonyl , leading to dna alkylation . for example , in duocarmycin , upon binding dna , a conformational change positions the cyclopropane for attack by the n3 of adenine . however , the proposed alkylated ring - opened intermediate contains a michael acceptor ( or an analogous conjugated iminium acceptor ) ( fig . this could allow the second strand of the duplex dna to attack , forming an interstrand crosslink between the two strands of dna . a model precolibactin shunt product , which served as a mimic of the open chain product , was shown to exhibit weak dna interstrand crosslinking activity in in vitro assays , supporting this notion . based on this data , we proposed several possible modes of action for colibactin toxicity ( open chain and closed chain molecules ) , which are shown in ( fig . the balskus group identified the same model compound and subjected it to a clbp protease cleavage assay in lysogeny broth growth conditions ( lb , 10 g / l nacl ) . a mass consistent with cl addition to the cyclopropyl moiety of the closed - chain structure based on this data , they similarly proposed a colibactin activity consistent with dna alkylation ( fig . , we did not detect cl adducts , but we could see masses consistent with model colibactin cleavage products in both the open - chain ( primary amine ) and closed - chain ( imine ) forms , from freshly prepared organic extracts . alkylation and crosslinking activities can result in mutagenesis , activation of apoptotic pathways , and downstream dna double strand breaks . in considering the above possible modes of action , several structural and biosynthetic features need to be considered . as discussed earlier , during export out of the cell , precolibactins are cleaved releasing a primary amine . the amine would largely be protonated under physiological conditions , and may help colibactin bind dna . alternatively , the amine could undergo an intramolecular cyclization , forming a reversible cyclic imine ( fig . the calculated pka for its conjugate acid iminium species is predicted to be approximately 5 . with a nuclear ph of 7.2 , less than one percent of the reactive iminium while its neutrality may reduce inter- the colibactin warhead contains a ring - strained cyclopropane . nucleophilic attack by dna could lead to opening of the ring and dna - alkylation ( a ) . cleavage of precolibactin liberates a primary amino group , which may be involved in dna binding or modulating the warhead activity by forming a cyclic imine ( bottom ) . a michael addition ( or analogous iminium addition ) into the alkylated warhead could result in a dna - interstrand crosslink ( b ) . formation of alternative cyclization products ( pyridones ) could compete with the five - membered cyclic imine cyclization route . no biological activities have yet to be reported for the stable pyridone - containing molecules ( c ) . action with the dna phosphate backbone , it may alternatively participate in dna binding , and transient protonation ( or dna binding - induced protonation , pka perturbation ) of the imine may serve to activate the cyclized warhead and promote attack by dna . additionally , a cyclopropane - containing metabolite arising from an alternative cyclization mode featuring a pyridone scaffold was recently proposed from the colibactin pathway by ms ( fig . 2 and fig . this compound was isolated in low yields ( 0.1 mg/ 200 l ) from a clbp strain overexpressing the colibactin pathway . in a wildtype strain , protease clbp would cleave the n - acyl - d - asn moiety , and consequently , two competing cyclization routes can explain the structural differences ( fig . 4 ) . as no functional data was reported for the pyridone - containing colibactin metabolites , it is currently unclear if these molecules are stable shunt metabolites or advanced biosynthetic products ( i.e. , precolibactin b ? ) . precolibactin a has a predicted thiazolinyl- and thiazole containing moiety that presumably participates in dna binding and poises the clbp - cleaved warhead ( open- or closed - chain ) for electrophilic attack . a bithiazole - containing structure from the colibactin pathway has also been proposed ( fig . bleomycin , an antitumor compound produced by streptomyces , contains a c - terminal bithiazole and is capped with a c - terminal cationic group , whereas phleomycin contains a thiazolinyl - thiazole moiety similarly capped with a cationic group . these structural motifs are required for the action of bleomycin and phleomycin , presumably by mediating dna binding . while the diverse colibactin products proposed to date may have distinct activities , further mechanistic studies are required to assess the dominant molecular route(s ) for colibactin genotoxicty in mammalian cells . the continued expansion of microbial genome sequence information has provided enormous promise of much more to come in novel biosynthesis and secondary metabolite discovery . based on an overabundance of historical precedence , novel structural scaffolds to come will continue to serve as leads for the development of new - in - class ( ant)agonists , molecular probes , and pharmaceuticals . discovery and characterization of novel biocatalysts will continue to expand our arsenal of biocatalytic reactions while assigning functions to the unannotated majority , hypothetical proteins . multidisciplinary and systematic genes - to - molecules approaches have been effective at aiding in these overall efforts . in particular , molecular networking has provided a route to begin to overcome current metabolite database inadequacies , especially for groups focused on novel secondary metabolite discovery from functional or activated pathways . pathway - targeted molecular networking , as highlighted for the genotoxic colibactin pathway , enables a detailed systems biosynthesis - level view of a targeted secondary metabolic pathway within a complex metabolome , and ties into existing molecular networking workflows . the colibactin pathway provides a nice example of the sequence first , structures to follow paradigm in the human microbiome . deciphering the unexpected structural and functional outcomes for the colibactin pathway and determining its mode of action in vivo remain subjects of rigorous inquiry .

adult intussusception ( ai ) in the western countries is usually caused by tumors and requires resection . ( 1 ) in africa and other tropical locations , ai is frequently due to other causes . ( 210 ) . the purpose of this case report of ai , encountered by the authors ( rsg and alk ) during a surgical mission to the democratic republic of congo ( drc ) and review the literature , serves to emphasize these differences . a 35-year - old woman gravida 11 para 10 was referred to the maternity department of a district hospital in eastern drc , with intermittent dull lower abdominal pain . she reported no vomiting or nausea , no burning or discharge on urination and normal stools . her past medical history was significant for partial thickness burns to her chest when she was 4-years - old and recent treatment with albendazole for abdominal discomfort secondary to possible ascaris infection . her abdomen was soft , non - distended , with no palpable masses but with suprapubic tenderness without rebound or guarding . the available laboratory tests revealed a hemoglobin level of 12.6 g / dl , blood group b+ , and a positive urine pregnancy test . an abdominal ultrasound revealed minimal fluid in the pouch of douglas , no intra abdominal masses and no evidence of an intrauterine pregnancy . cervicitis , salpingitis and tubal or extra uterine pregnancy were considered and since she was stable and had only mild symptoms , it was initially elected to observe her while treating her with intravenous fluids and antibiotics . after one day her abdominal pain increased and a right para - umbilical mass was noted . after reduction of the intussusception the bowel appeared viable but a cecal mass was identified and a right hemicolectomy was subsequently undertaken . upon transection of the bowel , a 22x1 cm smooth sub mucosal mass was noted in the cecum opposite the ileo - cecal valve . bisection of the mass showed collagen - like tissue , but no definite histology was possible to obtain . antibiotics for treatment of cervicitis were continued while on follow up the urine pregnancy test became negative and a course of albendazol was repeated . ai in the developed world has a high association with malignancy and resection is recommended . ( 1 ) in africa and other tropical locations a variants of ai of unknown etiology has been reported . ( 210 ) cook states in his review on colonic intussusception : although children may be afflicted , the vast majority of patients affected are adults . the aetiology of this condition , as with volvulus , is unclear ; while intestinal polyps or amoebomas account for a minority , there is no obvious clue in most cases . gangrene is about three times more common with the ileo - ileal and ileo - caecal varieties compared with the caeco - colic type ( 2 ) in a review , vanderkolk et al showed that ceco - colic intussusception was the most common cause of intestinal obstruction at a district hospital in rwanda . of the 43 patients with the authors hypothesized , that the etiology of ai was irritation from secretions or diet . they recommended simple reduction by careful milking of the bowel in much the same manner as pediatric cases and resection only for gangrenous bowel . ( 3 ) cole , in 1966 , described similar ceco - colic variant of ai in 100 cases of intussusception . ( 4 ) however , a later review from the same area ( 2002 ) suggested that the pattern of obstruction had changed considerably from that previously described by cole . these authors concluded that a change to a more western lifestyle , particularly diet , may have altered the prevalence of this condition . ( 5 ) other authors , from haiti , sri lanka and papua new guinea , described a similar , so called tropical intussusception . ( 6,7,8 ) greco and lepreau reported 15 cases from haiti , where there was a high incidence of gangrenous bowel , requiring resection in almost all patients . ( 6 ) rasaretnam presented a series of 76 cases seen between 1964 and 1974 in sri lanka and also recommended resection . ( 7 ) melcher and safadi suggested that that 40 - 50% of ileo - colic intussusception in adults was due to a malignancy . ( 1 ) cotton , a general surgeon in zimbabwe , in response to that conclusion asserted that enteric intussusception in adults in africa was a relatively common condition and instead of being associated with a malignancy was usually associated with lymphadenopathy from various causes . he concluded that ai was recognized as a presenting complication of human immunodeficiency virus disease ( hiv ) , and not infrequently found as a result of ileo - cecal tuberculosis . ( 9 ) although a growing literature links intussusception to hiv , most of the reports focus on ai of the small intestine rather than on the ileo - colic or cecal - colic variety . ( 10 ) given the worldwide prevalence of hiv , but particularly in africa , and its relation to ai , one could argue for testing all such patients for hiv ; we could not do so in our patient since the testing and therapy was not available . it is most likely that our patient developed an adult intussusception secondary to a cecal lipoma as the lead point . the cervicitis and ascaris infestation were probably not related to the new onset of this ai . the value of presenting this case is to make other surgeons aware of the complex differential diagnosis of ai in africa , which differs from that found in the western countries .

necrotic tissues , bacteria , and biofilms have been identified , along with coronal leakage , vis - a - vis poor restorations , recurrent caries , tooth fractures , or extensive periodontal disease , as the causes for persistent periapical disease following root canal treatment . elimination of these etiologies is essential to reestablish an environment conducive to repairing and healing . this implies that if nonsurgical revision is the treatment of choice then not only must these etiologic factors be removed , but also the filling material present in the root canal system must be eliminated [ 2 , 3 ] . the literature is replete with studies that have discussed the techniques for removal of the causative factors for persistent periapical disease [ 1 , 4 ] . the ability to remove root filling materials is oftentimes most difficult due to anatomical constraints that may prevent thorough cleaning . while a wide range of anatomical complexities may be encountered during root canal retreatment / revision procedures , including fins , webs , cul - de - sacs , isthmuses , ribbon- and dumbbell - shaped canals , dilacerations , and c- and s - shaped canals , the most commonly encountered anatomical challenge may be the curved canal . furthermore , clinicians often forget that even though the roots may appear straight on a radiograph , curvatures in the third dimension are quite common . the main concern with the removal of filling materials from curved root canals is using instruments with shape memory ( nickel titanium niti ) without altering the integrity of the root canal walls in an adverse way . this is of special importance knowing that canal curvatures are widely variable in all dimensions [ 6 , 7 ] . canal curvatures exceeding 30 lead to complications in root canal preparation and cases are considered more complex . morphology of curved root canals is of great importance in determining the outcome of instrument application , and , in cases of retreatment , inability to remove material adequately from the root canal can invite repeated failures . specific rotary niti file designs for root filling material removal are the protaper universal rotary retreatment system ( ptus , dentsply maillefer , ballaigues , switzerland ) and the r - endo ( micro - mega , besanon , france ) . the manufacturers claim that these systems , in addition to shaping and finishing the root canal , are also effective in the removal of the root filling material from root canals . both systems have been evaluated as to their ability to remove the previous root filling materials and retained debris from canals systems , with neither system demonstrating 100% effectiveness [ 3 , 811 ] . from a general perspective , most rotary instrument techniques that are designed specifically for the removal of root filling materials will benefit somehow from the use of heat , which they can generate during applications , or solvents that are added to the canal for ease of initial penetration , although variations in outcomes have been noted and the outcomes are inconclusive [ 1214 ] . while one study showed that niti rotary instruments removed more gutta - percha when used with a solvent , another study found no difference when the same technique was used to remove gutta - percha with and without chloroform . during the use of heat or solvents , an amorphous melt or mixture of the filling material is produced that can be pushed even further into unclean canal irregularities or into the dentinal tubules . this may then require a greater removal of dentin to remove filling materials from within the tubules and enhance the cleanliness of the canal walls [ 14 , 17 ] . this may result in weakened root canal walls in the apical third of the canal . regardless of the technique used , thorough debridement has not shown to be achieved [ 19 , 20 ] . here again , operator influence on the outcomes has not been considered when performing these technical evaluations , especially when studied in curved canals . while studies have been performed that address the removal of gutta - percha and zinc - oxide - eugenol and resin sealers using newer niti rotary systems , these were done primarily on teeth with straight roots [ 8 , 1416 , 2123 ] . though few studies have addressed the removal of root filling material from curved canals [ 24 , 25 ] , the ability of instruments to remove resin sealers has not been evaluated thus far . this is clinically important considering the fact that epoxy resin sealers are the most commonly used in contemporary endodontics and that they set to a hard mass . it was the aim of this study to evaluate the cleanliness of root canal walls after retreatment using two root canal files or systems specifically designed for retreatment and compare the outcomes to the use of hedstrm files in severely curved single - rooted human teeth , obturated with gutta - percha and an epoxy resin or zinc - oxide - eugenol sealer . the null hypotheses in this study were as follows : ( a ) there is no difference between the file systems in removal of the root filling materials and ( b ) there is no influence of the type of sealer in terms of susceptibility to be removed by the three file systems . human single - rooted mandibular first premolars ( n = 90 ) were collected and thoroughly cleaned by removing the hard deposits using curettes and the soft deposits by soaking in 5.25% naocl for 5 minutes . the specimens were scanned by a cone - beam computed tomography ( cbct ) scanner ( 3d accuitomo , j. morita corporation , osaka , japan ) , and only teeth with root curvatures between 40 and 45 degrees radius of curvature < 10 mm were included . the teeth were decoronated at the cementoenamel junction using a diamond disc , under water cooling . working length was established using size 10 k - file ( mani inc , tochigi , japan ) to the root canal terminus and subtracting 0.5 mm from this measurement . all procedures were performed by a single trained and calibrated operator who demonstrated the same level of proficiency in all the techniques of instrument application for retreatment purposes . the root canals were instrumented using nickel titanium rotary instruments ( mtwo , vdw gmbh , munich , germany ) up to size number 25 , 0.07 taper . irrigation was performed with 3% sodium hypochlorite , using a 5 ml disposable plastic syringe with a polypropylene capillary tip ( ultradent products inc . , south the tip was placed passively into the canal , up to 2 mm from the apical foramen without binding . all root canals were irrigated with 5 ml of 17% edta ( pulpdent , mass , usa ) for 1 minute to remove the smear layer and then rinsed with 5 ml of distilled water . the roots were randomly divided into two groups ( n = 45 ) with the aid of a computer algorithm ( http://www.random.org/ ) , based on the material used for obturation : group 1 , obturated with gutta - percha and a zinc - oxide - eugenol sealer ( pulp canal sealer , sybron endo , calif , usa ) ; group 2 , obturated with gutta - percha and an epoxy resin sealer ( ah plus , dentsply detrey , gmbh , germany ) . the root canals were dried with sterile paper points and obturated with gutta - percha and sealer using continuous wave of warm gutta - percha technique . a system b compactor 0.04 taper tip size 30 ( analytic technology , redmond , wash , usa ) was inserted 3-mm short of the working length , with the unit set at 200c and power 10 . after searing off the points at the canal orifices , the activated compactor was pushed apically into the gutta - percha until just short of the premeasured length . the compactor was seated to length without heat and apical pressure was maintained for approximately 10 s. a second burst of heat was used to remove the compacting instrument . backfilling was performed by injecting thermoplasticized gutta - percha ( obtura ii , obtura corp , fenton , mo , usa ) . the teeth were radiographed ( dsx 730 , owandy dental imaging , champs sur marne , france ) at different angulations to verify the quality of filling procedure . following 1 month of storage , the specimens of each group were randomly subdivided into three subgroups ( n = 15 ) based on the technique of retreatment . specimens of subgroup a were retreated using a combination of gates glidden drills numbers 3 and 4 ( mani inc , tochigi , japan ) in the coronal and middle thirds up to 7 mm in depth , and the remaining portion of the canal was cleaned with h files ( mani inc , tochigi , japan ) in a crown - down fashion up to an apical size of 30 . the canals were instrumented in a crown - down sequence using protaper d1 and d2 files to remove the root filling material . the debris was rinsed from the canal with 1% sodium hypochlorite . in subgroup c , r - endo instruments ( rm , re , r1 , r2 , r3 ) were used in a gentle in - and - out motion on canal walls according to the manufacturer 's instructions . a manual file was used first to relocate the canal orifices , then the re instrument removed the first 2 - 3 mm of the filling . r1 and r2 were used on one - third and two - thirds of the estimated working length , respectively . finally , r3 was used at the working length to complete the removal of filling material from the root canal , and the rs file was used at full working length to finish the preparation . all instruments were used only for one specimen , and removal of filling materials was judged complete when the working length was reached , and no more gutta - percha could be seen on the last instrument used . all the teeth were grooved buccolingually with a diamond disc just enough to weaken the tooth to be split into longitudinal sections with a chisel . during the grooving , both the access opening and apical foramen were covered with sticky wax to prevent any debris that may have been generated during the grooving process from getting into the canal and contaminate the root walls . both halves of the root canal were photographed ( nikon coolpix 4500 ; nikon , melville , ny , usa ) under a stereomicroscope at 40x magnification and analyzed with autocad 2007 software ( autodesk inc . , san rafael , calif , usa ) . the evaluation of coded specimens was performed by 2 operators blinded to the techniques and the devices used for retreatment . the arithmetical means of the area of the canal and remaining gutta - percha and sealer ( in millimeters ) , obtained by the 2 operators , were used to measure the percentage of remaining filling materials for all specimens . the intraclass correlation coefficient was calculated to estimate the reliability of the measurements recorded by the 2 examiners . the percentage of remaining filling material and the mean time of gutta - percha removal were evaluated for each group . the values were compared statistically by one - way anova with bonferroni adjustment for multiple comparisons , considering p = 0.05 as the level of significance . the mean percentage of filling material of the retreatment protocols in the different root thirds was tabulated ( table 1 ) , and the value of the intraclass correlation coefficient was very high ( = 0.99 ) . the apical third of roots obturated with group ii ( gp / ah plus ) and retreated with subgroup a ( h files ) showed significantly higher ( p < 0.05 ) percentage of filling material ( 65.50 1.17 ) ( figure 1 ) . the least percentage of filling material was found in the middle third of roots obturated with group i ( gp / zoe ) and retreated with subgroup c ( r - endo ) ( 13.93 0.94 ) ( figure 2 ) , although this was not significantly different from the coronal and middle third subjected to the same protocols ( p > 0.05 ) . analysing the percentage of root filling material considering root thirds as variable , the apical third always had significantly more material than the middle third and coronal third ( p < 0.05 ) , except in group i subgroups b and c. disregarding the root third as grouping variable , the r - endo system performed significantly better than the other two file systems ( p < 0.05 ) . the present study determined the efficacy of two rotary retreatment systems in comparison with hedstrm files to remove gutta - percha obturated in conjunction with an epoxy resin or zinc - oxide - eugenol - based sealer . while studies using niti instruments have shown the ability to clean and shape curved root canals with a reasonable degree of safety , thereby preventing or minimizing ledging , transportation , and zipping , their use in the removal of root filling materials and biologic debris has not been investigated extensively [ 27 , 28 ] . although newer instruments have been introduced for the specific removal of gutta - percha , sealer , core carriers , and paste fillings , a thorough evaluation of their efficacy in canal debridement and cleaning is lacking in two particular areas : ( i ) their use in curved canals and ( ii ) their use relative to the calibration and standardization of the clinician or clinicians who are evaluating the procedures . only too often studies cite that a specific instrument or technique has failed to achieve an objective , when the application of a specific instrument and the outcome were totally operator dependent . historically and contemporarily , techniques used to remove root canal filling materials have included the use of hand ( k - files and hedstrm file ) or rotary instruments ( gates glidden burs , peeso reamers , and niti instruments ) with or without the use of heat , solvents , and/or ultrasonic applications [ 10 , 2931 ] . the use of hand files for root canal retreatment has generally been found to be less effective in canal cleaning procedures [ 10 , 32 ] , although some deviations from this finding have been noted with the removal of gutta - percha and synthetic polymer - based materials , such as epiphany / resilon ( resilon research llc , madison , conn , usa ) or endo rez ( ultradent products inc , south jordan , utah , usa ) [ 25 , 33 ] , and when evaluated using a dental operating microscope . before the advent of niti rotary instruments and the development of specific niti instruments for root canal retreatment , the use of gates glidden burs and peeso reamers was commonplace in the removal of gutta - percha , sealers , and pastes . even recent studies have focused on this approach with variable outcomes being reported [ 31 , 35 ] . the ptus has three files , each of different lengths , tapers , and apical tip diameters . d1 ( length = 16 mm , tip diameter = 0.30 mm , 9% taper ) , d2 ( length = 18 mm , tip diameter = 0.25 mm , 8% taper ) , and d3 ( length = 22 mm , tip diameter r = 0.20 mm , 7% taper ) serve to remove filling material from the coronal , middle , and apical root thirds , respectively . the d1 instrument serves to flare the canal walls and has an active tip to facilitate initial penetration into the filling material . the manufacturer recommends that d1 and d2 be used in the coronal and middle thirds , respectively , while d3 should be used to full working length . in the present study , d2 was used up to full working length because the canal was initially prepared to an apical size of 25 with mtwo instruments . the r - endo files are a system of four files re ( size 25 , 0.12 taper ) which serves to flare the first few millimetres of the canal , while the other three files r1 , r2 , and r3 ( size 25 with 0.08,0.06 , and 0.04 tapers , resp . ) are dedicated to the coronal , middle , and apical root thirds , respectively . the results of the present study support the results of previous studies in that no retreatment instrumentation protocol is able to completely remove the root filling material [ 810 , 13 , 16 , 21 ] . though the protaper universal system was more effective than hedstrm files in removal of root filling material , it was less effective than the r - endo system . while the cross - sectional design of the ptus files favors removal of large amounts of gutta - percha in spirals around the instruments , the same cross - sectional design and the high - centering ability prevent it from contacting all the walls of the root canals , thereby deterring complete removal of filling material from the root canals [ 10 , 22 , 36 ] . the r - endo files , on the other hand , have a triangular cross - section with three equally spaced cutting edges and no radial land . these results are in contrast to earlier reports where r - endo was compared with ptus or h files . however , this difference in results may be attributed to the use of solvents in those reports . solvents may soften the root filling material and thereby compact the material into the irregularities along the root canal wall and dentinal tubules , after which removal may not be possible . the method used to evaluate the filling remnants plays an important role on the results obtained in each study . since radiographs are limited to two dimensions , longitudinal cleavage of roots was performed to observe residual remnants on the root canal walls . in order to measure the remnants , commercial software was used to calculate the dentinal wall surface and sealer and gutta - percha remnants . in general all systems were able to remove gutta - percha obturated with zinc - oxide - eugenol more than gutta - percha with resin sealer . furthermore , the presence of gutta - percha remnants was higher in the apical third of the root for all three groups . this finding suggests the necessity to increase the size of the apical preparation when rotary instruments are used . however , the remaining gutta - percha in the apical third was significantly less in the r - endo group than ptus and h - files . this may be because of the increased tip diameter of the rs file ( 0.30 mm ) as compared to the d2 instrument ( 0.25 mm ) . furthermore , there is evidence to show that ptus and h files may cause damage to the root canal dentin during retreatment and that the debris extruded by ptus during retreatment is less than that by h files . future studies should focus on comparing the effects of different rotary retreatment systems on damage to root dentin and debris extrusion when used in severely curved canals . this in vitro study showed that none of the systems were able to completely remove filling material from the root canals . more specifically , application of the r - endo system resulted in less percentage of root filling material on the walls when compared to the protaper retreatment system and hedstrm files ; the residual root filling remnants were greater when samples where obturated with an epoxy resin sealer ; the apical third always had more filling remnants compared to the coronal and middle thirds .

motor unit number estimation ( mune ) was originally described by mccomas et al . over three decades ago . the original technique was a modification of the compound muscle action potential ( cmap ) recording technique that employed a gradual increase of stimulation to obtain submaximal increments . these increments were summed and averaged to determine an estimated size of a single motor unit potential ( smup ) . this size was divided into the cmap response to estimate the number of motor units innervating the muscle being tested . following the original description , numerous variations using both electrophysiological responses and incremental force ( mechanical ) measurements have been used in both human studies and animal models . the mune technique was modified by shefner and colleagues to investigate mouse models of amyotrophic lateral sclerosis ( als ) . in the current description , experienced individuals can perform these measures in 1020 min per animal , and repeated measures are feasible which permits the acquisition of longitudinal data . in the current studies , , clinical electrodiagnostic systems are optimized for rapid and efficient capture of electrophysiological data in vivo , nevertheless standard electrophysiological rigs can easily be adapted for this application . this protocol was approved by and adheres to the animal care and ethics guidelines of the ohio state university wexner medical center . following induction of anesthesia , maintain anesthesia at 23% and 1 l per minute o2 flow rate . adjust o2 flow and isoflurane percentage for adequate anesthesia according to animal s disease state , age , and respiration rate . smaller or weaker animals may require less isoflurane for adequate anesthesia ( i.e.1.52.5% isoflurane).confirm adequate anesthesia by applying light hindlimb footpad pressure with an object such as forceps to demonstrate a lack of withdrawal response . adjust o2 flow and isoflurane percentage for adequate anesthesia according to animal s disease state , age , and respiration rate . smaller or weaker animals may require less isoflurane for adequate anesthesia ( i.e.1.52.5% isoflurane ) . confirm adequate anesthesia by applying light hindlimb footpad pressure with an object such as forceps to demonstrate a lack of withdrawal response . maintain temperature at 37 c surface temperature with a thermostatic warming plate as variation in temperature can affect cmap size and duration . apply veterinarian petroleum - based ointment to eyes to prevent dryness . monitor level of anesthesia observing respiration rate and assessing for withdrawal responses following pressure applied to the foot pad via forceps . after removal of hair from the hindlimb(s ) to be studied , lightly extend the hindlimbs at the knee , abduct at the hips and affix to the working surface using adhesive tape ( as shown in figure 1 ) . following the cmap and mune recordings and discontinuation of anesthesia , do not leave animal unattended until it has regained sufficient consciousness to maintain sternal recumbency . place the active ( e1 ) ring electrode on the skin overlying the proximal portion of the gastrocnemius muscle of the hind limb , at the knee joint , and the reference ( e2 ) ring electrode on the skin over mid - metatarsal portion of the foot.in order to reduce impedance , coat the skin underlying the ring electrodes with gel to sufficiently saturate residual hair and maximize electrode - skin contact . avoid excessive application of electrode gel as this may cause an electrical bridge between electrodes and could prevent accurate recording . place the active ( e1 ) ring electrode on the skin overlying the proximal portion of the gastrocnemius muscle of the hind limb , at the knee joint , and the reference ( e2 ) ring electrode on the skin over mid - metatarsal portion of the foot . in order to reduce impedance , coat the skin underlying the ring electrodes with gel to sufficiently saturate residual hair and maximize electrode - skin contact . avoid excessive application of electrode gel as this may cause an electrical bridge between electrodes and for stimulation of the sciatic nerve at the proximal hind limb , use two insulated 28 g monopolar needles as the cathode and anode . insert the cathode at the region of the proximal hind limb and insert the anode more proximally in the subcutaneous tissue overlying the sacrum . avoid inserting the stimulating electrodes overly close to the sciatic nerve or too deep that it would directly injury the sciatic nerve or other structure . avoid inserting the stimulating electrodes overly close to the sciatic nerve or too deep that it would directly injury the sciatic nerve or other structure . figure 1 illustrates electrode placement . for the ground electrode , place a disposable surface electrode on the contralateral hind limb or tail . sciatic cmap obtain sciatic cmap responses by stimulating the sciatic nerve with square - wave pulses of 0.1 ms duration and intensity ranging from 110 ma.acquire cmap responses with increasing stimulus intensity until the amplitude of the response no longer increases . then , in order to ensure supramaximal stimulation , increase the stimulation to ~120% of the stimulus intensity utilized to obtain a maximal response and obtain an additional response . if there is no further increase in the cmap size , record this response as the maximal cmap.record baseline - to - peak and peak - to - peak cmap amplitudes in mv ( figure 2 ) . obtain sciatic cmap responses by stimulating the sciatic nerve with square - wave pulses of 0.1 ms duration and intensity ranging from 110 ma . acquire cmap responses with increasing stimulus intensity until the amplitude of the response no longer increases . then , in order to ensure supramaximal stimulation , increase the stimulation to ~120% of the stimulus intensity utilized to obtain a maximal response and obtain an additional response . if there is no further increase in the cmap size , record this response as the maximal cmap . record baseline - to - peak and peak - to - peak cmap amplitudes in mv ( figure 2 ) . average single motor unit potential ( smup ) size and mune calculation determine the average single motor unit potential ( smup ) size with an incremental stimulation technique . to obtain incremental responses , deliver submaximal stimulation of 0.1 ms duration at a frequency of 1 hz while increasing the intensity in 0.03 ma steps to obtain the minimal all - or - none responses . if the initial response does not occur with stimulus intensity between 0.21 ma and 0.70 ma , adjust the stimulating cathode position either closer or farther away from the position of the sciatic nerve in the proximal thigh to decrease or increase the required stimulus intensity , respectively.if the initial incremental response is obtained with a stimulus intensity between 0.21 ma and 0.70 ma and fulfills the criteria noted below ( 3.2.2 ) , store and record additional increments with increasing stimulus intensities adjusting by steps of 0.03 ma to obtain a total of 9 additional increments that meet the established criteria . determine the average single motor unit potential ( smup ) size with an incremental stimulation technique . to obtain incremental responses , deliver submaximal stimulation of 0.1 ms duration at a frequency of 1 hz while increasing the intensity in 0.03 ma steps to obtain the minimal all - or - none responses . if the initial response does not occur with stimulus intensity between 0.21 ma and 0.70 ma , adjust the stimulating cathode position either closer or farther away from the position of the sciatic nerve in the proximal thigh to decrease or increase the required stimulus intensity , respectively.if the initial incremental response is obtained with a stimulus intensity between 0.21 ma and 0.70 ma and fulfills the criteria noted below ( 3.2.2 ) , store and record additional increments with increasing stimulus intensities adjusting by steps of 0.03 ma to obtain a total of 9 additional increments that meet the established criteria . if the initial response does not occur with stimulus intensity between 0.21 ma and 0.70 ma , adjust the stimulating cathode position either closer or farther away from the position of the sciatic nerve in the proximal thigh to decrease or increase the required stimulus intensity , respectively . if the initial incremental response is obtained with a stimulus intensity between 0.21 ma and 0.70 ma and fulfills the criteria noted below ( 3.2.2 ) , store and record additional increments with increasing stimulus intensities adjusting by steps of 0.03 ma to obtain a total of 9 additional increments that meet the established criteria . during measurements of the incremental responses , ensure that the initial negative peak of the incremental responses is aligned temporally within the negative peak of the maximal cmap response shown as the shaded portion of the cmap illustration in figure 2.ensure that each incremental response is stable and without fractionation , established by observing three duplicate responses . distinguish visually incremental responses in real - time ( superimposed on the previously recorded increments).note : each increment should be visually distinct and larger compared with the preceding response ( figure 3 ) . analysis in real - time allows recognition of larger amplitude ( incremental ) responses compared to prior responses , and small changes attributable to background noise can be disregarded . a superimposed view of 10 increments is shown in figure 4 ( b and d ) to further illustrate this point.after visually confirming each increment , ensure that the measured amplitude difference ( confirmed response - amplitude of the prior response = amplitude difference ) is at least 25 v.if the increment is less than 25 v , discard and re - measure the response . after recording 10 incremental responses , assess the increments to ensure that the amplitude of each individual incremental response is not greater than 1/3 of the sum of all ten increments ( i.e. the total amplitude of the final response ) . ensure that the initial negative peak of the incremental responses is aligned temporally within the negative peak of the maximal cmap response shown as the shaded portion of the cmap illustration in figure 2 . ensure that each incremental response is stable and without fractionation , established by observing three duplicate responses . distinguish visually incremental responses in real - time ( superimposed on the previously recorded increments ) . note : each increment should be visually distinct and larger compared with the preceding response ( figure 3 ) . analysis in real - time allows recognition of larger amplitude ( incremental ) responses compared to prior responses , and small changes attributable to background noise can be disregarded . a superimposed view of 10 increments is shown in figure 4 ( b and d ) to further illustrate this point . after visually confirming each increment , ensure that the measured amplitude difference ( confirmed response - amplitude of the prior response = amplitude difference ) is at least 25 v . if the increment is less than 25 v , discard and re - measure the response . after recording 10 incremental responses , assess the increments to ensure that the amplitude of each individual incremental response is not greater than 1/3 of the sum of all ten increments ( i.e. the total amplitude of the final response ) . average the 10 incremental values to give an estimation of the average single motor unit potential ( smup ) amplitude ( figure 3 ) . note : figure 3 details the basis of the average smup calculation , but the average smup amplitude can be simply calculated by dividing the entire amplitude of the final incremental response by the total number of increments ( i.e. , 10 ) . example individual smup calculations ( illustrated in figure 3 ) : smup 1=peak - to - peak amplitude of increment 1smup 1=0.050 mvsmup 2= ( peak - to - peak amplitude of increment 2 ) ( peak - to - peak amplitude of increment 1)smup 2=0.150 mv-0.050 mv=0.100 mv calculate each subsequent increment ( up to a total of 10 ) , and make an average of the ten increments . smup 1=peak - to - peak amplitude of increment 1 smup 2= ( peak - to - peak amplitude of increment 2 ) ( peak - to - peak amplitude of increment 1 ) smup 2=0.150 mv-0.050 mv=0.100 mv calculate each subsequent increment ( up to a total of 10 ) , and make an average of the ten increments . calculate each subsequent increment ( up to a total of 10 ) , and make an average of the ten increments . calculate mune by dividing the maximum cmap amplitude ( peak - to - peak ) by the average smup amplitude ( peak - to - peak ) . when necessary , convert cmap and smup results to similar units prior to mune calculation . wear gloves while handling mice . anesthetize mice with inhaled isoflurane and place in the prone position . following induction of anesthesia , maintain anesthesia at 23% and 1 l per minute o2 flow rate . adjust o2 flow and isoflurane percentage for adequate anesthesia according to animal s disease state , age , and respiration rate . smaller or weaker animals may require less isoflurane for adequate anesthesia ( i.e.1.52.5% isoflurane).confirm adequate anesthesia by applying light hindlimb footpad pressure with an object such as forceps to demonstrate a lack of withdrawal response . adjust o2 flow and isoflurane percentage for adequate anesthesia according to animal s disease state , age , and respiration rate . smaller or weaker animals may require less isoflurane for adequate anesthesia ( i.e.1.52.5% isoflurane ) . confirm adequate anesthesia by applying light hindlimb footpad pressure with an object such as forceps to demonstrate a lack of withdrawal response . maintain temperature at 37 c surface temperature with a thermostatic warming plate as variation in temperature can affect cmap size and duration . apply veterinarian petroleum - based ointment to eyes to prevent dryness . monitor level of anesthesia observing respiration rate and assessing for withdrawal responses following pressure applied to the foot pad via forceps . after removal of hair from the hindlimb(s ) to be studied , lightly extend the hindlimbs at the knee , abduct at the hips and affix to the working surface using adhesive tape ( as shown in figure 1 ) . following the cmap and mune recordings and discontinuation of anesthesia , do not leave animal unattended until it has regained sufficient consciousness to maintain sternal recumbency . place the electrodes for the cmap and mune recordings as pictured in figure 1 . use two fine ring electrodes for the recording electrodes . place the active ( e1 ) ring electrode on the skin overlying the proximal portion of the gastrocnemius muscle of the hind limb , at the knee joint , and the reference ( e2 ) ring electrode on the skin over mid - metatarsal portion of the foot.in order to reduce impedance , coat the skin underlying the ring electrodes with gel to sufficiently saturate residual hair and maximize electrode - skin contact . avoid excessive application of electrode gel as this may cause an electrical bridge between electrodes and could prevent accurate recording . place the active ( e1 ) ring electrode on the skin overlying the proximal portion of the gastrocnemius muscle of the hind limb , at the knee joint , and the reference ( e2 ) ring electrode on the skin over mid - metatarsal portion of the foot . in order to reduce impedance , coat the skin underlying the ring electrodes with gel to sufficiently saturate residual hair and maximize electrode - skin contact . avoid excessive application of electrode gel as this may cause an electrical bridge between electrodes and could prevent accurate recording . for stimulation of the sciatic nerve at the proximal hind limb , use two insulated 28 g monopolar needles as the cathode and anode . insert the cathode at the region of the proximal hind limb and insert the anode more proximally in the subcutaneous tissue overlying the sacrum . avoid inserting the stimulating electrodes overly close to the sciatic nerve or too deep that it would directly injury the sciatic nerve or other structure . avoid inserting the stimulating electrodes overly close to the sciatic nerve or too deep that it would directly injury the sciatic nerve or other structure . figure 1 illustrates electrode placement . for the ground electrode , place a disposable surface electrode on the contralateral hind limb or tail sciatic cmap obtain sciatic cmap responses by stimulating the sciatic nerve with square - wave pulses of 0.1 ms duration and intensity ranging from 110 ma.acquire cmap responses with increasing stimulus intensity until the amplitude of the response no longer increases . then , in order to ensure supramaximal stimulation , increase the stimulation to ~120% of the stimulus intensity utilized to obtain a maximal response and obtain an additional response . if there is no further increase in the cmap size , record this response as the maximal cmap.record baseline - to - peak and peak - to - peak cmap amplitudes in mv ( figure 2 ) . obtain sciatic cmap responses by stimulating the sciatic nerve with square - wave pulses of 0.1 ms duration and intensity ranging from 110 ma . acquire cmap responses with increasing stimulus intensity until the amplitude of the response no longer increases . then , in order to ensure supramaximal stimulation , increase the stimulation to ~120% of the stimulus intensity utilized to obtain a maximal response and obtain an additional response . if there is no further increase in the cmap size , record this response as the maximal cmap . record baseline - to - peak and peak - to - peak cmap amplitudes in mv ( figure 2 ) . average single motor unit potential ( smup ) size and mune calculation determine the average single motor unit potential ( smup ) size with an incremental stimulation technique . to obtain incremental responses , deliver submaximal stimulation of 0.1 ms duration at a frequency of 1 hz while increasing the intensity in 0.03 ma steps to obtain the minimal all - or - none responses . if the initial response does not occur with stimulus intensity between 0.21 ma and 0.70 ma , adjust the stimulating cathode position either closer or farther away from the position of the sciatic nerve in the proximal thigh to decrease or increase the required stimulus intensity , respectively.if the initial incremental response is obtained with a stimulus intensity between 0.21 ma and 0.70 ma and fulfills the criteria noted below ( 3.2.2 ) , store and record additional increments with increasing stimulus intensities adjusting by steps of 0.03 ma to obtain a total of 9 additional increments that meet the established criteria . determine the average single motor unit potential ( smup ) size with an incremental stimulation technique . to obtain incremental responses , deliver submaximal stimulation of 0.1 ms duration at a frequency of 1 hz while increasing the intensity in 0.03 ma steps to obtain the minimal all - or - none responses . if the initial response does not occur with stimulus intensity between 0.21 ma and 0.70 ma , adjust the stimulating cathode position either closer or farther away from the position of the sciatic nerve in the proximal thigh to decrease or increase the required stimulus intensity , respectively.if the initial incremental response is obtained with a stimulus intensity between 0.21 ma and 0.70 ma and fulfills the criteria noted below ( 3.2.2 ) , store and record additional increments with increasing stimulus intensities adjusting by steps of 0.03 ma to obtain a total of 9 additional increments that meet the established criteria . if the initial response does not occur with stimulus intensity between 0.21 ma and 0.70 ma , adjust the stimulating cathode position either closer or farther away from the position of the sciatic nerve in the proximal thigh to decrease or increase the required stimulus intensity , respectively . if the initial incremental response is obtained with a stimulus intensity between 0.21 ma and 0.70 ma and fulfills the criteria noted below ( 3.2.2 ) , store and record additional increments with increasing stimulus intensities adjusting by steps of 0.03 ma to obtain a total of 9 additional increments that meet the established criteria . during measurements of the incremental responses , ensure that the initial negative peak of the incremental responses is aligned temporally within the negative peak of the maximal cmap response shown as the shaded portion of the cmap illustration in figure 2.ensure that each incremental response is stable and without fractionation , established by observing three duplicate responses . distinguish visually incremental responses in real - time ( superimposed on the previously recorded increments).note : each increment should be visually distinct and larger compared with the preceding response ( figure 3 ) . analysis in real - time allows recognition of larger amplitude ( incremental ) responses compared to prior responses , and small changes attributable to background noise can be disregarded . a superimposed view of 10 increments is shown in figure 4 ( b and d ) to further illustrate this point.after visually confirming each increment , ensure that the measured amplitude difference ( confirmed response - amplitude of the prior response = amplitude difference ) is at least 25 v.if the increment is less than 25 v , discard and re - measure the response . after recording 10 incremental responses , assess the increments to ensure that the amplitude of each individual incremental response is not greater than 1/3 of the sum of all ten increments ( i.e. the total amplitude of the final response ) . ensure that the initial negative peak of the incremental responses is aligned temporally within the negative peak of the maximal cmap response shown as the shaded portion of the cmap illustration in figure 2 . ensure that each incremental response is stable and without fractionation , established by observing three duplicate responses . distinguish visually incremental responses in real - time ( superimposed on the previously recorded increments ) . note : each increment should be visually distinct and larger compared with the preceding response ( figure 3 ) . analysis in real - time allows recognition of larger amplitude ( incremental ) responses compared to prior responses , and small changes attributable to background noise can be disregarded . a superimposed view of 10 increments is shown in figure 4 ( b and d ) to further illustrate this point . after visually confirming each increment , ensure that the measured amplitude difference ( confirmed response - amplitude of the prior response = amplitude difference ) is at least 25 v . if the increment is less than 25 v , discard and re - measure the response . after recording 10 incremental responses , assess the increments to ensure that the amplitude of each individual incremental response is not greater than 1/3 of the sum of all ten increments ( i.e. the total amplitude of the final response ) . average the 10 incremental values to give an estimation of the average single motor unit potential ( smup ) amplitude ( figure 3 ) . note : figure 3 details the basis of the average smup calculation , but the average smup amplitude can be simply calculated by dividing the entire amplitude of the final incremental response by the total number of increments ( i.e. , 10 ) . example individual smup calculations ( illustrated in figure 3 ) : smup 1=peak - to - peak amplitude of increment 1smup 1=0.050 mvsmup 2= ( peak - to - peak amplitude of increment 2 ) ( peak - to - peak amplitude of increment 1)smup 2=0.150 mv-0.050 mv=0.100 mv calculate each subsequent increment ( up to a total of 10 ) , and make an average of the ten increments . smup 1=peak - to - peak amplitude of increment 1 smup 2= ( peak - to - peak amplitude of increment 2 ) ( peak - to - peak amplitude of increment 1 ) smup 2=0.150 mv-0.050 mv=0.100 mv calculate each subsequent increment ( up to a total of 10 ) , and make an average of the ten increments . calculate each subsequent increment ( up to a total of 10 ) , and make an average of the ten increments . calculate mune by dividing the maximum cmap amplitude ( peak - to - peak ) by the average smup amplitude ( peak - to - peak ) . when necessary , convert cmap and smup results to similar units prior to mune calculation . the techniques of cmap and mune described in this report allow recording of neuromuscular function of the sciatic innervated hind limb muscles utilizing minimally invasive electrode placement ( figure 1 ) . supramaximal cmap size , which represents the total output from a muscle group , can be described using the parameters of amplitude and area ( figure 2 ) , however , in the current methods , we use amplitude to quantify the cmap and the smup sizes . since the cmap response measures summated depolarization of muscle fibers within a muscle , pathology anywhere from the motor neuron to the muscle fiber can result in reduction in cmap size . due to compensatory changes that can occur following denervation ( i.e. collateral sprouting ) , cmap size may be maintained despite processes of motor neuron or motor axonal loss . therefore , the technique of mune is required to determine the motor neuron or axon input to the muscle or group of muscles being tested . recording of individual increments ( figure 3 ) allows estimation of the average output of single motor units ( smup size ) to give more detailed information about functional status of motor units . cmap and mune can be utilized to measure neuromuscular function in various mouse models of neuromuscular disease . in figure 4 , findings in an adult control mouse and an adult mouse 11 weeks following sciatic nerve crush are contrasted . following sciatic nerve crush , mune is severely reduced at 50 estimated functional motor units compared with normal findings of 278 functional motor units in the control mouse . in contrast , the cmap amplitude in the crushed animal ( 39.6 mv baseline - to - peak , 74.9 mv peak - to - peak ) shows only mild reduction compared to control ( 49.0 mv baseline - to - peak , 84.2 mv peak - to - peak ) due to collateral sprouting . mune and cmap are clinically relevant measures frequently utilized in research studies and in monitoring patients with neuromuscular disorders such as als and spinal muscular atrophy ( sma ) . for instance , in sma , cmap and mune correlate well with age , severity and clinical measures of function . both measures are minimally invasive and allow assessment of function longitudinally in the same individual . importantly , these measures can not measure activation or recruitment of the motor unit by cortical motor neurons , but they provide a clinicallyrelevant assessment of the integrity of the motor neuron and its functional counterpart , the motor unit . animal models of neuromuscular disease are critical to an understanding of pathogenic mechanisms of human disease and to the preclinical development of potentially effective therapeutic agents . the ability to translate outcome measures and biomarkers that can be utilized across species can facilitate and hasten the translation of promising preclinical findings to human clinical trials . several groups have previously utilized both electrophysiological and force ( mechanical ) measurements to estimate motor unit function in mouse models . due to the relative complexity of the measures , we have refined these techniques in a visual format to allow more widespread use and implementation in mice . the format of video demonstration and instruction , allows key steps of the procedure to be highlighted and potential pitfalls to be addressed . the application of these techniques to preclinical testing of potential therapies in motor neuron diseases may improve the translation of putative therapies from mice to human disease . there are several critical steps in the process of acquiring the cmap and mune responses . proper and consistent recording electrode placement and sufficient electrode contact with the hind limb are critical for reproducible measurement of amplitude and to decrease background noise . therefore , close contact between hind limb skin and electrodes should be consistently confirmed . we have found that surface electrodes offer more consistent cmap and mune recordings than needle electrodes . due to very thin subcutaneous tissues , small movements of needle recording surface may lead to wide variation in cmap amplitudes . additionally , the more invasive nature of needle electrodes is not optimal for neonatal mice or longitudinal studies due to potential muscle disruption and injury . one potential drawback of non - selective , surface electrode recordings relates to the possibility of diminished phenotype resolution if a particular muscle is more or less involved compared with another , and this has been reported in an als mouse model . acquiring the average smup size is technically more challenging compared to the cmap . due to the smaller response size ( in the range of v rather than mv ) background noise background noise can be reduced by adjusting the ground electrode , cathode , anode , and checking other electrical equipment near the experimental setup . a faraday cage , typically used for intracellular electrophysiology applications , is not required . visual determination of the individual smup responses is the most difficult skill to acquire and takes practice for consistent results with adequate repeatability . it is important to ensure that the smups that are being recorded initiate within the duration of the maximal cmap response . we have defined criteria for acceptance of individual incremental responses to make this process more straightforward to perform and to increase intra- and inter - rater reliability . one potential drawback of the incremental mune technique includes the possibility of overestimating the number of functional motor units due to alternation of motor units . each response should be reproducibly seen a total of 3 times to reduce the impact of this phenomenon . in our experience , clinical electrodiagnostic systems are optimized for the studies described herein due to improved examiner - electrodiagnostic system interface ergonomics allowing ease of control . the two - channel system utilized in our lab is equipped with two non - switched amplifier channels using an amplifier with 24 bit analog to digital converter and a sampling rate of 48 khz per channel . the low frequency filter has a range from 0.2 hz-5 khz , and the high frequency filter settings range from 30 hz-10 khz . a constant - current stimulator is used ( intensity : 0100 ma ; duration : 0.021 ms ) . most clinical systems have similar appropriate features and can be adjusted to adequately record cmap and mune responses . additionally , standard electrophysiological rigs can be assembled to adequately record cmap and mune , but the interface may need to be adjusted for ease of stimulation adjustment and rapid identification of cmap and smup responses . we have previously utilized the techniques of cmap and mune described here to allow rapid and reproducible assessment of the sciatic innervated muscle of the hind limb in mice during the early postnatal period to adulthood . these techniques allow assessment in mouse models when behavioral testing for motor function is not feasible or is less reliable . application of this technique to neonatal mice facilitates the study of motor unit development and has the potential to expand our understanding of motor neuron innervation and pruning . for instance , we have shown that the number of functional motor units recorded with mune will increase during pruning from polyneuronal to mononeuronal innervation during the first two weeks of life in neonatal mice . the ability to test mice over long periods of time with this technique lends itself to the study of motor unit response to peripheral nerve injury , hereditary neuromuscular disorders and aging .

we used the data collected at the baseline visit for the renal insufficiency and cardiovascular events ( riace ) italian multicenter study ( registered with clinicaltrials.gov , nct00715481 ; http://clinicaltrials.gov/ct2/show/nct00715481 ) , an observational , prospective cohort study on the effect of egfr on cvd morbidity and mortality in type 2 diabetes . the riace population consisted of 15,933 caucasian patients with type 2 diabetes ( defined by the american diabetes association criteria ) attending consecutively 19 hospital - based diabetes clinics of the national health service throughout italy ( see supplementary data ) between 2007 and 2008 . the quality and completeness of data were controlled , and 160 patients were excluded due to missing or implausible values . all patients underwent a structured interview to collect the following information : age , smoking status , and known diabetes duration ; current glucose- , blood pressure ( bp ) , and lipid - lowering therapy , with indication of the class of drug ; and previously documented major acute cvd events , including myocardial infarction , stroke , foot ulcer , gangrene or amputation ; coronary , carotid , and lower limb revascularization ; and surgery for aortic aneurysm . cvd events were adjudicated based on hospital discharge records or specialist visits by an ad hoc committee in each center . at physical examination , weight and the presence of retinopathy was assessed by an expert ophthalmologist by ophthalmoscopy or retinography and classified as absent , nonadvanced , or advanced , the latter comprising maculopathy , preproliferative and proliferative retinopathy or history of previous photocoagulation , and blindness ( if less than 1/10 normal vision or 20/200 on the snellen test ) . glycated hemoglobin ( hba1c ) was measured by high - performance liquid chromatography using diabetes control and complications trial aligned methods ; fasting levels of triglycerides and total and hdl cholesterol were determined by standard analytical methods ; and ldl cholesterol was calculated by the friedewald formula . albumin excretion rate ( aer ) was obtained from timed ( 24-h ) urine collections or calculated from the albumin - to - creatinine ratio in early - morning , first - voided urine samples , in the absence of symptoms and signs of urinary tract infection or other interfering clinical conditions . a conversion formula developed in type 1 diabetes ( 20 ) and preliminarily validated in a subgroup from the riace cohort albuminuria was measured in fresh urine samples by immunonephelometry or immunoturbidimetry ( 21 ) . as recommended , the analytical coefficient of variation ( cv ) of both methods was largely < 15% ( i.e. , 2.15.2% for immunonephelometry vs. 3.48.1% for immunoturbidimetry ) , with a detection limit of 1.7 and 3.0 mg / l , respectively . to ensure an external quality control of urinary albumin assays , 50 samples from each center were reanalyzed at the reference laboratory of the coordinating center . results showed that the cvs between the peripheral and central values were lower than 15% in 94% of samples included in the 15500 mg / l interval . one to three measurements for each patient were obtained , and in case of multiple measurements , the geometric mean was used for analysis . in subjects with multiple measurements ( 4,062 with at least two and 2,310 with three values ) , concordance rate between the first value and the geometric mean of multiple measurements was > 90% for all classes of albuminuria ( 21 ) . patients were then assigned to one of the following classes of albuminuria ( mg/24 h ) : normoalbuminuria ( aer < 30 ) , microalbuminuria ( aer 30299 ) , or macroalbuminuria ( aer 300 ) . in addition , normoalbuminuric subjects were further classified as having normal ( aer < 10 ) or low albuminuria ( aer 1029 ) , according to the recent definition of the nkf ( 22 ) . one to three measurements were obtained for each patient , and egfr was calculated by the four - variable modification of diet in renal disease ( mdrd ) study equation ( 23 ) using the mean serum creatinine value in case of multiple measures . as recommended ( 24 ) , one of two versions of this equation was used , depending on whether serum creatinine methods had been calibrated to be traceable to an isotope dilution mass spectrometry ( idms ) reference method . gfr was also estimated using the recently developed chronic kidney disease epidemiology collaboration ( ckd - epi ) equation , which was found to be more accurate and to provide lower estimates of ckd prevalence than the mdrd study equation ( 25 ) . to this end , non - idms serum creatinine values were standardized using the following equation : 0.166 + 1.10 ( measured serum creatinine [ mg / dl ] ) . to derive this equation , 201 frozen samples previously analyzed using a non - idms method were reassayed by an idms reference method . patients were then assigned to one of the following classes of egfr ( ml / min/1.73 m ) : 1 ( 90 ) , 2 ( 6089 ) , 3 ( 3059 ) , 4 ( 1529 ) , or 5 ( < 15 ) . finally , subjects were classified as having no ckd ( egfr 60 ml / min/1.73 m without micro- or macroalbuminuria ) or stages 15 ckd , based on the presence or absence of micro- or macroalbuminuria and the value of egfr according to the nkf kdoqi ( 5 ) . patients assigned to ckd stages ( and gfr classes ) 4 and 5 were pooled together . data are expressed as median and mean sd or number of cases and percentages . the kolmogorov - smirnov normality test , followed by mann - whitney test for continuous variables or pearson test for categorical variables , were applied . logistic regression analyses with stepwise variable selection were performed to identify factors independently associated with major acute cvd events , either total or by vascular bed ; that is , coronary events , including myocardial infarction and/or coronary revascularization ; cerebrovascular , including stroke and/or carotid revascularization ; and peripheral , including ulcer / gangrene / amputation and/or lower limb revascularization . separated analyses for events ( myocardial infarction , stroke , and ulcer / gangrene / amputation ) and revascularization procedures were also performed . covariates were individual values , deciles , or categories of egfr and albuminuria or ckd phenotypes , such as reduced egfr ( stage 3 ckd ) , with or without albuminuria ( micro or macro ) , and albuminuria with nonreduced egfr ( stages 12 ckd ) , together with age , male sex , and smoking status , known diabetes duration , hba1c , hypertension , triglycerides , hdl and ldl cholesterol , lipid - lowering treatment , bmi , nonadvanced and advanced retinopathy , and when applicable , previous cvd event(s ) in other vascular beds . hypertension was defined by systolic bp 140 and/or diastolic bp 90 mmhg and/or antihypertensive treatment . dyslipidemia was defined as high ldl cholesterol and/or lipid - lowering treatment , whereas high triglycerides and low hdl cholesterol were considered separately . results of these analyses were expressed as odd ratios ( ors ) with their 95% ci . all p values were two - sided , and a p value of < 0.05 was considered statistically significant . statistical analyses were performed using spss 13.0 software ( spss inc . , chicago , il ) . we used the data collected at the baseline visit for the renal insufficiency and cardiovascular events ( riace ) italian multicenter study ( registered with clinicaltrials.gov , nct00715481 ; http://clinicaltrials.gov/ct2/show/nct00715481 ) , an observational , prospective cohort study on the effect of egfr on cvd morbidity and mortality in type 2 diabetes . the riace population consisted of 15,933 caucasian patients with type 2 diabetes ( defined by the american diabetes association criteria ) attending consecutively 19 hospital - based diabetes clinics of the national health service throughout italy ( see supplementary data ) between 2007 and 2008 . the quality and completeness of data were controlled , and 160 patients were excluded due to missing or implausible values . all patients underwent a structured interview to collect the following information : age , smoking status , and known diabetes duration ; current glucose- , blood pressure ( bp ) , and lipid - lowering therapy , with indication of the class of drug ; and previously documented major acute cvd events , including myocardial infarction , stroke , foot ulcer , gangrene or amputation ; coronary , carotid , and lower limb revascularization ; and surgery for aortic aneurysm . cvd events were adjudicated based on hospital discharge records or specialist visits by an ad hoc committee in each center . at physical examination , weight and the presence of retinopathy was assessed by an expert ophthalmologist by ophthalmoscopy or retinography and classified as absent , nonadvanced , or advanced , the latter comprising maculopathy , preproliferative and proliferative retinopathy or history of previous photocoagulation , and blindness ( if less than 1/10 normal vision or 20/200 on the snellen test ) . glycated hemoglobin ( hba1c ) was measured by high - performance liquid chromatography using diabetes control and complications trial aligned methods ; fasting levels of triglycerides and total and hdl cholesterol were determined by standard analytical methods ; and ldl cholesterol was calculated by the friedewald formula . albumin excretion rate ( aer ) was obtained from timed ( 24-h ) urine collections or calculated from the albumin - to - creatinine ratio in early - morning , first - voided urine samples , in the absence of symptoms and signs of urinary tract infection or other interfering clinical conditions . a conversion formula developed in type 1 diabetes ( 20 ) and preliminarily validated in a subgroup from the riace cohort albuminuria was measured in fresh urine samples by immunonephelometry or immunoturbidimetry ( 21 ) . as recommended , the analytical coefficient of variation ( cv ) of both methods was largely < 15% ( i.e. , 2.15.2% for immunonephelometry vs. 3.48.1% for immunoturbidimetry ) , with a detection limit of 1.7 and 3.0 mg / l , respectively . to ensure an external quality control of urinary albumin assays , 50 samples from each center were reanalyzed at the reference laboratory of the coordinating center . results showed that the cvs between the peripheral and central values were lower than 15% in 94% of samples included in the 15500 mg / l interval . one to three measurements for each patient were obtained , and in case of multiple measurements , the geometric mean was used for analysis . in subjects with multiple measurements ( 4,062 with at least two and 2,310 with three values ) , concordance rate between the first value and the geometric mean of multiple measurements was > 90% for all classes of albuminuria ( 21 ) . patients were then assigned to one of the following classes of albuminuria ( mg/24 h ) : normoalbuminuria ( aer < 30 ) , microalbuminuria ( aer 30299 ) , or macroalbuminuria ( aer 300 ) . in addition , normoalbuminuric subjects were further classified as having normal ( aer < 10 ) or low albuminuria ( aer 1029 ) , according to the recent definition of the nkf ( 22 ) . one to three measurements were obtained for each patient , and egfr was calculated by the four - variable modification of diet in renal disease ( mdrd ) study equation ( 23 ) using the mean serum creatinine value in case of multiple measures . as recommended ( 24 ) , one of two versions of this equation was used , depending on whether serum creatinine methods had been calibrated to be traceable to an isotope dilution mass spectrometry ( idms ) reference method . gfr was also estimated using the recently developed chronic kidney disease epidemiology collaboration ( ckd - epi ) equation , which was found to be more accurate and to provide lower estimates of ckd prevalence than the mdrd study equation ( 25 ) . to this end , non - idms serum creatinine values were standardized using the following equation : 0.166 + 1.10 ( measured serum creatinine [ mg / dl ] ) . to derive this equation , 201 frozen samples previously analyzed using a non - idms method were reassayed by an idms reference method . patients were then assigned to one of the following classes of egfr ( ml / min/1.73 m ) : 1 ( 90 ) , 2 ( 6089 ) , 3 ( 3059 ) , 4 ( 1529 ) , or 5 ( < 15 ) . finally , subjects were classified as having no ckd ( egfr 60 ml / min/1.73 m without micro- or macroalbuminuria ) or stages 15 ckd , based on the presence or absence of micro- or macroalbuminuria and the value of egfr according to the nkf kdoqi ( 5 ) . patients assigned to ckd stages ( and gfr classes ) 4 and 5 were pooled together . data are expressed as median and mean sd or number of cases and percentages . the kolmogorov - smirnov normality test , followed by mann - whitney test for continuous variables or pearson test for categorical variables , were applied . logistic regression analyses with stepwise variable selection were performed to identify factors independently associated with major acute cvd events , either total or by vascular bed ; that is , coronary events , including myocardial infarction and/or coronary revascularization ; cerebrovascular , including stroke and/or carotid revascularization ; and peripheral , including ulcer / gangrene / amputation and/or lower limb revascularization . separated analyses for events ( myocardial infarction , stroke , and ulcer / gangrene / amputation ) and revascularization procedures were also performed . covariates were individual values , deciles , or categories of egfr and albuminuria or ckd phenotypes , such as reduced egfr ( stage 3 ckd ) , with or without albuminuria ( micro or macro ) , and albuminuria with nonreduced egfr ( stages 12 ckd ) , together with age , male sex , and smoking status , known diabetes duration , hba1c , hypertension , triglycerides , hdl and ldl cholesterol , lipid - lowering treatment , bmi , nonadvanced and advanced retinopathy , and when applicable , previous cvd event(s ) in other vascular beds . hypertension was defined by systolic bp 140 and/or diastolic bp 90 mmhg and/or antihypertensive treatment . dyslipidemia was defined as high ldl cholesterol and/or lipid - lowering treatment , whereas high triglycerides and low hdl cholesterol were considered separately . results of these analyses were expressed as odd ratios ( ors ) with their 95% ci . all p values were two - sided , and a p value of < 0.05 was considered statistically significant . statistical analyses were performed using spss 13.0 software ( spss inc . , chicago , il ) . clinical characteristics of subjects with or without prior cvd event(s ) are reported in table 1 . compared with subjects without cvd , patients with prior event(s ) were older , with a higher prevalence of men , a longer diabetes duration , and worse metabolic control ; moreover , a significantly higher percentage was receiving insulin treatment . concerning the other cvd risk factors , these patients had higher triglycerides and lower hdl cholesterol , but also lower ldl cholesterol and diastolic ( but not systolic ) bp levels . however , the percentage of patients treated for dyslipidemia and/or hypertension was higher among those with prior cvd event(s ) , who also showed a higher prevalence of retinal involvement . finally , with regard to renal function , patients with prior cvd event(s ) had higher serum creatinine and albuminuria levels and lower egfr whether by mdrd or ckd - epi than patients without . correspondingly , the two groups differed for egfr and albuminuria class and ckd stage distribution . clinical characteristics of subjects with or without prior cvd event(s ) multiple logistic regression indicated that age , male sex , diabetes duration , smoking status , and presence of dyslipidemia and hypertension were positively associated with total cvd , whereas hdl cholesterol levels were inversely correlated ( supplementary table 1 and table 2 ) . independent of these factors , pharmacologic treatment of diabetes and presence of retinal involvement were graded risk indicators ( supplementary table 1 and table 2 ) . compared with normal egfr and albuminuria categories , subnormal egfr and low albuminuria were nonsignificantly associated with total cvd , with the association becoming significant for egfr < 60 ml / min/1.73 m ( a 65% risk increase , which was only marginally greater for egfr < 30 ml / min/1.73 m ) and micro- or macroalbuminuria ( supplementary table 1 ) . after accounting also for the use of angiotensin - converting enzyme inhibitors ( aceis ) and/or angiotensin receptor blockers ( arbs ) , association with cvd events remained significant at the same threshold values of egfr and albuminuria ( data not shown ) . results were virtually identical when separate analyses were conducted for cvd events alone ( with or without revascularization ) , whereas a significant association of revascularization procedures alone was found for egfr but not albuminuria categories ( data not shown ) . when egfr and albuminuria values were included as continuous variables , the age- and sex - adjusted risk for a cvd event increased by 6.8% for every 5 ml / min/1.73 m egfr decrease ( or 1.068 [ 95% ci 1.0581.078 ] ; p < 0.0001 ) and by 0.4% for every 10 mg/24 h albumin increase ( 1.004 [ 1.0031.005 ] ; p < 0.0001 ) . the risk associated with albuminuria ( 1.006 [ 1.0041.007 ] ) and reduced egfr ( 1.074 [ 1.0641.085 ] ) increased further if the other variable was excluded from the model . when deciles of egfr and albuminuria were considered , age- and sex - adjusted risk for a cvd event increased linearly by 12% ( 1.12 [ 1.101.14 ] ) for each decreasing decile of egfr and by 9% ( 1.09 [ 1.081.10 ] ) for each increasing decile of albuminuria . excess risk was significant for egfr values < 78 ml / min/1.73 m and albuminuria values 10.5 mg/24 h ( fig . 1 ) ; the egfr cutoff became 84 ml / min/1.73 m , whereas that of albuminuria did not change when the two variables were included simultaneously . age- and sex - adjusted risk of major acute cvd events ( or [ 95% ci ] ) according to egfr deciles ( ml / min/1.73 m ) ( a ) and albuminuria deciles ( mg/24 h ) ( b ) . , event ; , reference category ( rc ) . when ckd phenotypes were introduced as covariates in the model , reduced egfr without albuminuria was more strongly associated with events than normal egfr with micro- or macroalbuminuria ; however , the combination of reduced egfr and albuminuria marked an increased risk of cvd events in an additive manner ( table 2 ) . logistic regression analysis of all cvd events ( n = 3,654 ) with ckd phenotypes as covariates when analysis was carried out separately for coronary events ( table 3 ) , albuminuria alone was no longer an independent predictor , even after accounting for the use of aceis and/or arbs ( data not shown ) , and the association was stronger with nonalbuminuric than with albuminuric renal impairment . no association was observed between coronary events and retinopathy , whereas , as expected , the coexistence of a cerebrovascular or peripheral event carried additional quotas of risk . in contrast , the analysis of the relationship between cerebrovascular or peripheral events and ckd phenotypes ( table 3 ) showed that the association with nonalbuminuric ckd was somewhat weaker than that with the albuminuric forms . again , a previous event in another vascular bed was an additional risk determinant , whereas the presence of retinopathy was an independent risk factor for peripheral events . a strong association of reduced egfr with coronary events and a weak relation to cerebrovascular and peripheral disease also emerged when egfr and albumin categories were introduced as covariates ( supplementary tables 24 ) . logistic regression analysis of coronary ( n = 2,405 ) , cerebrovascular ( n = 1,298 ) , or peripheral ( n = 894 ) events with ckd phenotypes as covariates data from this large multicenter italian cohort extends previous reports on the association of major acute cvd events with increased albuminuria and/or reduced egfr in subjects with type 2 diabetes ( 1416 ) . both markers of renal dysfunction , independent of each other and of the cluster of traditional cvd risk factors , were associated with cvd in a graded manner . although total cvd was nonsignificantly associated with subnormal egfr ( i.e. , 6089 ml / min/1.73 m ) and also with low albuminuria values ( i.e. , 1029 mg/24 h ) , the analysis by deciles of egfr and albuminuria showed thresholds within these ranges , consistent with the observation that an albuminuria cutoff of 10 mg/24 h predicts all - cause and cvd mortality in the general population ( 8) . interestingly , in the presence of albuminuria , the egfr cutoff for cvd risk almost reached the lower limit of the normal range , whereas that of albuminuria was not influenced by egfr levels . the weaker association of total cvd with albuminuria alone , compared with reduced egfr alone , supports the assignment of subjects with increased albuminuria and normal or subnormal egfr to earlier ckd stages than patients with reduced egfr , irrespective of albuminuria . however , the significantly higher cvd burden associated with the combination of increased albuminuria and reduced egfr suggests that subjects presenting with albuminuric stage 3 ckd should be considered at higher cvd risk than those with the nonalbuminuric phenotype of renal impairment , in keeping with recent reports from general population ( 7,8 ) and type 2 diabetes ( 14,15 ) cohorts . this would also imply that all stages of ckd should be stratified by the presence or absence of albuminuria , as recently suggested ( 26 ) , for better prediction of both cvd and renal outcome . more importantly , our data suggest that the relation of cvd with renal dysfunction is more complex and varies with the vascular bed affected , although a history of an event in one vascular bed is associated with a two- to threefold risk of event in another . at variance with previous reports ( 15,27 ) , this might be only partly explained by a survivor or inclusion bias , due to the higher risk of cvd death and progression toward ckd associated with albuminuria than with reduced egfr in subjects with type 2 diabetes ( 16 ) . however , in the action in diabetes and vascular disease : preterax and diamicron - mr controlled evaluation ( advance ) study , although the multivariable - adjusted hazard ratio ( hr ) for total cvd events was 2.48 for every 10-fold increase in baseline albuminuria and 2.20 for every halving of baseline egfr , the hrs associated with microalbuminuria alone , macroalbuminuria alone , and reduced egfr alone were 1.48 , 1.18 , and 1.33 , respectively ( 14 ) . on the other hand , the strong association of coronary events with reduced egfr is in keeping with the finding that a moderate - to - severe reduction in egfr is related to the number of narrowed coronary arteries in subjects undergoing coronary angiography for suspected coronary artery disease ( cad ) ( 28 ) . a possible explanation of our finding is that patients with coronary events were treated more aggressively than subjects with cerebrovascular or peripheral events , with a higher use rate of drugs known to reduce albuminuria , such as aceis / arbs ( 71.8% vs. 65.1% ; p < 0.0001 ) and statins ( 71.2% vs. 48.1% ; p < 0.0001 ) . in this view , regression of micro- or macroalbuminuria to aer values within the normal range due to intensive treatment might have occurred in a percentage of subjects with a previous coronary event , although lack of association with albuminuria remained after accounting for acei / arb use . moreover , in a cross - sectional analysis , this powerful association between cad and impaired egfr might likely reflect the unique bidirectional nature of heart kidney interactions in the context of the cardiorenal syndrome ( 29 ) , with chronic cardiac dysfunction resulting from cad causing progressive egfr impairment or renal dysfunction favoring coronary atherosclerosis . the latter scenario is consistent with the role of renal dysfunction in promoting vascular calcification , the extent of which was inversely related to egfr ( 30 ) and predicted cad and death ( 31 ) . finally , the stronger association of coronary events with reduced egfr than with albuminuria may also reflect differences in renal pathology underlying the albuminuric and nonalbuminuric ckd phenotypes . in fact , microalbuminuria is considered a reliable biomarker of renal microvascular disease ( 32 ) , although it may occur in the absence of significant kidney damage ( 33 ) , whereas egfr was inversely related with indexes of intrarenal as well as systemic atherosclerosis ( 34 ) , although this association occurred independent of albuminuria ( 35 ) . thus , nonalbuminuric renal impairment , as a manifestation of prevailing renal macrovascular involvement , would be more frequently associated with coronary atherosclerosis than the albuminuric forms , which might conversely indicate the presence of microangiopathy . this interpretation is supported by the lack of association of coronary events with retinopathy . at variance with reports from other large datasets of subjects with type 2 diabetes ( 14,15 ) , data showed that involvement of both vascular beds was associated more strongly with albuminuria than coronary events , with a weaker relation to nonalbuminuric ckd . this is consistent with reports that albuminuria , but not egfr , is associated with maladaptive carotid artery remodeling ( 36 ) and that reduced egfr is a risk factor for hemorrhagic but not ischemic stroke ( 37 ) . this also in keeping with data from the nhanes iii cohort showing a higher relation of peripheral artery disease with albuminuria than with reduced egfr ( 38 ) , although this was not the case in diabetic subjects ( 39 ) . however , this might argue against the concept that the albuminuric phenotypes underlie predominantly microangiopathic lesions within the kidney and , therefore , should be less frequently associated with atherosclerosis . indeed , events in noncoronary vascular beds , at variance with coronary events , were significantly related with retinopathy , even if no relation was found between cerebrovascular disease and advanced retinopathy . the main limitation of this study is the cross - sectional design , which does not allow us to derive any cause effect relationship , although data clearly indicate an association of reduced egfr and/or albuminuria with cvd , with unique differences by vascular bed . however , data did not change when analyzed separately by the method for measuring albuminuria ( immunonephelometry vs. immunoturbidimetry ) or serum creatinine ( idms - traceable vs. non idms - traceable ) . in conclusion , this large - cohort study indicates that in subjects with type 2 diabetes , 1 ) cvd is linearly associated with egfr reduction and albuminuria ; 2 ) this association becomes significant for an egfr < 78 ml / min/1.73 m and an albuminuria 10.5 mg/24 h ; and 3 ) the relation with reduced egfr , independent of albuminuria , is stronger for coronary than for cerebrovascular or peripheral events , thus suggesting that nonalbuminuric renal impairment carries significant risk for cad and vice versa . these observations also highlight the usefulness of ckd screening by gfr estimation , which was claimed to be effective in high - risk populations , such as subjects with type 2 diabetes , though not advisable in the general population ( 40 ) . even considering the above - reported limitations , our survey describes the relationship of cvd with renal dysfunction in the largest cohort of type 2 diabetic patients referring to outpatient diabetes clinics in europe .

recently , authors have argued that prevention is the most effective strategy for protecting native biodiversity and avoiding unwanted economic damages from invasive species . further , prevention focused on dispersal pathways ( e.g. , waterway connections , canals , ships ballast , organisms in trade ) is likely to be more cost - effective than that focused on individual species because a given prevention strategy can be effective against multiple species . however , for most pathways , little information exists on the effectiveness of prevention strategies because of the expense or infeasibility of experimental tests at realistic field scales . therefore we use structured expert judgment ( sej ) to evaluate the uncertainty around the effectiveness of alternative strategies to prevent invasions through the chicago area waterway system ( caws ) that connects the mississippi river and laurentian great lakes watersheds ( figure 1 ) . the chicago area waterway system ( caws ; white star ) , located in northern illinois at the southern tip of lake michigan , is a man - made hydrological connection of the two watersheds . while human - made waterway connections have clear socio - economic benefits , these hydrologic connections are often also conduits for species invasions and disease outbreaks worldwide . in europe , canals are the most important pathway for aquatic nonindigenous species along the rhine river . in north america , the canals that connect the mississippi river and great lakes basins have enabled the interbasin passage of a number of species such as sea lamprey ( petromyzon marinus ) and zebra mussel ( dreissena polymorpha)which have significantly damaged ecosystems in both watersheds . as the great lakes and mississippi river basins have the highest diversity of freshwater fishes and mussels in the world , the continued interbasin transfer of nonindigenous species presents a major threat to global aquatic biodiversity . two of the species of greatest concern as potential invaders into the great lakes from the mississippi river basin via the caws are asian carps ( bighead ; hypophthalmichthys nobilis , and silver ; h. molitrix ) . originally introduced in arkansas in the 1970s , their invasion throughout the mississippi river basin has been characterized by exponential increases in biomass and damages to ecosystems , human safety , and fisheries . should asian carps gain access to the great lakes , there is potential for establishment and concern they may change food webs and significantly impact the multibillion dollar fishing industry . to prevent passage of other prevention strategies are also under consideration , including physical hydrologic separation of the mississippi and great lakes basins , which would cost $ 3.59.5 billion usd . whether any fish deterrent strategy can provide an effective and long - term method for preventing passage between watersheds research on fish deterrence technologies for asian carps has been conducted in laboratory confinements , outdoor raceways , and a small river system , with limited , short - term studies in the caws . the empirical information required to rigorously evaluate the long - term effectiveness of prevention strategies at the relevant field scale does not exist and is likely not feasible to acquire with field experiments . further , the time required to develop , implement , and conduct multiple large - scale field trials even they were feasible and ethical might preclude the utility of this information because an invasion may occur within that time frame . despite extensive analysis and review , great lakes natural resource managers remain unsure as to the optimal approach for asian carp prevention between the mississippi and great lakes watershed . with this study we use a performance - based method of sej , which aggregates expert knowledge to quantify the uncertainties associated with invasive species pathway management . sej has been widely used in a variety of applications including consequence assessment for chemical substances , nuclear accidents , probabilistic hazard and risk assessment and increasingly in conservation biology . sej has been successfully used to quantify uncertainty in scenarios when scientific consensus is confounded by divergences of opinion or lack of data . thus providing the information required for decision makers to reduce economic damages and in some cases , loss of human life associated with urgent hazards . specifically , we use sej to assess the efficacy of alternative strategies to prevent asian carp dispersal via the caws , one of the largest human - made water connections in north america . this analysis not only provides important information for the management of the caws , but results are also applicable to many canal systems globally . in addition , we provide a novel and tractable approach for using sej to address the management of species invasions under great uncertainty . several approaches are available for the elicitation and aggregation of individual experts judgments , most of which seek to find homogeneity or create a single combined aggregate of a group of expert assessments . here we used a structured , performance - based method to elicit and aggregate expert judgments ( the classical model ) . assessments by individual experts were obtained with 23 elicitors present , in which each expert quantified his response as the 5th , 50th , and 95th percentiles of his subjective probability distribution for each target variable . each expert was then given different scores according to individual performance on a set of calibration variables , that is , variables from the experts field of specialization whose values were not known at the time of elicitation , but were realized post hoc . for example , we asked experts to quantify the asian carp biomass removed from the caws by commercial fishing crews during 2012 ; the effort was ongoing at the time of elicitation , and results were published afterward by the u.s . expert assessments were then aggregated into a single combined outcome , determined through methodologies described below . to select experts , we identified individuals with expertise in fisheries biology and specific experience in the great lakes , or asian carp species , or both . from this list , we contacted 11 experts ( table 1 ) , all of whom agreed to participate . the choice of the number of experts is supported by simulations showing that adding experts above 10 does not lead to increased performance of combined assessments . prior to an in - person interview , each expert received the elicitation questionnaire ( supporting information part a ) , background information concerning expert elicitation , and a booklet containing biological information about the great lakes , and bighead and silver carp ( all materials are available upon request ) . experts were encouraged to utilize any additional information sources or considerations not included in the provided background information or booklet that would help them estimate their responses . we interviewed each expert individually , in person , between may 1 and june 15 , 2012 . each interview began with a brief presentation explaining the goals of the study , a statement of assumptions , and an explanation of calibration and target variables . the experts are listed in alphabetical order which does not correspond to responses presented in this study . the elicitation questionnaire consisted of 84 questions about bighead and silver carp establishment in the great lakes . for the present study , we focused on a subset of questions about the efficacy of 17 asian carp deterrence strategies proposed for use in the caws ( table 2 ) . we asked experts to quantify the percentage of asian carps that would be prevented from accessing lake michigan as a result of implementing one of the 17 deterrent strategies at a time . of the 84 questions in the overall questionnaire , 20 were calibration variables used to evaluate the performance of each expert . the product of the calibration and information scores determines the performance - based weight that each expert receives . the calibration score is the probability that the divergence between the expert s probabilities and calibration variable realizations might have arisen by chance . for the set of calibration variables , each expert provides quantile information to test the hypothesis h0 : this expert is well calibrated . if an expert gives 90% confidence bands for the set of calibration variables , then it might be anticipated that about 10% of actual realizations ( e.g. , the true outcomes of the calibration variables ) will fall outside his chosen bands . thus , for an expert assessing 20 calibration variables for which realizations become known post hoc , three or four outcomes outside the confidence bands is expected . however , if 10 or more of the 20 variables fell outside the expert s bands , it is not likely that so many outliers resulted by chance . if an expert is perfectly accurate statistically , then his / her calibration score is uniformly distributed on the interval , where scores near 1 indicate strong agreement between the expected number of realizations falling into the inter quantile intervals , and scores near zero indicate strong disagreement . in this sense , on which the classical model is based , a score of 0.05 would be sufficient to reject the hypothesis that an expert is statistically accurate . the information score is the degree to which the expert s distribution is concentrated , relative to a user - selected background measure . this is to say that the width of an expert s confidence band determines his or her information score . this information score is calculated for each expert at each variable and then averaged over all variables to get the overall information score . in addition to evaluating each expert s performance , we also derived two alternative aggregates of expert responses : equally weighted ( eq ) , in which the score of each expert is weighted equally ; and performance - based ( pb ) in which weighted experts ( those whose calibration score is above a specified cutoff level , ( see supporting information part b for derivation of ) are pooled . additionally , we recorded notes on what each expert revealed during the interview about the rationale associated with quantitative responses provided for each target variable . expert names and affiliations are provided herein , but not associated with their responses . thus , ordering of the experts presented in table 1 is alphabetical and does not correspond to the ordering of results . the identification of responses associated with individual experts are privately maintained by the authors of this study . the experts calibration scores ranged from 2 10 to 0.53 , with 9 of 11 experts scoring above 0.05 ( table 3 ) . higher calibration scores indicate better performance in assessing the calibration variables accurately . all experts assessed 15 calibration variables , with the exception of expert 8 who assessed only 11 , reducing the effective number of items for all experts to 11 . the eq had a calibration score of p = 0.3126 ( table 3 ) , indicating that we would not reject the hypothesis that eq s probability assessments were accurate . both weighting schemes ( eq , pb ) returned good calibration and high information ( mean relative information ; all variables : 0.5789 , 3.798 ) . higher mean relative information scores indicate better performance in assessing the calibration variables precisely . the pb combination was better calibrated and more informative than the eq combination ; therefore we put the greatest emphasis below on pb results . the pb combination was comprised of responses from a single expert ( expert 4 ) due to this expert s high calibration ( table 1 ) and the strictly proper scoring rule ( see supporting information part b ) . calibration score ( 2nd column ) is the likelihood that the realizations of calibration variables correspond with the expert assessments . mean relative information measures the degree to which an expert s uncertainty distribution is concentrated around the true answers to a set of variables ( either to all variables ( 3rd column ) , or to the calibration variables 4th column ) . decision makers : eq = equally weighted combination , where all experts responses are pooled with equal weights ; and pb = performance - based combination , where experts are weighted according to a selected cut - off level for calibration for which the normalized weight ( 5th column ) of the combination is maximal . both combinations ( pb , eq ) indicated that hydrologic separation at the caws is the most effective prevention strategy , with median estimates of 99% for the pb and 98% for the eq result ( figure 2a , b ) . uncertainty ranges ( 5 , 95th percentiles ) for the pb ( 95 , 100 ) and eq ( 78 , 100 ) results for hydrologic separation were all small compared to any other prevention strategy considered , indicating high certainty among experts . equally weighted ( eq ; a ) and performance - based ( pb ; b ) expert assessments of the percentage of asian carp prevented access to the great lakes as a result of implementing 17 proposed fish deterrent strategies in the chicago area waterway system ( strategy acronyms from table 2 ) . similarly , both combinations indicated that electric barrier was the second most effective strategy , ranging from a median of 88% to 92% of asian carps prevented . the pb indicated that the acoustic - bubble - strobe ( abs ) combination of deterrent technologies was similarly effective to the electric barrier option , with a median result of 92% prevented , but with a wide uncertainty range in comparison to electric barrier or hydrologic separation ( figure 1b ) . the equally weighted median result for abs indicated a much lower proportion of fish prevented ( 60% ) . pheromone treatment was indicated as the fourth best option according to the pb , with co2 and abs being similarly low in their expected effectiveness for the eq decision maker . the similarity in the pb decision maker among the third to sixth approaches ( thr , co2 , hyp , chl , ph , gun ) is due to this combination being comprised of the responses of a single expert , whose estimates of uncertainty for all of those strategies were equivalent . this suggests that the expert did not view the effectiveness of any of these strategies as being meaningfully distinguishable from one another . according to experts , none of the fish deterrent strategies would be 100% effective in preventing asian carp passage between watersheds . all experts indicated that hydrologic separation would be the most effective at reducing the greatest proportion of asian carps , due to a lack of mechanical failures other than extreme flooding events . all experts also acknowledged and considered as part of their rationale that asian carp introduction to lake michigan could occur through other pathways ( e.g. , bait contamination , movement by birds ) . experts indicated that an electric barrier the only method currently in place is likely to be less effective than hydrologic separation . this expectation was attributed to the possibility of mechanical failures of the electric barrier , which is also expected for other strategies that require a constant power source ( i.e. , abs , thermal ) . likewise , in situ materials such as block nets and associated maintenance were expected to be subject to breakdown . experts indicated that chemical , thermal or other curtain barriers ( i.e. , chlorine , bubble , acoustic , strobe ) were highly dependent on two factors : the ability to consistently maintain the barrier with appropriate magnitude , and the behavioral responses of fishes . variation in hearing or vision of asian carp , their tolerance to physical or chemical cues , and habituation to these cues were identified as the main sources of failure even if all barriers functioned full time . uncertainties about the success or failure rates of species prevention strategies may lead to delayed decision making by managers or policy makers potentially enabling further damages from invasion . here we have provided a previously missing and key aspect of the information needed for risk management the expected effectiveness of prevention strategies . experts identified hydrologic separation as the most effective strategy for preventing asian carp passage , with alternative prevention strategies having greater uncertainty around lower estimated effectiveness . sej provides a relatively rapid and transparent way to provide essential information to managers and policy makers . given the similar biological and engineering issues present in many of the world s canals , the experts responses here may also be useful to the management of waterways elsewhere . to manage risk , natural resource managers often seek to evaluate the value of any management action against the likelihood of invasion , and the potential damages to ecosystem services that may be caused by the invader . while this study identifies and quantifies the uncertainties associated with the effectiveness of asian carp deterrent strategies , it does not address three important aspects of any species invasion that managers would desire : ( 1 ) the relationship between the number of asian carps ( i.e. , propagule pressure ) and the probability of establishment , ( 2 ) whether asian carp , if established , would cause ecological damage in the great lakes , and ( 3 ) the costs associated with the alternative prevention strategies we considered . the caws is the primary focus of efforts to prevent asian carp introduction into the great lakes because the caws provides a direct connection to the illinois river , home to the densest populations of asian carp known to exist . proximity to a source of dispersing individuals increases propagule pressure , and therefore the likelihood that an invasive species will establish . however , it is the dynamics of initial populations that actually reach the great lakes that would determine establishment success . demographic stochasticity , density dependence and interaction with the environment are some factors that complicate the ability to estimate invasion risk from propagule pressure alone . despite evidence that abundant spawning habitat , food availability and suitable climate exist for asian carps in the great lakes , major uncertainties remain about the likelihood of how persistence , survival and recruitment within the great lakes would be quantitatively related to propagule pressure . it is unlikely that additional empirical work can be done at appropriate spatial scales to resolve this question within a time useful to management decisions . whether asian carp would cause significant ecological or economic damage in the great lakes is also uncertain . observations of rapid growth and dispersal , and reductions in planktivore populations of river systems indicate that asian carps may also similarly impact great lakes food webs , and subsequently , commercial or recreational fisheries . in contrast , assimilations with little or no measurable impact to the great lakes ecosystem and resource limitation have been suggested as reasons why asian carps may not cause ecological harm . the uncertainties remaining about how damaging asian carps would be may be narrowed by additional research using empirical studies and foodweb models . costs associated with the implementation of some of these alternative strategies are known or would be fairly easy to quantify , as suggested by these examples : one electric barrier located in the caws cost $ 13 million ( usd ) to install and $ 0.9 million per year to maintain ; the minnesota department of natural resources requested $ 1219 million to install an abs barrier at lock and dam 1 on the mississippi river with an annual operating cost of $ 0.25 million ; maintaining a hypoxic barrier in the caws would cost approximately $ 0.25 million per day , or over $ 91 million per year ; and hydrologic separation is estimated to cost $ 3.59.5 billion with low annual maintenance cost . thus hydrological separation , the strategy identified with high certainty as the most effective strategy , is also likely to be the most expensive strategy , at least in initial expenditures . however , at least two other considerations suggest that the initial expense of hydrological separation would be at least partially offset by other advantages relative to the other strategies . first , initial expenditures of hydrological separation would be effective over a much longer time frame relative to all the other methods , which would have shorter lifespans and/or higher periodic maintenance costs . the time frame over which decision - makers assess costs and benefits will be a major determinant of cost - benefit calculations . second , we asked experts to estimate the effectiveness of the 17 fish deterrent strategies against asian carps only , but many other animals and plants are poised to disperse through the caws in one or the other direction . in contrast to hydrologic separation , the other 16 strategies are designed primarily to deter the movement of juvenile or adult fishes ( e.g. , abs , electric barriers , pheromones , block nets ) , and are not suitable to prevent the passage of mollusks , crustaceans , macrophytes , or gametes or larvae of fishes and other taxa suspended in the water column . the implementation of prevention strategies that inhibit the movement of multiple species within a pathway , rather than strategies that focus on single species , are likely to be more effective and have greater societal benefits . thus the costs of each of the alternative strategies reviewed above would more reasonably be weighed against the avoided damages from many species not just asian carps . with projected increases in species introductions , global transportation and canal construction , the management of these pathways ( including the coupling of deterrent strategies with surveillance programs and response plans to reduce the likelihood of invasion ) is necessary to reduce the unwanted effects of species invasions . although the results presented here are specific to asian carp passage from the mississippi river basin to the great lakes , this approach can serve as the framework for addressing the scientific unknowns associated with any species invasion , or uncertainties associated with new environmental risks . for example , sej is valuable method for identifying key uncertainties that may point the way to future empirical research programs . rather than allocating resources to understand whether less effective methods are marginally more effective than one another ( i.e. , hydrogun vs bubble curtain ) , natural resource managers can instead focus efforts on maximizing the projected effectiveness of hydrologic separation or electric barriers . by separating expert responses and the identity of experts , the classical model of sej provides a means for experts to quantify scientific uncertainties on important societal issues without the risk of political confrontation , and for policy makers to utilize this information to make risk - based decisions in a transparent and defensible way .