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due to lack of evidence from prospective clinical trials, the diagnostic procedures, their frequency, as well as the length of the follow - up period in cutaneous melanoma patients should be based on the individual risk of disease recurrence, which is strongly dependent on the stage of disease at the time of diagnosis. in the paper we propose the current recommendations for follow - up strategy. nowadays, new effective treatment options with biological agents justify the closer monitoring of high risk melanoma patients.

after obtaining approval by the institutional review board, a retrospective medical record review was conducted using records from 106 consecutive patients (110 eyes) who were treated with intravitreal anti - vegf agents for pcv at kim's eye hospital, konyang university college of medicine from november 2008 to june 2010. patients were included if they met all of the following criteria: 1 ) confirmation of pcv with fluorescein angiography (fa) and indocyanine green angiography (icga) performed using a confocal laser scanning system (hra-2 ; heidelberg engineering, dossenheim, germany) at the first visit. we only included patients whose icga showed a branching vascular network with polypoidal shaped choroidal vascular lesions, 2 ) patients who were treated with only one type of anti - vegf agent (either bevacizumab or ranibizumab), and 3 ) a minimum follow - up period of 6 months. exclusion criteria were the following: 1 ) combination therapy of more than one anti - vegf agent, 2 ) prior treatment with pdt, 3 ) pathologic myopia, 4 ) idiopathic choroidal neovascularization (cnv), 5 ) other secondary cnv, 6 ) other ocular disease that could affect visual acuity, 7 ) trauma during the study or in the fellow eye, 8) aphakia, or 9 ) previous vitreoretinal surgery. after the initial loading injections at the time of diagnosis, retreatment for each patient was planned as a' retreat as needed' protocol. anti - vegf agents were re - injected on a monthly basis if objective visual deterioration of more than two lines, persistent exudates and hemorrhage, or evidence of an active pcv lesion were observed on fa, icga, or optical coherence tomography (oct), were observed. best - corrected visual acuity (bcva) was obtained every one or two months using the snellen chart. patients also underwent an ophthalmic examination, including a slit - lamp evaluation and fundus examination, as well as oct (spectral oct / slo ; oti ophthalmic technologies inc ., miami, fl, usa), fa, icga, or a combination thereof. foveal center thickness (fct) was assessed by oct using six diagonal fast and slow 6-mm scans. the off - label nature of the treatment and its potential risks and benefits were discussed in detail with all patients, and signed informed consent was obtained from all patients. patients received 1.25 mg of bevacizumab or 0.5 mg of ranibizumab. prior to administration of the injection, topical anesthesia was applied, and 10% povidone - iodine was used for scrubbing of eyelid and lashes. following the application of povidone - iodine eyedrops (1.25%), a sterile lid speculum was placed into place. intravitreal injection was performed with a 30-gauge needle at 3.5 to 4 mm from the inferotemporal limbus. pressure was applied to the injection site using a sterile cotton swab, for 1 minute. the changes in bcva and fct between baseline and the 6 month follow - up were analyzed with a 1-tailed, paired t - test. for continuous variables, medians for baseline, final values, change, and percent change were compared between bevacizumab and ranibizumab treatment groups using a t - test or a wilcoxon rank sum tests. the off - label nature of the treatment and its potential risks and benefits were discussed in detail with all patients, and signed informed consent was obtained from all patients. patients received 1.25 mg of bevacizumab or 0.5 mg of ranibizumab. prior to administration of the injection, topical anesthesia was applied, and 10% povidone - iodine was used for scrubbing of eyelid and lashes. following the application of povidone - iodine eyedrops (1.25%), a sterile lid speculum was placed into place. intravitreal injection was performed with a 30-gauge needle at 3.5 to 4 mm from the inferotemporal limbus. pressure was applied to the injection site using a sterile cotton swab, for 1 minute. the changes in bcva and fct between baseline and the 6 month follow - up were analyzed with a 1-tailed, paired t - test. for continuous variables, medians for baseline, final values, change, and percent change were compared between bevacizumab and ranibizumab treatment groups using a t - test or a wilcoxon rank sum tests. bevacizumab - treated and ranibizumab - treated patients had similar baseline characteristics for age, sex, distribution of baseline bcva, location of polyps, or fct (table 1). all patients were korean, and no systemic adverse events were recorded for any of the patients treated with intravitreal injection. no complications, including issues such as endophthalmitis, traumatic lens injury, or retinal detachment, were associated with intravitreal injections. the average number of injections was 3.31 1.25 in the bevacizumab group and 3.44 0.92 in the ranibizumab group. at baseline, the mean bcva (standard deviation, sd) in the bevacizumab and ranibizumab groups was 0.92 (0.54 ; snellen equivalent, 20 / 166) and 0.96 (0.57 ; snellen equivalent, 20 / 182), respectively. six months after treatment, both bevacizumab and ranibizumab groups had significantly increased bcva to 0.74 (0.51 ; snellen equivalent, 20 / 110 ; p = 0.03) and 0.78 (0.43 ; snellen equiva lent, 20 / 120 ; p = 0.01) respectively (table 2, figs . 1 and 2). there was no statistically significant difference in bcva improvement achieved between these two groups (p = 0.83). six (10.3%) eyes out of 58 eyes in the bevacizumab group and 5 (10.0%) eyes (10.0%) out of 52 eyes in the ranibizumab group showed a loss of 3 lines of visual acuity. in either group, no significant difference in proportion of more than 3 lines of visual acuity loss was observed (p = 0.82). there was also no significant difference in proportion of more than 3 lines of visual acuity gain in either group (p = 0.12) (table 3). mean (sd) fct at baseline in the bevacizumab and ranibizumab groups was 322 (62.48) m and 338 (50.79) m, respectively. six months after treatment, fct of both the bevacizumab and ranibizumab groups were significantly decreased to 274 (40.77) m (p = 0.02) and 286 (36.93) m (p = 0.02), respectively. there was no statistically significant difference in reduction of fct in either group (p = 0.74) (table 2). twenty four of 58 eyes (41.4%) in the bevacizumab - treated group showed a decrease of more than 10% from baseline fct. thirty one eyes out of 52 eyes (59.6%) in the ranibizumab - treated group showed a decrease of more than 10% from baseline fct. no significant difference in the proportion of decrease greater than 10% from baseline fct was observed in either group (p = 0.35). however, a greater number for 10% decreased fct was observed in the ranibizumab group, and the difference was statistically significant (p = 0.02) (table 3). polyp regression was found in 12 of 58 eyes (20.7%) in the bevacizumab group and in 13 of 52 eyes (25.0%) in the ranibizumab group. no significant difference in polyp regression rate was observed between groups (p = 0.87) (table 3). pcv is increasingly recognized as a major cause of vision loss throughout the world; however, the incidence of pcv is especially high in asian countries and in asian people throughout the world. the most important causes of severe visual loss include repeated subretinal hemorrhage and leakage from pcv lesions, repeated injuries ing in atrophy of rpe, or scar change. although the pathophysiology of pcv is poorly understood, resolving subretinal hemorrhage and decreasing leakage from pcv lesions may be an important and reasonable approach to stabilize visual acuity. additionally, vegf concentrations in the aqueous have been found to be markedly increased in pcv eyes, and a strong expression of vegf was observed in pre cells of pcv specimens. collectively, evidence appears to support the success of anti - vegf treatment for pcv eyes. ranibizumab, which is specifically for intraocular use, has several theoretical advantages over bevacizumab. ranibizumab is a humanized anti - vegf antibody that inhibits all forms of biologically active vegf - a; treatment with ranibizumab appears to significantly decrease bleeding and exudation in pcv. bevacizumab (a humanized full length anti - vegf antibody) also appears to have a treatment effect in pcv eyes. considering the molecular weight of each medication (ranibizumab is a 48-kda fab fragment, whereas bevacizumab is a complete 149-kda antibody), ranibizumab may be more effective in treatment of pcv because of its smaller molecular weight and possible deeper penetration to choroidal vascular abnormality lesions in those with pcv. moreover, ranibizumab is affinity - matured and may provide better vegf inhibition through stronger molecular binding, compared with bevacizumab. in comparison, penetration of the neural retina up to the choriocapillaries by intravitreal bevacizumab has been demonstrated. and therefore, it may be possible that the clinical efficacies of these two drugs might differ. in the current study, both drugs significantly improved visual acuity in the short term, and showed similar increases in their mean visual acuity from baseline. however, despite the lack of power to determine small changes in visual acuity between bevacizumab and ranibizumab, our revealed a trend toward a greater number for 3-line improvements in the ranibizumab group. although it is uncertain what factors made this difference in visual acuity between the two groups, these showing a significantly greater number for 10% decreased cft in the ranibizumab group could have an association. macular edema based on fct significantly improved in both groups and showed similar decreases in mean fct from baseline. a similar decrease in macular edema was noted after three monthly bevacizumab injections in a study by lai et al.. additionally, kokame et al. reported improvement in macular edema after 6 monthly ranibizumab injections. findings from these previous reports as well as those of our study collectively suggest that short - term continuous anti - vegf therapy doses do have a significant effect in reducing macular edema but not in all pcv eyes. in this study, our revealed a significantly greater number of patients who showed 10% decrease in fct in the ranibizumab group compared to that in the bevacizumab group. it may be that the difference in changes of fct between the two groups ed from differences in penetration ability; ranibizumab may have been more anatomically effective than bevacizumab in reducing macular edema with pcv lesions. further, these imply that there is a distinct difference in short - term biologic activity between bevacizumab and ranibizumab. the choroidal vascular branching network and polypoidal complexes have been resistant and poorly responsive to anti - vegf therapy with bevacizumab or ranibizumab. in the gomi et al. and the gomi et al. studies, choroidal vascular abnormalities remained in ten of 11 eyes after one to three intermittent injections of bevacizumab. kokame et al. found that polypoidal complex decreased in four of 12 eyes (33%) after 6 continuous monthly ranibizumab injections. in the current study , polypoidal lesions appeared to be resistant to both anti - vegf agent, and the polypoidal complex showed a decrease in only 12 of 58 eyes (20.7%) in the bevacizumab group and 13 of 52 eyes (25.0%) in the ranibizumab group. even though ranibizumab has a theoretically better ability to penetrate through the retina and rpe to the choroidal vascular abnormalities of pcv the location of the pcv vessels beneath the rpe may prevent sufficient penetration of anti - vegf drugs to induce pcv regression. this suggests that pcv may be a different inner choroidal vascular abnormality and not just a variant of choroidal neovascularizatoin (cnv). in recent studies , pdt has shown good in reduction of leakage and regression of polyps in pcv eyes. particularly, in the everest study, the first randomized and prospective study of pcv treatment, a pdt combination of ranibizumab and pdt monotherapy was effective in completely regressing polyps at month 6 than was ranibizumab monotherapy. however, there was no significant difference in improvement of visual acuity from baseline between pdt combination with the ranibizumab group and the ranibizumab monotherapy group. in addition, severe visual loss due to extensive subretinal hemorrhage is not uncommon after pdt, and pdt itself can in a temporary increase in vegf. in terms of visual outcome, despite weakness in polyp regression, anti - vegf monotherapy could be considered for pcv in cases with minimal polyp lesions or in cases with only a branching vascular network. we await long - term of the everest trial, which can confirm which modality is superior for treatment of pcv. more clinical and basic science studies are necessary to clarify the pathogenesis of pcv and to provide therapeutic guidelines. because of the retrospective nature of the study the inherent bias that exists in this study. and because the treatment choice was left to the discretion of the patient and treating physician, some potential for bias does exist. however, in our institute, the preferred pcv treatment with anti - vegf (except for pdt) shifted from bevacizumab to ranibizumab from 2008 to 2009. almost all patients were treated with bevacizumab from 2008 to the first half of 2009 and with ranibizumab from the second half of 2009 to date. as a , this study could be more comparative. moreover, the similarity in baseline characteristics between the two groups suggests that the groups were well balanced. another limitation in this study was the absence of a strict protocol for measuring visual acuity, which led to some of the variances in visual acuity that were noted in the two groups and may limit interpretation of these visual acuity . however, we were able to significantly identify trends in visual improvement after anti - vegf injection for pcv eyes. a planned randomized, controlled study would be necessary for a more precise determination of the differences between these two treatments. in summary, bevacizumab and ranibizumab have similar effects on stabilization of visual acuity and macular edema with pcv eyes in the short term. however, ranibizumab appears to be superior to bevacizumab with regard to short - term ability to decrease exudation. additionally, there is a trend suggesting that ranibizumab may also provide superior visual acuity. although this study could not provide definite proof that there were significant differences in the visual acuity as of pcv eyes treated with bevacizumab or ranibizumab, these appear to demonstrate a possible difference in the biologic activities of the two anti - vegf agents. these should be considered clinically when performing combination therapy with pdt or when deciding on a course of anti - vegf agent for treatment of pcv eyes.

purposeto compare the effectiveness of intravitreal injections of bevacizumab and ranibizumab in patients with treatment - naive polypoidal choroidal vasculopathy (pcv).methodsrecords from 106 consecutive patients who received intraviteral bevacizumab (n = 58, 1.25 mg) or ranibizumab (n = 52, 0.5 mg) for treatment of pcv were retrospectively reviewed. after three initial monthly loading injections, injection was performed as needed. the main outcome measures included best - corrected visual acuity (bcva), foveal central thickness (fct) as assessed by spectral domain optical coherence tomography, and the changes in polypoidal lesions based on an indocyanine green angiography.the average number of injections was 3.31 1.25 in the bevacizumab group and 3.44 0.92 in the ranibizumab group. mean logarithm of the minimum angle of resolution of bcva from baseline to 6 months after injection improved by 0.17 in the bevacizumab group (p = 0.03) and by 0.19 in the ranibizumab group (p = 0.01). average fct decreased from 322 62.48 m to 274 40.77 m in the bevacizumab group (p = 0.02) and from 338 50.79 m to 286 36.93 m in the ranibizumab group (p = 0.02). polyp regression rate was 20.7% (12 of 58 eyes) in the bevacizumab group and 21.2% (11 of 52 eyes) in the ranibizumab group. there was no statistically significant difference between groups in bcva improvement achieved, fct improvement achieved, and polyp regression rate between groups.intravitreal injections of bevacizumab and ranibizumab have similar effects in stabilizing of visual acuity, macular edema, and regression of polypoidal complex in pcv eyes over the short term.

to test the hypothesis that human infections with s. suis occur more commonly than currently recognized, we examined archived serum samples from 73 swine - exposed and 67 non swine - exposed adults living in iowa. these persons had all previously completed occupational history questionnaires detailing their pig exposure and use of personal protective gear. use of materials was approved by the institutional review board at the university of iowa. antibodies to serotype 2 s. suis were measured by an elisa that used whole s. suis cells (serotype 2, strain 3033606) as antigen. the elisa optical density readings for the 73 swine - exposed study participants and 67 non swine - exposed participants were first compared to the positive - control mean option density read per plate per dilution. optical densities greater or equal to mean positive - control optical density were considered positive. to be conservative, again, when a conservative approach was used, the lowest titer among duplicates was considered as the final antibody titer. we used the fisher exact test to test the null hypothesis that the exposed group does not have higher incidence of antibody titer > 10 than the non swine - exposed group does. we tested a similar hypothesis for specific risk groups exposed to swine (such as nursing or finishing swine, use of gloves) compared with groups not exposed to swine. seven (9.6%) of 73 swine - exposed study participants were positive, and 1 (1.5%) of the 67 non swine - exposed participants was positive. study participants who work with both finishing and nursery swine had 8.8 the odds of having a titer > 10 when compared to nonexposed study participants (exact 95% confidence interval 1.1406.3). we identified no positive persons in the group that worked solely with nursery swine, a somewhat unexpected finding because most s. suis disease occurs in young pigs. however, our study had relatively few persons who worked exclusively with nursery swine (11/73); most participants worked with both nursery and finishing swine. additionally, no farm - level data on prevalence of s. suis where these persons were employed were collected; therefore, whether those persons worked on farms where s. suis had been confirmed is not known. other factors such as age, gender, use of tobacco products, and use of gloves when working with animals were not statistically significant (table). * an antibody titer was considered positive when its optical density was greater or equal to mean positive control optical density. in this cross - sectional pilot study, we found that more swine - exposed persons had higher titers of antibodies to s. suis than did non these data suggest that human infection with s. suis is more common in the united states than currently thought. two possible reasons stand out regarding the lack of human s. suis disease in the united states. one possibility is underdiagnosis or misdiagnosis, rather than a true absence of the disease. supporting this hypothesis are reports showing that s. suis has been mistaken for enterococci, listeria spp., viridans streptococcus, or streptococcus pneumoniae. because of this potential misclassification , previous publications have asserted that s. suis should be considered in the differential diagnosis of septicemia, especially when complicated by meningitis in adults with a recent history of contact with pigs or unprocessed pork. a second possibility is that s. suis strains colonizing swine in the united states may be less virulent than asian strains and therefore unlikely to cause overt human disease even when transferred between species. this possibility is supported by molecular analyses showing that many us strains belong to sequence type (st) 25, whereas most virulent serotype 2 isolates have been st1. finally, these hypotheses are not mutually exclusive; both underdiagnosis / misdiagnosis and the circulation of lower - virulence strains may be occurring, ing in fewer diagnoses of human s. suis infections in north america. s. suis infection is an important occupational disease in humans in many countries. in research conducted in s. suis endemic countries, the annual incidence of s. suis meningitis was 3 cases/100,000 swine - exposed people: roughly 1,500 higher than the rate in the nonexposed population. because of this risk, it has been recommended that persons in daily contact with pigs or pig meat should use protective gloves to avoid skin trauma and subsequent risk for exposure to the bacterium. because no human vaccine against s. suis exists, suitable preventive measures coupled with education and supervision of those who come in contact with live swine or unprocessed pork are important to decrease the transmission of this organism to humans. however, few studies have been conducted to detect subclinical cases of s. suis infection; therefore, the true incidence of infection among the swine - exposed is unknown. because our findings only examined 1 serotype of s. suis, our may not accurately reflect antibody prevalence. because we used a whole - cell elisa , some antibody reactions may be due to cross - reacting antibodies to other serotypes of s. suis or other species of streptococcus. however, using a slightly different method and population, other investigators found a higher seroprevalence, particularly among farmers and meat inspectors. this difference may stem partly from the fact that we used a conservative criterion for considering a sample positive, which may further underestimate seroprevalence in our group. for example, 4 study participants (3 swine exposed) were classified as having a titer < 10 because the first dilution (1:10) was negative in 1 repeat test. acquisition of human positive control serum as a standard to test our assay would enable us to make more definitive comparisons. finally, because the samples analyzed for this pilot study were not collected to specifically assess s. suis infections, more definitive future prospective studies seem indicated. one limitation of this serologic study is that it does not enable us to distinguish antibodies generated as a of true infection versus exposure to s. suis antigens present in manure or dust in the facility, for example. additionally, because the questionnaire did not include information on pork consumption or handling of raw pork, those factors could not be examined as potential risks. future studies might include targeted questionnaires, attempts of bacterial isolation, and serial sera collections to examine serologic evidence of infection.

despite numerous cases of human infection with streptococcus suis worldwide, human disease is rarely diagnosed in north america. we studied 73 swine - exposed and 67 non swine - exposed us adults for antibodies to s. suis serotype 2. serologic data suggest that human infection with s. suis occurs more frequently than currently documented.

from the time of adam's creation, god has gifted humans with delivery to have offspring. it is a spontaneous process that needs no intervention, and cesarean section (cs) is conducted only when natural delivery is contraindicated to protect mother's and infant's health. nowadays, unfortunately, having a cs has become a culture for escaping pain and has influenced public health. this is when cs imposes more risk to the mother, compared to vaginal delivery, and includes complications such as endomyometritis, bleeding, thromboembolism, preterm labor and mortality (in mother), and respiratory distress syndrome, resistant pulmonary hypertension, and damages like injury, bruise, or other traumas in infant. the incidence of cesarean delivery (cd) is 1015% worldwide, while in some iranian cities it is reported as 47%. some studies report that 65% of cs conducted in iran are unnecessary and optional (elective cesarean), which may be due to lake of knowledge, negative attitude toward normal delivery, fetal health, prevention of urogenital lacerations, fear of pain, change in sexual relationships, persistence of spouse, insurance for expenses, and others experiences. nowadays, in most of the developed countries , it has been tried to reduce cs and its complications by interventions such as physicians education and change in their attitude. in iran for instance, tavasoli et al. reported a reduction in rate of cs by 15% through mothers education and their psychological and mental preparation to have a natural delivery. but despite these interventions, the rate of cs is growing in developing societies. one of the important factors that play an unnecessary role in increase of cs is fear of childbirth. there are notable evidences on the primary role of fear of childbirth in the demand for cs, as well as the effect of fear on negative experiences of delivery and emergency cs. about 36% of swedish women claimed fear of pain to be the main reason for selecting cs. the main challenge of a pregnant woman that in their hesitation is taking decision on the mode of delivery. a person whose decision - making is a part of his / her existence experiences fear as a major problem in life. humans naturally fear of what is going to happen the next day or what he / she can achieve. fear, anxiety, and pain are three factors that play a key role at the time of delivery. some reports in sweden and finland showed that half of the women experiencing the fear of childbirth cancelled their request for cs and had a successful vaginal delivery after attaining the ability to express their anxiety and fear and having an interview with a trained person. one of the most effective ways to cope with fear is non - medicational methods including education and giving proper information to the pregnant women. research showed that an increase in knowledge and information influences individuals ability to recognize key points and enhances their understanding and perception. meanwhile, shortage of knowledge causes fear and anxiety which negatively affect decision - making. melender reported an association between absenteeism of primiparous women in delivery preparation classes and pregnancy anxiety, fear of childbirth, and consequently, a request for cs. recently, selection of the most efficient educational method with emphasis on learners skills, abilities, and active participation has been reported as one of the main and basic principles in health care. one of these methods is use of stories and scenarios which are adopted in role play education. role play is one of the new educational methods that tries to help individuals find their meaning in the social world and get help from a social group to make a decision on the solution of a dilemma. in this method, also, the learners discover human - related issues by facing problematic situations and discussing about these situations. nowadays , role play has found numerous applications such as acting as a tool for emotional drain and expression of fears and feelings, and a method for attitude change or formation of insight in referrals and a method for education of new behaviors. therefore, with regard to high prevalence of cs in iran and the effect of fear of childbirth on selection of this method, as well as the restricted research in this field and the mode of research (descriptive research), this study aimed to investigate the effect of role play education on primiparous women's fear of natural delivery and their decision on the mode of delivery. this blind clinical trial was conducted on 67 primiparous women (35 in role play group and 32 in lecture education group) referring to the health care centers in three cities of mashhad province after obtaining the approval of ethics committee of mashhad university of medical sciences. as no report has been published in relation to the effect of role play on the fear of childbirth, based on a pilot study, sampling was by cluster sampling conducted among the health care centers of region number three. two centers were assigned to role play method, two to lecture, and from the remaining centers, three subcenters were assigned to role play and three to lecture method. to prevent bias in education, it was tried to conduct education in identical conditions concerning the place of education, level of noise, educational equipments, time of the day, etc. as the routine educational method in health care centers is lecture, there was no need for a control group. inclusion criteria were: no experience of acute psychological emotions, delivery and childbirth fear score > 28, primiparous, single pregnancy, gestational age of 3436 weeks, age of 1835 years, no history of infertility, no indication for cs, and not having passed educational course for delivery methods. exclusion criteria were: some medical condition in pregnant woman, diagnosis of abnormal fetus / no possibility for delivery of fetus on sonography, and abnormal volume of amniotic fluid or placenta. to investigate pregnant women's decision, a researcher - made checklist containing one question and to investigate the fear of childbirth, harman childbirth attitude questionnaire (caq) which was revised by lowe were adopted. firstly, two english language experts separately translated the questionnaire into persian and two others back - translated that into english. after finalization, the version obtained by the bilingual translator was compared with the original version of the questionnaire. the questionnaire has 14 items rated on a four - point likert's scale (never, seldom, sometimes, and often). as there was no cut - off point for the fear of childbirth, similar international studies were reviewed to determine the cut - off point and a median score of 28 was considered for fear of childbirth. for checking the validity of caq questionnaire, content validity, and reliability, cronbach's alpha and internal consistency of the questionnaire were used (a = 0.84). validity of the decision - making checklist before intervention, 2 weeks after intervention, and of decision - making on the mode of delivery at admission in the labor were confirmed through content validity and their reliability was obtained by evaluators consensus (r = 0.974). the two groups of lecture and role playing were divided into four subgroups after taking pre - test. the role playing group was divided into two subgroups of 10 subjects each and another two subgroups of 9 subjects each (38 subjects). each group was instructed in a 90-min session about the advantages and disadvantages of normal delivery and cs. in this method, the researcher with two other co - researchers played three scenarios in seven steps (for each scenario) including warm up, selecting the participant, preparing the scene, preparing observers, play, discussion and evaluation, and generalization to education about the advantages and disadvantages of normal delivery and cs. in the warm - up stage, the researcher narrated two true stories about the individuals who were wondering about the selection of the mode of delivery due to fear of childbirth and asked the participants to voluntarily accept to play the role of pregnant woman with the researcher and two co - researchers. then the participants helped the researcher to prepare and process the scene (scene preparation was conducted with the needed equipments for role play in two scenarios), and the observers were asked to pay close attention to the scenarios, taking important notes, and discuss them at the end of scenario. in scenarios, the reasons for mothers fear of natural delivery and cs were discussed. in the first scenario, one of the participants (a pregnant woman) played the role of a woman who referred to a midwife's office to select the mode of delivery and witnessed the events occurring in the office. the midwife talked to her about the two types of delivery impractically and asked her to express her decision after the scenario about choosing the type of delivery. after choosing the type of delivery, participants discussed the pregnant woman's selection (same researcher gave no help) and justified each other to come to a . the second scenario was about a woman with a normal delivery and the benefits and complications experienced by her. the next step was similar to the first scenario. in the third scenario, one of the co - researchers defended cs and another defended normal delivery at judge (natural delivery and cs appeared like a human and judge is researcher). after these three scenarios, participants were asked to talk about their friends/relatives experiences of the two types of delivery. lecture group (two subgroups of 10 subjects each and two subgroups of 9 subjects each) was instructed using powerpoint presentation, marker, and whiteboard in a 90-min session. at the end of the session, two weeks after administration of educational session in each group (lecture and role play), the caq was filled by the pregnant women to measure their fear, and further follow - ups concerning women's decision on delivery mode continued at the time of admission and in the maternity unit. finally, the researcher recorded the favorite mode of delivery of participants through phone calls. data were analyzed by spss, and paired t - test, independent t - test, and chi - square were used for analysis. participants were aged 24 4 years, of whom 58% were housewives and 14.9% were employees. educational level of most of the participants (51.4%) was up to high school. majority of their husbands had an educational level up to junior high school (38.8%) and they were workers (44.3%). two groups showed no significant differences in terms of age, educational level of pregnant woman and her husband, income, job of pregnant woman and her husband, income, source of information about the type of delivery, insurance, time of referring for pregnancy care, and suggestion of mother, husband, friends, relatives, and gynecologist about the type of delivery (p < 0.05). table 1 shows a significant reduction in the mean score of fear about the mode of delivery after intervention in both groups of role play and lecture, compared to before intervention (p = 0.001 in role play group and p = 0.047 in lecture group). the obtained also showed a significant difference in the mean score of fear after intervention in both groups of role play and lecture (p = 0.007). this difference was not significant before intervention (p = 0.074). comparison of primiparous women's mean scores of fear before and two weeks after intervention in two groups of lecture and role play fischer's exact test was used for comparing the frequency of primiparous woman's decision - making about the type of delivery before, 2 weeks after intervention, and at admission to maternity department. as to the , no significant difference was found; however, it was significant at admission (p = 0.001). comparing frequency of primiparous woman's decision making about type of delivery before, two weeks after intervention and at admission to maternity department chi - square test showed no significant difference in the frequency of mode of delivery (pregnant women 's function) in the two groups (p = 0.117). comparison of frequencies of primiparous women's mode of delivery in two groups of role play and lecture the obtained showed that education through role play is effective on reduction of fear of childbirth and increase of natural delivery. although there was no significant difference in the mode of delivery in the present study (p = 0.117), the rate of elective cs in the lecture group was fivefold more compared to that in the role play group. the goal of antepartum education is to reduce mother's request for elective cs, which was achieved in the present study. as no study was found on the effect of role play on pregnant women's decision - making and fear, the will be compared with the effects of other educational methods on fear and selecting the type of delivery. in the present study, the mean score of fear was higher in the lecture group after intervention, compared to the role play group. ryding states that the fear of delivery in third trimester can be associated with the occurrence of emergency cs. johnson and stlid showed no difference in the fear of delivery between women with natural delivery and those with elective or emergency cs, revealing the effect of fear of childbirth in various cultures. in a study on the effect of group therapy in reducing the fear of childbirth, reported that 85% of women cancelled their request for cs after intervention. mehdizadeh et al., in a study on the effect of delivery preparation classes on reduction of cs, obtained similar . meanwhile, fahami, in a study on the effect of lazmar exercises on the outcome of primiparous women's pregnancy, found no significant difference in the rate of cs between the two educational groups of lazmar technique and control. a person whose decision - making is a part of his / her existence experiences fear as a major problem in his / her life. a fear - captivated mind is always wondering and in conflict, and is the main concern of pregnant women which brings about hesitation in decision - making on their mode of delivery. being bombarded with negative information and losing power, they experience high fear, which reduces their ability of correct decision - making. khorsandi et al., in a study on the effect relaxation on reduction of cs, showed that relaxation is effective on reducing the fear of childbirth and increasing natural delivery. in khorsandi's study, the mean scores of childbirth fear in relaxation and control (receiving conventional care) groups were 40.71 (6.23) and 39.35 (6.96), respectively, before intervention and 38.03 (9.27) and 29.82 (7.18), respectively, after intervention. the difference between the present study and that of khorsandi et al. was that in their study, each group separately received educational program (including nine 90-min sessions) from the second trimester and used actual play, role play, lecture, and educational cd for education on relaxation, but the control group received only routine care. meanwhile, in the present study, role play method was conducted just in one group to reduce pregnant women's fear of childbirth and the education included one 90-min session. then, the obtained were compared with those of lecture method of education. in the present study, the difference in fear score was significant in the two groups of role play and lecture, and the score of pregnant women's fear was reduced leading to a reduction in an elective cs request at admission in the maternity unit and, consequently, a lower number of elective css. therefore, the present study managed to obtain acceptable with less time consumed in educating mothers and lower educational costs, compared to the study of khorsandi. nowadays, role play has applications such as emotional drain, expression of fears and feelings, attitude change, making an insight in referrals, and education of new behaviors. in the present study , role play could drive pregnant women to select the best mode of delivery by giving them a way to empty their emotions and reduce their fear. in the lecture group, 25% of women probably and 40.6% two weeks after intervention, the possibility of a decision on natural delivery was reduced by 6.2% and its absoluteness increased by 31.3%. but at the time of admission in the labor ward, this possibility decreased by 3.2%, the absoluteness of natural delivery decreased by 12.5%, and the decision on cs increased, compared to before intervention. on the contrary, in role play group, 31.4% and 57.1% of women before intervention would possibly and absolutely select natural delivery, respectively. two weeks after intervention, 11.4% decrease of probable decision and 22.9% increase to absolute decision were observed. at the time of admission in the labor ward, therefore, role play could change pregnant women's hesitation to decisiveness. in fathian's study, behavioral intent model (based on this theory, people firstly make decision and behave based on rational and logical review of available information, and secondly, people consider the outcomes and of their function before making a decision) was found to be effective in changing pregnant women's attitude toward normal delivery, in comparison with routine pregnancy care (p = 0.007). in fathian's study, 74.3% of pregnant women of the experimental group (25.7% absolute and 48.6% probable) and 71.4% of the control group (30.0% absolute and 41.4% probable) intended to undergo normal delivery before intervention. after intervention, normal delivery intention was 92.9% in the experimental group and 49.8% in the control group. so, intention to normal delivery has increased by 18.6% in the experimental group and has decreased by 21.6% in the control group. this could be indicated as insufficient routine pregnancy care which has decreased by approaching to delivery time. the difference between the present study and fathian's study is that in our study, pregnant women's decision - making was studied in three sections (before intervention, 2 weeks after intervention, and at admission) while fathian studied it before and after intervention. at admission , many factors could affect the decision on the type of delivery. in this study, although increase in decision - making for normal delivery after intervention in role play in comparison with behavioral intent model was 11.518.6%, this increase led to 100% final decision for normal delivery, which remained 100% up to admission in the maternity department, while fathian did not compare the effect of behavioral intent model after intervention and at admission. in fathian's study, the type of delivery performed in the two groups was not significant (p = 0.002); however, the elective css in the presence and absence of midwifery problems were not studied. in the present study, although no significant difference was found in the type of performed delivery (p = 0.17), the elective cesarean section in the lecture group was 5 times as much as in the role play group. the aim of education during pregnancy period was to bring about a decrease in elective cesarean by mothers, which was achieved in this study. lashgari et al. conducted a study entitled effect of training programs of pregnant women on their delivery type selection: a single blind, randomized control trial. the test group used different structural methods such as film, booklet / lecture notes, visiting the maternity department and interviewing the women who had delivery. showed significant different between the test and control (no education) groups in term of the type of delivery selected (p = 0.03). education could increase the decision taken toward normal delivery to 14%; however, in the present study, role playing could increase the decision for normal delivery to 100%, which remained unchanged up to time of delivery. in fact, the role play method could increase the decision for normal delivery in a short time (90 min) in comparison with the study of lashgari (180 min) and sustain the decision made. in lashgari's study, pregnant women's decision at admission to the maternity department was not studied, so there was no data to be compared in this part. in rahimi kian et al.'s study entitled the effect of education based on health belief model on selecting type of delivery, the test group used different structural methods such as lecture, question and answer, film presentation of normal delivery and cs, and pamphlets about normal delivery and cs. the showed a significant difference between the two groups in terms of the type of delivery selected (p = 0.001) (control group received routine pregnancy care). as rahimi kian did not report the rate of normal delivery and cs before intervention, there was no data to compare with the present study. although the educational intervention of rahimi kian, fathian, and lashgari could increase the rate of normal delivery, none of them studied the sustainability of this decision at the beginning of labor that is the main situation for decision - making, and finally, the type of performed delivery. of the strengths of the present study is 100% decision - making for normal delivery, sustained decision up to admission to the maternity department, and decrease in elective cesarean in the role play group in comparison with the lecture group. in role play education, it is tried to help people find their internal meaning in the social world and get help from it to make a decision while facing a dilemma. the methods, which need active involvement, including role play, increase the individuals association to the message and lead to individuals emotional drain. therefore, this issue can be one of the reasons for an increase in pregnant women's decision to undergo natural delivery and reduction of elective cs in role play method, compared to lecture method. it is suggested to conduct further studies on the effect of role play education on pregnant women's fear of childbirth and selection of mode of delivery, and compare it with other educational methods in order to use the most efficient educational method to reduce elective cs. our emphasize on the effect of role play education in reducing the fear of childbirth and unnecessary cs. application of active educational methods such as role play, accompanied with lecture method, is suggested to reduce primiparous women's fear of childbirth and, consequently, reduce unnecessary cs.

: the number of women who select cesarean section due to fear of childbirth has increased. role play education seems to be a helpful method to remove or reduce the fear of childbirth. therefore, this study aimed to investigate the effect of role play education on primiparous women's fear of natural delivery and their decision on the mode of delivery.materials and methods: in this blind clinical trial, 67 primiparous women with natural pregnancy at 3436 weeks of gestational age and with no indication of cesarean section were selected from the health care centers in mashhad. they were randomly assigned to two groups who underwent pre - test and post - test with the help of delivery attitude questionnaire to investigate their fear of childbirth and a researcher - made pregnant women's decision investigation questionnaire. education through role play was conducted in the form of three scenarios during seven stages. the findings were analyzed by fisher's exact test and independent t - test through spss.:the two groups were significantly different concerning the fear of childbirth after the intervention (p = 0.007), and the fear score showed a higher reduction in the role play group compared to the lecture group. there was a significant difference between the two groups concerning the reduction of elective cesarean section and the decision on the mode of delivery at the time of admission in the labor room (p = 0.000). about 75% in the lecture group and 100% in the role play group selected natural delivery.:in the present study, the effect of role play was more in making a decision on natural delivery, reducing the fear of childbirth, and reducing the rate of elective cesarean section. it is suggested to use role play method to educate pregnant women to reduce the rate of cesarean sections.

gastric volvulus can be acute, acute on chronic or chronic, and may be primary or secondary to anomalies that affect the fixity of the stomach like hiatus hernia, eventration of diaphragm or congenital diaphragmatic hernia (cdh). the association of bronchopulmonary foregut malformations such as congenital cystic adenomatoid malformation of lung and bronchopulmonary sequestration (bps) with cdh has also been well described in the literature. herein, we describe a 4-year - old girl with a rare presentation of gastric volvulus and pulmonary sequestration in association with cdh. a 4-year - old girl presented to our casualty with upper abdominal pain, distension, and nonbilious vomiting for the last two days. her mother gave a history of recurrent episodes of similar symptoms every month for the last six months, and each episode lasted for 4 - 5 h. the symptoms used to diminish spontaneously. her perinatal history was unremarkable. a preliminary work up for her past symptoms in another hospital revealed that she had a suspected left sided eventration as seen on the chest x - ray. on examination, the child was lethargic, obtunded, had cold peripheries with tachycardia, hypotension, and feeble pulse. a nasogastric tube insertion was attempted that failed initially but subsequently drained hemorrhagic gastric contents. a skiagram at this stage revealed a gastric bubble, with a paucity of distal bowel gas and a raised left hemi diaphragm. (a) radiograph of the abdomen prior to presentation at our institute showing evidence of viscus in left hemithorax. note the paucity of bowel gas excluding the single stomach gas shadow suggesting the possibility of a volvulus after adequate resuscitation, the child was explored, and the intra - operative findings revealed a mesoaxial gastric volvulus. after successful reduction, it was noted that the stomach was grossly healthy except the greater curvature, which was congested. there was a large defect in the posterolateral aspect of the left hemi - diaphragm, which was covered with a sac and the diaphragm had a reasonably well - developed rim of muscle along the anterior segment. (a) defect in the diaphragm is seen with well - defined anterior lip (arrows). (b) intraoperative image of the mass seen to occupy the cranial aspect of the diaphragmatic hernia sac (cut edges indicated by arrows) to repair the defect the sac was excised and it was noted that the cephalic surface of the sac had a lobular 6 cm 3 cm fleshy pedicled mass flimsily attached to it. the pedicle of the blood supply was traced and it was found to be entering the mediastinum. the repair was completed by primary interrupted sutures, and a three - point gastropexy was done to prevent recurrences. the histopathology report of the mass indicated that it was an extra - pulmonary bps. the stomach is prevented from rotating along its axis by the ligamentous supports like the gastrocolic, gastrohepatic, gastrophrenic and the gastrosplenic ligaments. the gastroesophageal junction and the pylorus are two additional fixed points that afford protection against volvulus. when there is a laxity in these supports and the stomach rotates more than 180, a primary gastric volvulus . other conditions like hiatus hernia and cdh predisposes to secondary volvulus. based on the two axes along which this rotation takes place it could produce either an organo - axial, mesentero - axial or a mixed volvulus. it has been noted from the literature that the secondary gastric volvulus in children usually is associated with the mesentero - axial type of volvulus as was seen in our case. an astute clinician can diagnose acute gastric volvulus by the typical complaint of sudden onset upper abdominal pain with retching and inability to pass a nasogastric tube - the borchardt's triad. this symptom complex was also present in our case except for retching, but the radiographs endorsed our clinical suspicion. the exploration revealed a mass on the superior aspect of the sac, which looked like an extrapulmonary bronchopulmonary sequestration (ebps). these are non - functional pulmonary tissues with the systemic blood supply that seldom communicate with the tracheobronchial tree. bps comprise approximately 6% of all congenital malformations, and these are associated with cdh in 30 - 40% of cases. this distinction is made depending on whether or not the visceral pleura invests the sequestered lesion, with the latter referred to as ebps. although the above associations with cdh are common but cdh presenting with acute gastric volvulus and with an underlying ebps is very rare in pediatric age group and is described only once in english literature. had reported a case similar to ours except that the child had an eventration of the diaphragm. another case report in spanish describes an adult with bochdalek's hernia with stomach volvulus and extrapulmonary sequestration presenting as acute respiratory distress. the aim of this report is to reinforce pediatric surgeons to look for underlying secondary causes of gastric volvulus as most of them can be tackled simultaneously. an awareness of this triad of cdh, gastric volvulus, and bps can in early detection and safe resection of bps and thus avoid future diagnostic dilemmas.

congenital diaphragmatic hernia (cdh) is a known cause of secondary gastric volvulus. it is also known that bronchopulmonary sequestration (bps) may be associated with cdh. an extremely rare case of bps associated with gastric volvulus in a girl with left sided cdh is being reported.

most ice in nature forms because of impurities which boost the exceedingly low nucleation rate of pure supercooled water. however, the microscopic details of ice nucleation on these substances remain largely unknown. here, we have unraveled the molecular mechanism and the kinetics of ice formation on kaolinite, a clay mineral playing a key role in climate science. we find that the formation of ice at strong supercooling in the presence of this clay is about 20 orders of magnitude faster than homogeneous freezing. the critical nucleus is substantially smaller than that found for homogeneous nucleation and, in contrast to the predictions of classical nucleation theory (cnt), it has a strong two - dimensional character. nonetheless, we show that cnt describes correctly the formation of ice at this complex interface. kaolinite also promotes the exclusive nucleation of hexagonal ice, as opposed to homogeneous freezing where a mixture of cubic and hexagonal polytypes is observed.

in nature, the selective halogenation of nonactivated carbon atoms is performed by mononuclear nonheme fe(ii) (mnh), o2, and -ketoglutaric acid (-kg) dependent halogenases. the structural rationale of this chemically challenging reaction has been elucidated rather recently, and to date only a handful of mnh halogenases have been biochemically characterized. the molecular mechanism of these halogenases follows, in principle, that of the closely related -kg dependent hydroxylases. the -kg - dependent hydroxylases have a reactive fe(ii) center coordinated by a 2-his 1-carboxylate motif and three water ligands. upon coordination of the -kg cofactor, two water molecules are replaced but the six - coordinate (6c) geometry of the fe(ii) center is retained. only after substrate binding in the outer coordination sphere is the remaining water ligand displaced from the fe(ii) center and the ing five - coordinate (5c) species is activated for reaction with o2. the -kg cofactor is decarboxylated by the activated dioxygen species, which leads to the formation of a high - spin fe(iv)=o intermediate that abstracts a hydrogen atom from the substrate. while in mnh dependent hydroxylases the reaction proceeds via transfer of the hydroxyl group from the fe(iii)oh intermediate onto the substrate radical, in halogenases an iron - coordinated chloride replaces the carboxylate ligand and successfully competes with the hydroxyl moiety to yield the halogenated product (scheme 1). in the presence of -kg and substrate, o2 binds to the five - coordinate fe(ii) center and decarboxylates the -kg cofactor. this yields a highly reactive fe(iv)=o intermediate that abstracts a proton from the substrate. chloride rather than the hydroxyl moiety is rebound by the substrate radical, ing in a chlorinated reaction product. several reasons have been invoked for the strong preference of halogenation over hydroxylation that has generally been observed in halogenases: (i) the lower redox potential of cl compared to oh; (ii) possible bicarbonate formation between the metal bound hydroxyl group and the -kg derived co2 which prevents the transfer of the hydroxyl moiety; (iii) possible protonation of the hydroxyl group by a nearby glu - arg proton donor that in the formation of water, which makes hydroxylation unfavorable; (iv) the difference in binding strengths of the chloride and hydroxyl group to the metal ion and the ing energetic barrier for hydroxylation; or (v) the positioning of the substrate radical relative to the cl fe(iii)oh center. recently, it has been shown that the o2 reactivity of the -kg - bound halogenase syrb2 in a 5c trigonal bipyramidal fe(iv)=o intermediate with the fe o vector perpendicular to the c h bond of the substrate. h - atom abstraction by an fe(iv)=o * molecular orbital leads to an intermediate where the fe(iii)oh moiety is oriented away from the substrate carbon radical and the halide is primed for rebound halogenation. while substantial efforts have been made to investigate the second half of the halogenases reaction in order to rationalize the preference of substrate chlorination over hydroxylation after proton abstraction, there has not been much focus on the first half of the catalytic cycle. chloride - bound crystal structures of the substrate - free mnh halogenases syrb2, and cura - hal show that the halide can coordinate to the iron even before the substrate binds. on the other hand, in cytc3 no halide is present in the crystal structure with fe(ii) and -kg bound, despite the rather high chloride concentrations in the mother liquor (80 mm). no study has elucidated how the halogenases prevent alternative oxidation reactions in the absence of halide. recently, a fatty acyl - halogenase, hctb from l. majuscula, has been characterized in our laboratory. the enzyme that modifies middle - chain fatty acyl moieties displays an unprecedented three domain organization, which sets it apart from the monodomain amino acyl - halogenases and the multidomain ketide halogenases: an acyl - coenzyme a (acyl - coa) binding protein is n - terminally fused to a halogenase domain, while its c - terminus connects to an acyl - carrier protein (acp) domain. the acp domain bears an inherent thiolation site, where the substrate covalently binds via a phosphopantetheinyl bridge. this composition makes hctb self - sufficient in regard to a substrate binding entity and may be prototypical for fatty acyl - halogenases. the trifunctional enzyme introduces 5-oxo-, 5,5-dichloro-, and 5-chloro-4-vinyl moieties into the hexanoyl substrate under chloride saturating conditions. in the course of the enzyme s biochemical characterization we observed that o2 reduction was triggered by the presence of not only the substrate but also chloride under single turnover conditions. based on these observations, a suspected role of chloride in primary o2 activation in hctb is investigated in this study: circular dichroism (cd), magnetic cd (mcd), and variable - temperature, variable - field (vtvh) mcd spectroscopies directly observe the geometric and electronic structure of the enzymatic fe(ii) active site. in combination with stopped - flow kinetics the spectroscopic data reveal that the metal center is constituted in the absence of a halide ion but remains inert toward o2 and that the presence of chloride is essential for triggering o2 reduction at the metal center. molecular dynamics (md) simulations of the hctb halogenase domain are employed to gain insights into the role of the protein structure in chloride - dependent o2 activation. recombinant nonacylated hctb was expressed, purified, and acylated with the fatty acyl - coa substrate as described previously except the final enzyme solution was exchanged into 20 mm bis - tris buffer (ph 7.5) for stopped - flow analysis. the enzyme was made o2-free by 20 cycles of evacuation and n2-flushing in an airtight v - vial (wheaton, millville / usa) capped with a screw - top septum (supelco, bellefonte / usa), and subsequently 0.95 equiv of fe(ii)so47h2o and -kg (sigma - aldrich, st . louis / usa) were added from stock solutions (10 mm) in a n2-purged glovebox. the enzyme preparations (440550 m active sites) were mixed at 10 c with equal volumes of 20 mm bis - tris buffer, ph 7.5 that contained 1.4 mm o2 if not stated otherwise and either 0.1 or 1 m nacl if required, using an sx20 stopped - flow uv / vis spectrophotometer (applied photophysics, leatherhead / uk), which was equipped with a polychromatic light source. note that in a previous study the addition of 1 equiv of -kg to acylated hctb (100 m) yielded maximum initial o2 consumption rates and thus, in the enzyme kinetic measurements performed here, which used 95% cofactor - loaded active sites (> 100 m), saturating conditions were ensured. spectroscopic analyses were carried out by using an sx20 photodiode array detector, whereby absorptions from 270 to 730 nm were recorded with 400 collected data points in the first 0.4 s and 1 datum point per 100 ms in the remaining 40 or 60 s. absorbance traces of 4 measurements per condition were averaged and fit using the following methods: when rates of signal changes were so slow that they had linear characteristics over the monitored time, rates were determined based on the extinction coefficient of the -kg - fe(ii)-hctb complex (500 nm = 0.15 mm cm). rates were then related to the applied metal concentration to give specific rates. in the case of apparently bi- or triphasic curves, the pro - kineticist 1.0.10 software (applied photophysics) was used and the trace at the respective constant wavelength was fit to sequential models in the form of a b c and a b c d respectively, in order to obtain the respective apparent 1 order net rate constants. if not stated otherwise, for cd / mcd spectroscopy, protein samples were exchanged into deuterated 50 mm hepes / naod buffer pd 7.5, containing 1 m nacl if required, using 4 ml amicon ultra centrifugal filters and concentrated to 1.53.5 mm. o2 was removed from the samples by 20 cycles of evacuation and argon flushing. ferrous ammonium sulfate and -kg were added to the enzyme preparations in microliter quantities from deuterated, anaerobic stock solutions in a n2-purged glovebox, where the final sample was filled into a cd or mcd cell. cd measurements were carried out on a jasco j-730w spectropolarimeter at 283 k. spectra were corrected by subtracting the respective cofactor - free spectrum. mcd samples were prepared from cd samples by adding glassing agent to saturation at approximately 50% sucrose. samples were injected into mcd cells, frozen, and stored in liquid n2 until use. mcd spectra were recorded on a jasco j-730w spectropolarimeter equipped with an oxford instruments spectromag 4000 superconducting magnet and a liquid n2-cooled insb detector. to affirm the authenticity of the mcd signals, field dependencies at 7, + 3.5, and + 7 t were recorded at 5 k. the c - term origin of the signals was confirmed by tracking temperature dependencies at 5, 20, and 40 k at + 7 t (3 scans per condition were averaged). the data obtained were corrected by subtraction of the zero - field spectrum at the respective temperature. mcd spectra were compared to their respective cd counterparts in order to verify that transitions had the same energies in cd and mcd and with and without glassing agent. a structural model of the hctb halogenase md simulations were performed with the yasara structure suite, version 12.11.20 (yasara biosciences). a periodic simulation cell, which comprised the whole enzyme and an additional 5 in each dimension, was used with explicit solvent. the amber99 force field was employed and long - range electrostatic potentials were calculated with the particle mesh ewald (pme) method, with a cutoff of 7.864. force field parameters for -kg were generated with the autosmiles utility, which assigned a van der waals radius of 2.27 and a charge of + 2 to the iron cofactor. fe(ii) parameters were defined to reflect the principal octahedral geometry of the fe(ii) center. an equilibrium spring length that was derived from dft calculations was used for all ligands with a stretching force constant of 125 n m. ligand fe ligand angles of minimum energy were defined as 180 for opposite ligands and as 90 for all others, with angle bending force constants of 1000 kj mol rad. in this way, force field parameters for n- of his112, n- of his228, and -kg coordinating carboxylate and keto - oxygen were defined for the chloride - free hctb model. for the chloride - containing complex note that no force field parameters were defined for glu224. in order to investigate the impact of a putative strongly bound water at the halide position, in a second model the chloride atom was substituted by a water molecule and the respective parameters were adapted accordingly. in a third halide - free model a strongly bound water was positioned in the sixth coordination site of the iron, while the water in the halide binding position remained unrestricted. the oxidation state of the iron cofactor in all complexes was either + 2, as assigned by autosmiles, or in order to assess the impact of the effective iron charge on the simulation adjusted to values in the 0 to + 2 range (see table s2). the hydrogen bonding network was optimized by the method of hooft and co - workers, and yasara s pka values at ph 7.5 were assigned. the simulation cell was filled with water at a density of 0.997 g ml using nacl concentrations of 0.001 and 1 m, respectively. after relaxation of the solvent the system was energy minimized, whereby a steepest descent minimization was applied to remove conformational stress, followed by a simulated annealing minimization until convergence was reached (< 0.05 kj mol per 200 steps). integration time steps were set to 1.33 and 4 fs for intra- and intermolecular forces, respectively. md simulations at 298 k were initiated, whereby integration time steps for intramolecular and intermolecular forces were set to 1.25 and 2.5 fs, respectively. the oxidation of the fe(ii)--kg complex was monitored spectrophotometrically via single turnover stopped - flow measurements by recording the decay of its prototypical metal to ligand charge transfer (mlct) transition (500 nm = 150 m cm). therefore, anaerobic enzyme preparations of acylated hctb (ac - hctb), preloaded with 0.95 mol equiv fe(ii) and -kg, were mixed with o2-enriched (6501400 m) and, optionally, 1 m nacl - containing buffer at a 1:1 ratio. in the absence of nacl the -kg - fe(ii)-ac - hctb complex decayed slowly with a specific rate of 1.1 10 s (co2 = 700 m). by contrast, under analogous conditions but in the presence of chloride, the absorbance band disappeared within 20 s. the trace displayed three distinct phases that could be resolved: a lag phase of 60 ms (ksc1 = 35.1 s) was followed by a phase of fast signal decay, which gave an apparent first - order rate constant ksc2 of 6.74 s and accounted for approximately one - third of the total amplitude. the third phase of the reaction, a slower absorbance decrease (ksc3 = 0.22 s), equaled the o2 depletion rate (0.22 s) that had previously been determined using an o2 sensor under similar conditions (figure 1). (note that ksc1, ksc2, and ksc3 represent apparent first - order net rate constants .) stopped - flow absorption kinetic traces of the -kg - fe(ii)-ac - hctb complex decay. the conversion of the chromophoric -kg - fe(ii) pair was monitored in the absence (gray) and presence (black) of 0.5 m nacl at 500 nm, and average traces were fit with a linear regression (green) and via a three - phasic model using the pro - kineticist software (applied photophysics, see materials and methods section for details), respectively. rates were not significantly dependent on the o2 concentration (table s1, figure s1). the total amplitude of signal decay corresponded to 97% of the -kg - fe(ii) concentration, confirming a quantitative conversion of the complex. it is worth noting that in previous studies it was observed that the fe(iv)=o intermediate had a similar extinction coefficient at 520 nm as the fe(ii)--kg complex but additionally showed a significant absorbance increase at 318 nm (1500 m cm). an analogous signal increase was not detectable during substrate conversion by ac - hctb. instead, a slow signal increase at 320 nm in the absence (figure s2b) and presence (figure s2d) of chloride was obtained, which may indicate some fe(ii) oxidation as a side reaction. when nonacylated hctb (further on termed hctb) was subjected to stopped - flow analysis , the chloride - free complex displayed an almost stable signal at 500 nm over the measured time range with a specific rate of 0.1 10 s (figure s2a). in the presence of chloride, the velocity of precipitation was chloride dependent and led to an absorbance increase over the whole recorded wavelength range (figures s2c, s3, and s4). this phenomenon, which was not observed during o2 sensor measurements, prohibited the determination of these reaction rates. summarizing, according to stopped - flow measurements the decay of the -kg - fe(ii)-ac - hctb complex is accelerated 200-fold by chloride, while the presence of covalently bound substrate in the absence of chloride enhanced the rate only by an order of magnitude. in order to gain insight into the fe(ii) active site geometric and electronic structure, combined cd / mcd spectroscopic measurements of the enzymatic fe(ii) centers of hctb (i.e., no substrate) and ac - hctb in the presence and absence of -kg and saturating nacl concentrations were pursued. the methodology developed in ref allows for the determination of the fe(ii) center geometric structure, based on the energies of its associated d d transitions: free high spin fe(ii) possesses a d ground state, which upon the influence of an octahedral ligand field splits into a t2 g ground state and a eg excited state that are separated by 10 dq 10 000 cm. the eg state is further split in the distorted octahedral geometry of a protein environment, leading to two transitions split by 2000 cm. removal of an axial ligand forms a square pyramidal five - coordinated species ing in the splitting of the eg state by 5000 cm, yielding transitions at 10 000 and 5000 cm. rearrangement to a trigonal bipyramidal 5c geometry leads to transitions at < 10 000 and < 5000 cm (the latter frequently undetectable). thus, near - ir (nir) cd and mcd spectra give information on the number of different fe(ii) coordination environments present in a sample as well as their geometric structures. vtvh mcd complements these excited state data by providing information on the ground state splitting of the t2 g orbitals of a given site. a non - kramers doublet model developed previously for the fitting of vtvh mcd data provides ground state spin hamiltonian parameters and g|| for negative zero - field - split (zfs) systems or axial d and rhombic |e| zfs parameters for positive zfs systems, which in turn are used to determine the tetragonal splitting, , of the dxy orbital from the {dxz, dyz} pair, as well as the rhombic splitting, v, of the dxz and dyz orbitals. the relative energies of the five d orbitals of an fe(ii) active site can therefore be determined, and the site s geometric and electronic structures characterized. 283 k cd and 5 k mcd spectra of fe(ii)-hctb and fe(ii)-ac - hctb in the absence and presence of chloride are shown in figure 2. in cd, these four enzyme forms all show two transitions at 8100 and 10400 cm (figure 2a, c, e, and g) indicative of distorted octahedral fe(ii) sites, whereas in mcd two transitions are observed at 9200 cm and 11 400 cm for the four forms (figure 2b, d, f, and h). such shifts upon going to low temperature have been observed for clavaminate synthase 2 (cs2) and factor inhibiting hypoxia - inducible factor (fih) and are attributed to stronger ligand the similarity of the spectra for all four forms indicates that no significant change in the eg orbitals of hctb - bound fe(ii) has occurred in the presence of chloride or substrate. 283 k cd and 5 k mcd spectra of fe(ii)-hctb (a and b, respectively, dark blue), fe(ii)-ac - hctb (c and d, respectively, light blue), fe(ii)/cl - hctb (e and f, respectively, dark green), and fe(ii)/cl - ac - hctb (g and h, respectively, light green). component peaks and ant fits are in black and colored dashed lines, respectively. however, vtvh mcd data as shown in figure 3 indicate that the t2 g orbitals are perturbed when chloride is bound to fe(ii). the vtvh mcd data of fe(ii)-hctb and fe(ii)-ac - hctb in the absence of chloride (figure 3a and b) can be fit with the same parameters of a positive zfs system with d = + 10.8 0.1 cm and |e| = 2.6 0.1 cm, and corresponding t2 g orbital splittings of 500 100 cm and |v| 300 50 cm. upon addition of chloride, the vtvh mcd data change outside of error as shown in supporting figure s5a and b. fe(ii)/cl - hctb (figure 3c) fits to a positive zfs with parameters of d = + 10.2 0.1 cm and |e| = 2.6 0.1 cm, and corresponding t2 g orbital splittings of 700 100 cm and |v| 450 50 cm. the increased t2 g orbital splittings are attributed to chloride being a stronger donor ligand than the replaced water. in the presence of substrate and chloride (figure 3d), the vtvh mcd data fit to give a positive zfs with d = 3.7 0.1 cm and |e| = 2.4 0.1 cm, and corresponding t2 g orbital splittings of 850 100 cm and |v| 540 50 cm-1. that substrate binding only appears to perturb the fe(ii) site when chloride is also present is explored in the md simulation section below. vtvh mcd data and fitted curves for fe(ii)-hctb (a, 2.2, 3.3, 5, 7.5, 10, 15, and 20 k, dark blue), fe(ii)-ac - hctb (b, 2.2, 3.4, 5, 7.5, 10, and 15 k, light blue), fe(ii)/cl - hctb (c, 2.3, 3.3, 5, and 7.5 k, dark green), and fe(ii)/cl - ac - hctb (d, 2.2, 3.4, 5, 7.5, and 10 k, light green). error bars for the data are shown or otherwise are the size of the data points. cd and mcd spectra of -kg - fe(ii)-hctb and -kg - fe(ii)-ac - hctb in the absence and presence of chloride are given in figure 4. in the presence of -kg both hctb and ac - hctb mcd spectra show two nir transitions at 8800 and 11 800 cm as well as the rising edge of the fe(ii) -kg * metal - to - ligand charge transfer (mlct) starting at 16 000 cm (figure 4b and d). 283 k cd and 5 k mcd spectra of -kg - fe(ii)-hctb (a and b, respectively, red), -kg - fe(ii)-ac - hctb (c and d, respectively, pink), -kg - fe(ii)/cl - hctb (e and f, respectively, orange), and -kg - fe(ii)/cl - ac - hctb (g and h, respectively, purple). component peaks and ant fits are in black and colored dashed lines, respectively. the presence of substrate does not lead to a significant perturbation of the mcd spectra in the absence of chloride for the the relatively large eg splitting of 3000 cm is similar to that found in -kg / chloride - bound cytc3 (3660 cm) and may be attributed to a weaker water ligand than that found in a site containing the 2his/1-carboxylate facial triad, such as cs2. this has been explained by the lack of the h - bond between the facial triad carboxylate and the coordinated water, which gives this water partial hydroxide character and strengthens the fe the mcd spectrum of -kg - fe(ii)/cl - hctb (figure 4f, orange) shows three nir transitions at 7600, 9100, and 12 100 cm, in addition to the mlct shoulder at 16 000 cm. the presence of three transitions indicates that more than one fe(ii) site is present. in order to determine the nature of these species vtvh mcd data were collected on the three peaks at 7350, 9500, and 11 800 cm as shown in figure 5 (arrows in a, isotherms in b vtvh mcd data collected on the 7600 cm transition are fit to a negative zfs with 1.3 0.1 cm and g|| 8.6 0.1 . the very small value of is indicative of a trigonal bipyramidal site, as was found to be present in the reduced, naphthalene - bound form of the rieske - dependent naphthalene dioxygenase, which possessed a feature at 8165 cm with a 1.2 0.1 cm and g|| 8.5 0.1 . data collected at 9500 and 11 800 cm were fit to the same ground state to give a positive zfs with d = 9.3 0.1 cm and |e| = 2.1 0.1 cm, corresponding to t2 g orbital splittings of 900 100 cm and |v| 560 50 cm . note that the data at 11 800 cm required the addition of a b term of approximately 12% of the total intensity, due to overlap with the mlct transition . -kg - fe(ii)/cl - hctb is therefore a mixture of 6c and 5c trigonal bipyramidal sites. (a) mcd spectrum with arrows marking the energies at which vtvh mcd data were collected, at (b) 7350 cm (purple), (c) 9500 cm (red), and (d) 11 800 cm (orange). all data were collected at 2.2, 3.4, 5, 7.5, and 10 k. error bars for the data are shown or otherwise are the size of the data points. the mcd spectrum of -kg - fe(ii)/cl - ac - hctb (figure 4h, purple) shows four transitions: 5000, 7600, 9900, and 12 000 cm. the presence of four ligand field transitions indicates that -kg - fe(ii)/cl - ac - hctb is also a mixture in this case containing sites of square pyramidal (due to the 5000 cm transition) and octahedral sites. vtvh mcd data were collected on the low - energy band at 5750 cm as shown in figure 6 (the other bands were too weak for a meaningful signal - to - noise ratio). the vtvh mcd data for this band could be fit using negative zfs parameters of 2.6 0.1 cm and g|| 8.8 0.1, corresponding to t2 g splittings of 1100 100 cm and |v| 660 50 cm, the larger t2 g splitting being consistent with a square pyramidal fe(ii) center. thus, upon binding of chloride to -kg - fe(ii)-ac - hctb the site goes from 6c to 5c. (a) mcd spectrum with arrows marking the energy at which vtvh mcd data were collected at (b) 5750 cm (purple). data were collected at 2.3, 3.3, 5, and 7.5 k. error bars for the data are shown. to determine whether the maintenance of the six - coordinate fe(ii) site in the chloride - free -kg - fe(ii)-ac - hctb complex was due to a missing negative charge at the halide binding position, we recorded the spectrum of the equivalent sample at a pd of 9.1. however, the pd shift by 1.6 log units, which previously brought about water deprotonation in a noncarboxylate coordinated fe(ii) center, did not significantly alter the mcd spectrum (figure s6). consequently, to elucidate the structural basis of chloride mediated o2 activation, a model of the hctb halogenase domain was constructed based on the crystal structure of syrb2 and subjected to md simulations in the absence and presence of 1 m nacl in the simulation cell. in order to ensure a geometrically appropriate metal center structure, an octahedral iron force field was defined and applied to the ligands, namely his112, his228, -kg, and where appropriate chloride, as detailed in the experimental section. 100 ns md simulations of the 2-his 1-chloro -kg ligated fe(ii)-hctb model and its chloride - free analogue at 297 k showed overall structures that were stable over the simulation time. however, in the halide - free model, a rearrangement at the metal center was observed: residue glu224, which pointed away from the metal center in all starting structures, reoriented and coordinated to the fe(ii) ion, as demonstrated in a 90 ns snapshot of the active site (figure 7a). by contrast, the respective simulation in the presence of chloride roughly preserved the conformation of glu224 from the starting structure (figure 7b). when the simulation of the hctb domain under chloride - free conditions was repeated with a water molecule kept bound to the metal ion either at the axial, 6th position (figure s7) or at the equatorial position that is otherwise occupied by the chloride ion (figure s8), glu224 also reoriented in order to point toward fe(ii) in both cases, whereby the residue s carboxylate moiety h - bonded to the water molecule that occupied the cl binding site or coordinated directly (figures s7 and s8). (note that both a displacement and preservation of metal bound water are feasible in principle and, therefore, needed to be considered). trajectories of the fe(ii)-glu224 carboxylate oxygen distances show that this reorientation takes place instantaneously (< 100 ps), (figures 8a, left, s9a, s10a). by contrast, in the chloride containing structure the carboxylate moiety of glu224 remained pointing away from the hctb active site (figure 8a, right), presumably due to electrostatic interactions. 90 ns snapshots of the hctb halogenase domains derived from md simulations of the modeled structures at 297 k. while in the chloride - free structure (a) glu224 points toward fe(ii) (orange ball), in the chloride - containing structure (b) the halide (green ball) coordinates to fe(ii) and glu224 points away from the metal cofactor. this in a rearrangement of the neighboring residues val114, tyr115, asn140, and arg225. water molecules are not displayed. in order to assess the impact of a probable overestimation of electrostatics in our model on the orientation of glu224, 1 ns md simulations at varying reduced effective charges of fe(ii) were subsequently performed. showed that the observed two principal distinct orientations of glu224, toward and away from fe in the absence and presence of chloride, were not sensitive to the fe(ii) charge within the studied range of 0 to + 2. however, for cl - free models with no bound water in the halide binding pocket, increased charges roughly correlated with increased frequency of direct coordination of glu224 to fe (table s2). the reorientation of glu224 in the chloride - free hctb structures led to major structural changes. the residue s flipping ed in a h - bonding interaction between its backbone oxygen and the backbone nitrogen of val114 (figure 8c, left, dark red) in the model containing the water - free fe(ii) sphere and drew the respective strands closer together (figure 8b, left, teal). however, in the two models with a water tightly bound to the fe(ii) center either at the chloride - binding position or at the second water - binding position in the octahedral 2-his -kg complex, no interactions between these residues were observed (figures s9b and s10b). in all three chloride - free models (figures 7a, s7, and s8), the side chains of tyr115 and arg225 reduced their distance from an average of 10 to 46 (figures 8c, s9c, and s10c, pink and red traces) and became less flexible. in the case of the halide - free model without tightly bound water, the backbone nitrogen of glu224 formed a h - bond with the side chain oxo group of asn140 (figure 8c, left, orange). in all halide - free models, the backbone of asn140 and glu224 came closer together (figures 8b, s9b, and s10b, blue traces). thus, according to our models, the effect of chloride binding to fe(ii) is to induce conformational changes in hctb. trajectories from 100 ns md simulations depicting distances of residues involved in the chloride - triggered structural rearrangement in hctb in the absence (left panels) and presence (right panels) of 1 m chloride. panel b displays backbone distances from c-2 of asn140 to n-2 of glu224 (blue); n-2 of val114 to c-2 of glu224 (teal); and c-2 of tyr115 to c-2 of arg225 (green). panel c gives the distances of the putative h - bonding atoms o-7 of tyr115 to n- of arg225 (red); o-7 of tyr115 to n- of arg225 (pink); n-2 of val114 to o-1 of glu224 (dark red); and o-4 of asn140 to n-2 of glu224 (orange). this study demonstrates that although an enzymatic -kg bound fe(ii) center is constituted in the absence of a halide, the latter impacts the geometric structure of the metal center and thereby triggers o2 activation in hctb. cd / mcd spectroscopic studies of the metal centers in fe(ii)-hctb and fe(ii)-ac - hctb show a stable six - coordinate geometry in the absence of any cofactor. this sets hctb apart from the related -kg dependent halogenase cytc3, where -kg was a prerequisite for fe(ii) binding to the enzyme in solution. hctb samples with and without chloride show similar excited - state spectral features. however, the d (t2 g) orbitals are perturbed upon halide binding as shown by vtvh mcd. also, while the ground state splitting is the same for fe(ii)-hctb and fe(ii)-ac - hctb in the absence of halide, the splittings for halide - bound hctb in the absence of substrate differ from those where the substrate is bound. these provide experimental evidence for the effect of halide binding on protein conformation and thus are consistent with md simultations. it is interesting to note that in the presence of -kg and substrate, conditions in which most other -kg - dependent enzymes trigger water ligand dissociation and activation of o2, the fe(ii) site in hctb remains 6c (figure 4c and d). this is consistent with the idea that the substrate does not sterically interact with the active site until chloride is bound to fe(ii). evidence for a 5c fe(ii) site is found only upon binding of chloride to the -kg - fe(ii) site. for -kg - fe(ii)/cl - ac - hctb the vacated coordination site allows binding of o2 and in an active site that is competent for catalysis. this is supported by stopped - flow kinetics, which demonstrate that the presence of chloride in a 200-fold increase in the rate of -kg - fe(ii)-ac - hctb complex decay. interestingly, the presence of -kg and chloride but absence of substrate leads to the formation of a mixture of trigonal bipyramidal and octahedral sites. this directly reflects the slow but measurable uncoupled consumption of o2, observed only for this form (7% of the rate of -kg - fe(ii)-cl - ac - hctb ). these findings are in contrast to the for cytc3, which showed the presence of only 6c sites unless -kg, substrate, and chloride were all present, and kinetic findings for syrb2 which showed a 5000-fold loss in rate in the absence of substrate. in the absence of a crystal structure for hctb, stuctural models of its halogenase domain allowed some interesting insights into a putative role of cl in the activation of hctb for catalysis: md simulations of the hctb structural models suggest major structural differences between the chloride - bound and chloride - free forms. in the absence of chloride , the carboxylate moiety of glu224 is oriented toward the iron center (either directly binding to fe(ii) or h - bonding to a coordinated h2o ). this in h - bonding interactions between the backbones of glu224 and val114 in models containing a water - free fe(ii) center. additionally, hydrogen bonds form between glu224 and asn140 in all investigated halide - free models that bring the residues carbon atoms closer together. moreover the distances between the tyr115 and arg225 side chains are significantly reduced in all chloride - free models. notably, previous in silico docking studies suggested that the fatty acyl moiety accesses the active site between the strands that contain residues 114115 and 224225 (figure s11). as these residues converge in the chloride - free model, an analogous active site access by the substrate s acyl moiety could be severely hampered. such a scenario provides a rationale for the substrate s inability to affect mcd spectra (i.e., the 6c 5c conversion) and to markedly increase o2 consumption rates in the absence of chloride. the apparent transient stability of the -kg - fe(ii)-ac - hctb complex during single turnover kinetics, which is mirrored by the 60 ms lag phase in figure 1, may thus reflect the reorganization of the enzyme upon chloride binding, involving the opening of the substrate channel, the substrate entrance to the active site, and the formation of the five - coordinate iron center required for the o2 dependent catalysis to take place. in the absence of chloride a bound water could take the halide s position at the metal center, or alternatively, direct coordination of glu224 could take place. a corresponding acidic amino acid residue is present in the other so far annotated fatty acyl - halogenase psm3l, but it is not generally conserved in mnh halogenases. according to sequence alignments, the amino acyl - halogenases have a serine in the respective position and in the structure of syrb2 this residue occupies a similar space as glu224 in the hctb model. therefore, the proposed molecular mechanism may be specific to this subtype of fatty acyl - halogenases. however, a principal role of chloride in the activation of halogenases by triggering changes in the protein structure may be a general feature of the mnh halogenase mechanism. interestingly, in syrb2 a major rearrangement of polypeptide strands was obtained upon binding of fe(ii) and chloride to an -kg containing structure. in cura, concomitant coordination of -kg and chloride to the iron center also ed in a structural reorganization. in contrast, cytc3, which could not be crystallized with a metal coordinated halide, possesses the same conformation in its apo and fe(ii)+-kg - bound forms. even though structural data are scarce and the presence of charged buffer molecules may compromise their interpretation, it is noteworthy that in the available studies conformational rearrangements were accompanied by a switch from a halide - free to a halide - coordinating metal center. particularly for halogenases with low chloride affinity and those that perform multiple halogenations per substrate and therefore consume the bound halide, a chloride - dependent activation would prevent undesired hydroxylation after the primary halogenation step. note that a report was recently published that briefly described the chloride induced formation of an fe(iv) species in syrb2, supporting our notion that this may be a general mechanism in halogenases. in summary , the present study has shown that halide binding is key for the o2 reactivity of hctb, as neither is an open coordination position for o2 binding present nor is the substrate allowed to properly align above the active site unless halide is ligated to fe(ii). these effects help to maintain halogenase activity in hctb and may be factors involved in the mechanisms of halogenases in general.

mononuclear nonheme fe(ii) (mnh) and -ketoglutarate (-kg) dependent halogenases activate o2 to perform oxidative halogenations of activated and nonactivated carbon centers. while the mechanism of halide incorporation into a substrate has been investigated, the mechanism by which halogenases prevent oxidations in the absence of chloride is still obscure. here, we characterize the impact of chloride on the metal center coordination and reactivity of the fatty acyl - halogenase hctb. stopped - flow kinetic studies show that the oxidative transformation of the fe(ii)--kg - enzyme complex is > 200-fold accelerated by saturating concentrations of chloride in both the absence and presence of a covalently bound substrate. by contrast, the presence of substrate, which generally brings about o2 activation at enzymatic mnh centers, only has an 10-fold effect in the absence of chloride. circular dichroism (cd) and magnetic cd (mcd) studies demonstrate that chloride binding triggers changes in the metal center ligation: chloride binding induces the proper binding of the substrate as shown by variable - temperature, variable - field (vtvh) mcd studies of non--kg - containing forms and the conversion from six - coordinate (6c) to 5c/6c mixtures when -kg is bound. in the presence of substrate, a site with square pyramidal five - coordinate (5c) geometry is observed, which is required for o2 activation at enzymatic mnh centers. in the absence of substrate an unusual trigonal bipyramidal site is formed, which accounts for the observed slow, uncoupled reactivity. molecular dynamics simulations suggest that the binding of chloride to the metal center of hctb leads to a conformational change in the enzyme that makes the active site more accessible to the substrate and thus facilitates the formation of the catalytically competent enzyme substrate complex. are discussed in relation to other mnh dependent halogenases.

despite modern medical management, heart disease is still one of the most common chronic diseases and remains the leading cause of morbidity and mortality worldwide. accurate diagnosis of hd improve prognosis through early treatment. although, current strategy for prognosis of hd is good, however, are not enough to explain all coronary events. therefore, researches for potential novel its risk factors and diagnostic criteria are requirement to be fulfilled. adropin is a recently identified protein encoded by the energy homeostasis - associated gene (enho) identified during an investigation of obese insulin resistant mice as a novel factor linking with metabolic homeostasis. adropin appears to participate in the maintenance of energy homeostasis and insulin response, closely related to the development and progression of atherogenesis. endothelial dysfunction is a key early event in atherogenesis and onset of hd. furthermore, low serum adropin introduced as an independent predictor of clinically relevant coronary atherosclerosis. the aim of this present study was to investigate the effects of adropin on hd through systematic review. we performed a systematic search of pubmed, scopus, web of science, embase, google scholar and medline for studies examining the association between serum adropin levels and all type of heart disease. the prisma 2009 guidelines for systematic review and meta - analysis were considered throughout the study. the predetermined inclusion criteria were: 1 ) exposure introduced as serum adropin levels; 2 ) the outcome interest was all type of heart disease; 3 ) studies that conducted in adult population were not endocrine disorders. studies were excluded if they were pregnant and lactating women or reviews, editorial, letters, meeting abstract and animal study. studies screened independently by two investigators (sy, ss) based on the inclusion criteria with a low probability to exclude irrelevant abstract. overall, 220 abstracts were retrieved by searching in the electronic database. of those, 151 abstracts were duplicated, 27 did not study coronary heart disease as interesting outcome, 3 were editorial and review, 13 were non - human studies, 8 were meeting abstract and 12 conducted in children, pregnant and lactating women. after final screening of full text, six articles were eligible to address in this systematic review, which four of those were case - control studies and 2 articles were cross - sectional studies. all data extracted we rescanned two times. if there were discrepancies, group consensus and consulted a third reviewer were used to reach a ensure accuracy of data. the data extracted included: study characteristics (authors, year of publication, study location, study design) the participants age, number of allocated participants, serum adropin level, type of heart disease, adropin assessment method. the newcastle - ottawa scales, validated scale for non - randomized studies were applied to examine the quality of included evidences. we performed a systematic search of pubmed, scopus, web of science, embase, google scholar and medline for studies examining the association between serum adropin levels and all type of heart disease. the prisma 2009 guidelines for systematic review and meta - analysis were considered throughout the study. the predetermined inclusion criteria were: 1 ) exposure introduced as serum adropin levels; 2 ) the outcome interest was all type of heart disease; 3 ) studies that conducted in adult population were not endocrine disorders. studies were excluded if they were pregnant and lactating women or reviews, editorial, letters, meeting abstract and animal study. studies screened independently by two investigators (sy, ss) based on the inclusion criteria with a low probability to exclude irrelevant abstract. overall, 220 abstracts were retrieved by searching in the electronic database. of those, 151 abstracts were duplicated, 27 did not study coronary heart disease as interesting outcome, 3 were editorial and review, 13 were non - human studies, 8 were meeting abstract and 12 conducted in children, pregnant and lactating women. after final screening of full text, six articles were eligible to address in this systematic review, which four of those were case - control studies and 2 articles were cross - sectional studies. two authors conducted independently data extraction using a pre - formatted spreadsheet. all data extracted we rescanned two times. if there were discrepancies, group consensus and consulted a third reviewer were used to reach a ensure accuracy of data. the data extracted included: study characteristics (authors, year of publication, study location, study design) the participants age, number of allocated participants, serum adropin level, type of heart disease, adropin assessment method. the newcastle - ottawa scales, validated scale for non - randomized studies were applied to examine the quality of included evidences. table 1 exhibited detail of study and participants characterize. heart failure (hf), coronary atherosclerosis acute myocardial infarction and cardiac syndrome x (csx) all studies were conducted on both sex, two studies conducted in turkey, and four investigation were from china. characteristic of studies that evaluated of adropin status in patient with coronary artery disease there were significant differences in the plasma adropin level between heart disease patients with healthy participants. majority of evidences introduced low adropin as an independent predictor of heart disease. in a case - control study, the plasma level of adropin increased with the severity of hf (control : 6.00.3ng / ml ; ha functional class ii : 7.60.4 ; ha functional class iii : 9.80.5 ; ha functional class iv : 12.40.6 ng / ml ( p<0.01). in a similar design, reported the stable angina pectoris (sap) and acute myocardial infarction compare with control group have lower serum adropin. a decrease in adropin level found in patients with csx than control participants (1.70.8ng / ml and 3.41.8 ng / ml, respectively ; p<0.001). additionally, adropin was one of the predictor risk factors for csx (=0.104, p - value=0.005). patients with stablecoronaryarterydisease (scad) had lower serum adropin levels compared to the controls (59.219.3 versus 70.018.2 pg / ml, p<0.001). moreover, low serum adropin level was introduced as a predictor of scad. in a cross - sectional study, serum adropin was inversely associated with the score of severity of coronary atherosclerosis assessed by gensini, friesinger, and syntax scores. a decrease serum adropin indicated a negative independent predictor of coronary atherosclerosis (as syntax score>11). in similar design study introduced lower serum adropin as an independent predictor of saphenous vein graf disease (svgd) after coronary artery bypass grafting (or=0.558, 95%ci=0.4330.714, p<0.01). adropin is a newly identified protein that its protective role on endothelial cells has been shown previously, and has been referred to as a novel regulator of these cells. the key findings from the present review study were that there were significant differences in the plasma adropin level between patients with heart disease (ami, sap, atherosclerosis, cardiac syndrome x, occluded svg) and control subjects. serum adropin level may be referred to as a novel biomarker for evaluation and follow up in patients with hd. the endothelium plays a crucial role in the maintenance of vascular homeostasis, and endothelial dysfunction contributes to the development and progression of cardiovascular diseases. nitric oxide, a potent endogenous vasodilator formed in the endothelium by the endothelial isoform of enos, plays an important role in maintaining endothelial homeostasis. nitric oxide furthermore inhibits monocytes and leukocytes adhesion to the endothelium, aggregation of platelets, oxidation of low density lipoprotein, and smooth muscle cell proliferation. adropin could enhance the expression of endothelial nitric oxide synthase (enos), responsible for the production of vascular nitric oxide, in the endothelium, so adropin deficiency is associated with reduced nitric oxide bioavailability in the endothelium. loss of no bioavailability is a critical stage in formation of endothelial dysfunction that is an independent predictor of the onset of coronary artery disease. patients with cardiac syndrome x, characterized by endothelial dysfunction, compared to healthy subjects had significantly lower adropin levels. besides, the reduced circulating adropin concentrations participates in metabolic disorders such as insulin resistance related to the onset and progression of coronary atherosclerosis and increased incidence rate of cardiovascular events. c - reactive protein has been widely used for cardiovascular risk stratification. a negative correlation between adropin and hs - crp levels atherosclerosis has been regarded as a chronic inflammatory disease, so adropin as a potential anti - inflammatory protein play an important role in the prevention of atherosclerosis. however, plasma adropin levels were correlated positively with bmi and the severity of heart failure, which is in contrary to our . cardiac cachexia which characterized by weight loss , muscle wasting and cytokine activation frequently occurs in patients with advanced hf. liver enho expression decreases with either diet or genetically induced obesity. besides , cardiac cachexia or wasting, which could reflect a decrease in visceral fat, may be in the elevation of plasma adropin levels in patients with hf. adropin may serve as a potential biomarker for early diagnosis of and severity stratification hd. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc .) have been completely observed by the authors.

: heart disease is one of the most common chronic disease and leading cause of morbidity and mortality worldwide. adropin, a newly identified protein, is important for energy homeostasis and maintaining insulin sensitivity, and has been referred to as a novel regulator of endothelial cells. endothelial dysfunction is a key early event in atherogenesis and onset of hd. therefore, this review gives a systematic overview of studies investigating plasma adropin level in patient with heart disease.methods:data carried out in pubmed, scopus, web of science, embase, google scholar and medline, from the earliest available online indexing year through 2015. the search restricted to studies conducted in humans. the keyword search was adropin to apply in title, abstract and keywords. references lists of all original published articles were scanned to find additional eligible studies.:heart failure (hf), coronary atherosclerosis acute myocardial infarction and cardiac syndrome x (csx) were type of heart disease acknowledged in this study. majority of evidences introduced low adropin as an independent risk factor of heart disease. in a case - control study, the plasma level of adropin increased with the severity of hf.:adropinmay be a potential serum biomarker for early diagnosis of hd.

arachnoid cysts are prevalent among the general population and with increased access to brain imaging, incidental cysts are being increasingly diagnosed. surgical options for symptomatic arachnoid cysts include cyst fenestration, either open or endoscopic, insertion of a cysto - peritoneal shunt or marsupialization via a craniotomy. recent studies have shown areas of hypoperfusion related to patients with asymptomatic arachnoid cysts suggesting that these lesions may also require surgical intervention. we report a case where a large arachnoid cyst was treated by marsupialization and was complicated by remote intraparenchymal hemorrhage and a spinal subdural hematoma. a 6-year - old, otherwise healthy boy, presented with a chronic history of generalized headache, which had persisted despite strong analgesia. 1.5 t mr imaging (ge medical systems, milwaukee, usa) revealed a large left temporal arachnoid cyst causing mass effect with effacement of the left lateral ventricle, remodeling of the inner table of the calvarium, and mid - line shift. two smaller arachnoid cysts in the left parietal and right temporal regions were also noted. axial t2 fse images show the left arachnoid cyst causing midline shift (a) and effacement of left lateral ventricle (b) after a lengthy discussion with the family detailing the merits and disadvantages of the various treatment options, the patient was admitted for marsupialization of the large cyst. larger than usual cortical veins were noted postero - medially and were not disturbed during surgery. a subgaleal suction drain was inserted after closure of the dura was carried out. in the post - operative suite, the patient initially moved all four limbs equally and responded appropriately. at 1-h post - procedure , he dropped his conscious level and was flexing to pain with no verbal response and was not eye opening to pain. the drainage bag contained 200 ml of blood - stained fluid and the drain was then clamped. immediate axial computed tomography (ct) scan revealed significant decompression of the left middle cranial fossa arachnoid cyst and improvement in the degree of midline shift. focal areas of parenchymal hemorrhage were noted distant from the site of surgery in the left frontal lobe and left cerebellum. immediate post - op unenhanced axial ct shows reduction in arachnoid cyst volume (a) but also new left cerebellar (b) and frontal hemorrhages (c) an intracranial pressure (icp) monitor, inserted via a standard right frontal approach revealed the pressure to be 6 mmhg. the patient remained well sedated with an aim to keep the icp and cerebral perfusion pressure within normal limits. magnetic resonance imaging (mri) head at 18 h revealed a reduction in size of the left arachnoid cyst but also confirmed hemorrhage in the left frontal lobe and left cerebellar hemisphere. 18-h post - op axial t2 fse (a) and swan (b) show reduction in the arachnoid cyst and areas of parenchymal hemorrhage (as evidenced by the susceptibility artifact) the patient was managed post - operatively in the intensive care unit (icu) and gradually weaned from the ventilator over the following week. neurological examination confirmed a right - sided upper limb and bilateral lower limb weakness (mrc grade 4/5) with intact sensation and reflexes. ct head on day 8 revealed bi - frontal subdural collections and an extra - axial collection just beneath the bone flap. his power improved over the next 48 h. on day 10, he was noted to have difficulty in commencing micturition. subsequent mr spine revealed a subdural hematoma from the mid - thoracic region to the sacrum. day 10 post - op t1 images show the extensive subdural high t1 signal fluid collection in the lumbar (a) and thoracic (b) spine consistent with a hematoma he continued to improve with respect to the power in his lower limbs and the subdural hematoma was thereby managed conservatively. at 4 weeks post - operatively, the patient's bladder function, along with limb power, had returned to normal. he was seen in outpatients at 10 weeks post - operatively and his headaches had resolved and he was at school performing normal activities. larger than usual cortical veins were noted postero - medially and were not disturbed during surgery. in the post - operative suite, the patient initially moved all four limbs equally and responded appropriately. at 1-h post - procedure , he dropped his conscious level and was flexing to pain with no verbal response and was not eye opening to pain. the drainage bag contained 200 ml of blood - stained fluid and the drain was then clamped. immediate axial computed tomography (ct) scan revealed significant decompression of the left middle cranial fossa arachnoid cyst and improvement in the degree of midline shift. focal areas of parenchymal hemorrhage were noted distant from the site of surgery in the left frontal lobe and left cerebellum. immediate post - op unenhanced axial ct shows reduction in arachnoid cyst volume (a) but also new left cerebellar (b) and frontal hemorrhages (c) an intracranial pressure (icp) monitor, inserted via a standard right frontal approach revealed the pressure to be 6 mmhg. the patient remained well sedated with an aim to keep the icp and cerebral perfusion pressure within normal limits. magnetic resonance imaging (mri) head at 18 h revealed a reduction in size of the left arachnoid cyst but also confirmed hemorrhage in the left frontal lobe and left cerebellar hemisphere. 18-h post - op axial t2 fse (a) and swan (b) show reduction in the arachnoid cyst and areas of parenchymal hemorrhage (as evidenced by the susceptibility artifact) the patient was managed post - operatively in the intensive care unit (icu) and gradually weaned from the ventilator over the following week. neurological examination confirmed a right - sided upper limb and bilateral lower limb weakness (mrc grade 4/5) with intact sensation and reflexes. ct head on day 8 revealed bi - frontal subdural collections and an extra - axial collection just beneath the bone flap. his power improved over the next 48 h. on day 10, he was noted to have difficulty in commencing micturition. subsequent mr spine revealed a subdural hematoma from the mid - thoracic region to the sacrum. day 10 post - op t1 images show the extensive subdural high t1 signal fluid collection in the lumbar (a) and thoracic (b) spine consistent with a hematoma he continued to improve with respect to the power in his lower limbs and the subdural hematoma was thereby managed conservatively. at 4 weeks post - operatively, the patient's bladder function, along with limb power, had returned to normal. he was seen in outpatients at 10 weeks post - operatively and his headaches had resolved and he was at school performing normal activities. whilst our patient has made an eventual recovery, it is unclear as to the mechanism of the complications that occurred. sgouros and chapman studied three children with middle fossa arachnoid cysts using mri and single photon emission computerized tomography before and after surgery. in their paper, they have shown that these cysts may cause global impairment of the brain function by interfering with cerebral blood flow and perfusion. our patient had a large arachnoid cyst and may have had chronic impairment of his autoregulation response. after excision of an arteriovenous malformation (avm), hyperperfusion to the adjacent weakened capillary beds ing in hemorrhage has been described previously. the rapid decompression from the craniotomy could have led to a rapid rise in cerebral perfusion causing damage to the capillary bed and ing in parenchymal injury. changes in the intracranial dynamics due to brain shift can cause venous hyperemia. in our patient , the potentially rapid loss of cerebrospinal fluid (csf) may have led to brain shift and thus potentially cause the sites of hemorrhage noted distant from our operative site. the changes in venous and csf pressures causing the intracranial complications would equally be attributable to the hemorrhage seen in the spine. spinal subdural hematomas in children are usually associated with spinal puncture and non - accidental injury and/or in the presence of coagulation abnormalities, none of which was the case in our patient. brain shift and hyperemic states however, we believe that this is the first case of a spinal subdural hematoma as a complication from decompression of an intracranial arachnoid cyst. the subsequent brain shift may lead to a hyperperfusion injury. whilst we recognize that a craniotomy may provide the best long - term outcome for treating arachnoid cysts, more gradual decompression with programmable shunts or even endoscopic fenestration of the cyst into an adjacent basal cistern may be a safer approach. the use of a suction drain should be avoided in these cases even in the presence of macroscopic watertight closure of the dura.

arachnoid cysts are prevalent among the general population. the management options of symptomatic arachnoid cysts each have their own merits and disadvantages. we report a case where a large arachnoid cyst was treated by open fenestration and marsupialization that was complicated by remote intraparenchymal and spinal subdural hemorrhage. the potential physiological changes underlying these complications as well as the related literature are reviewed.

in 20072009, a population - based esophageal cancer cohort study was initiated in 9 villages in rural anyang, henan province, china. this hpv investigation was added to the original cohort study in 6 of the 9 villages. eligibility criteria were as follows: 1 ) male sex; 2 ) permanent residency in the target villages; 3 ) age 2565 years; and 4 ) no history of cancer, cardiovascular disease, or mental disorder. of 3,571 eligible men, 3,172 (89%) were enrolled. the main reason why the other 399, who were substantially younger, did not participate was loss of contact because they were employed outside anyang. participants were interviewed, and exfoliated cells were collected from the penile shaft, glans penis, coronal sulcus, and scrotum by using saline - soaked swabs. the sampling procedure was identical for circumcised and uncircumcised men. to assess the adequacy of the specimens positive specimens were subsequently tested for 13 oncogenic and 37 nononcogenic types of hpv by using pcr - based direct sequencing with a pair of spf1/gp6 + primers. samples with ambiguous hpv typing signals were subjected to further cloning and sequencing. to evaluate the associations between exposure variables and the presence of hpv dna, we used logistic regression analysis with stepwise backward elimination at p>0.1. to examine the association across the ordered categorical variables, we used a trend test. of 3,172 specimens tested, 2,236 were positive for -globin and were included in the analysis (median participant age 42 years ; interquartile range 3552 years). we excluded from the study men who were older and reported less risky sexual behavior than those who were included (technical appendix). of the 40 hpv types we tested for, we detected 36, including 13 oncogenic types oncogenic hpv was detected in 140 (6.3% ; 95% ci 5.3%7.3%) specimens, and nononcogenic hpv was detected in 251 (11% ; 95% ci 9.9%13%) specimens. among 15 hpv - positive specimens that had ambiguous direct sequencing signals and were further cloned and resequenced for genotyping , 3 had a second type and minor types were ignored. among these infections (table 1), the most common oncogenic type detected was hpv-16 (17.4%), followed by hpv-18 (7.2%). the most common nononcogenic type was hpv-3 (16.4%), followed by hpv-57 (7.9%). of 391 hpv - positive specimens, 15 displayed ambiguous typing signals by direct sequencing of pcr product. infection with > 1 hpv type was detected in only 3 of 15 specimens that were tested by cloning and sequencing; the predominant type is shown. oncogenic types tested in this study included hpv-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -66. on the basis of latest published literature and our knowledge of the detection method of hpv dna adopted in this study (spf1/gp6 + mediated pcr and sequencing), nononcogenic types that could be tested for were hpv-3, -6, -7, -10, -11, -26, -27, -29, -30, -32, -37, -40, -42, -43, -44, -53, -54, -55, -57, -61, -62, -67, -68, -69, -70, -72, -74, -75, -77, -81, -82, -84, -85, -87, -90, -91, and -94. prevalence of infection of any or nononcogenic hpv types decreased significantly with participant s increasing age (table 2). risk for infection with any hpv type was associated with being unmarried, having had multiple sex partners, and having had oral and anal sex. when the outcome was stratified by oncogenicity of hpv type, the association remained statistically significant for having had multiple sex partners. adjusted ors and 95% cis derived by multivariate logistic regression models including all the listed variables; backward method was used to select significant variables on the 0.10 level. p values for trend derived by logistic regression analyses, treating categorical variables as continuous variables. this study of genital hpv in a large sample of adult men from rural china addresses the paucity of data on male genital hpv infection in asia. prevalence rates for any type of hpv and oncogenic hpv were lower among these men in china than among heterosexual men elsewhere (asia pacific area and globally). this discrepancy might be partly explained by the relatively more conservative sexual behavior and higher median age of participants in this study. among populations of similar age, prevalence of a specific hpv type is usually lower among men than among women. however, the 17.5% prevalence found in this study exceeds estimates for married women 1559 years of age in china (14.8%16.8%). this finding is consistent with that of another study, which also reported higher hpv prevalence among men than among women. these inconsistent findings might be explained by differences in hpv type distribution between male genitalia and the female cervix and by variability of type - specific sensitivity in detection methods. although most studies of hpv infection in men worldwide have found no clear trend with regard to age, we found that infection mildly decreased with increasing age. a potential explanation for this age - related trend might be that in this population, young adults more commonly work for long periods outside the rural home area than do older adults (data not shown), and the increased mobility might be associated with more unprotected sexual behavior and consequent increased exposure to hpv. our finding that hpv-16 and -18 were the most commonly detected oncogenic hpv types is in keeping with findings of previous studies. however, our finding that hpv-3 and hpv-57 were the predominant nononcogenic types is in contrast to the findings of other studies that hpv-6 and hpv-11 were the most predominant. this discrepancy might partly be explained by the fact that in our in - house evaluation, the primer set spf1/gp6 + was more sensitive for hpv-57 but less sensitive for hpv-11 than was gp5+/gp6 +. completely opposite to our findings, dai et al. reported that among women in neighboring rural shanxi province, china, oncogenic types were more commonly detected than were nononcogenic types. one possible explanation is that in our study, a significant portion of the exfoliated cells were collected from skin tissue. therefore, a number of cutaneous hpv types, which are nononcogenic for mucosal lesion (cervical cancer), could be detected. we believe this might have led to the higher proportion of nononcogenic hpv than oncogenic hpv detected in our study. another possible explanation is that sequencing methods used in our study can detect more nononcogenic types, which escape identification in studies that use hybridization with preassigned probes. in contrast to previously reported rates of infection, in our study, infection with multiple types was rare. the previous studies used hybridization, an efficient way to identify co - infections, for genotyping. however, we used sequencing instead of hybridization to maximize demonstration of the spectrum of hpv types. because minor types would probably be covered by the predominant type in the sequencing process, sequencing the low proportion of -globin positivity might have partly ed from the lower efficiency of cell collection by use of a saline - moistened swab as opposed to other methods such as emery paper. another possible explanation is that a certain proportion of cells collected from male genitalia are keratinized and contain less nucleated human dna than cells collected from mucosal organs (e.g., the cervix). however, the biases potentially imposed by the nonparticipation of 400 younger men (because of mobility) and 936 older men (because of specimen inadequacy) must be noted. this nonparticipation of younger men might have neutralized the age - related association to some extent. although circumcision is extremely rare in rural china, the lack of accurate data for this variable is another study limitation, which rendered subgroup analysis by circumcision status impossible. as reported in other studies, having had multiple sex partners was associated with hpv infection in this population. this finding indicates that men with higher mobility (i.e., higher risk for multiple sex partners and unprotected sexual behavior) should receive more attention with regard to future hpv control in this region. characteristics of 3,172 men from rural henan province, by human papillomavirus -globin status of genital specimens, 20072009.

to determine prevalence of genital human papillomavirus (hpv) infection among men in rural china, we analyzed genital swab specimens. among 2,236 male residents of rural henan province, hpv infection prevalence was 17.5%. the most common oncogenic and nononcogenic types were hpv-16 and hpv-3, respectively. infection was associated with younger age and multiple sex partners.

acute myocardial infarction (ami) complicated by cardiogenic shock and left main coronary artery (lmca) disease is called left main shock syndrome (lmss). it is reported that the morbility and mortality of the syndrome is approximately 0.46% and 55%80%, respectively. , we present a patient with lmss who successively underwent emergency percutaneous coronary intervention (pci) and coronary artery bypass grafting (cabg) with hemodynamic support within 5 days. the patient is now on his three month uneventful out - patient follow - up. a 59 year - old male was admitted to our hospital experiencing recurrent chest pain for 6 years and severe pain for 10 h. he had been smoking for seven years and had prior 5 years history of hypertension (160/90 mmhg). on physical examination, the patient was clammy and normal blood pressure (115/60 mmhg), with normal heart sounds and bilateral pulmonary rales on the lower zone of lungs. his electrocardiogram demonstrated st segment elevation in leads i, avl, avr, v1 through v4, st segment depression in lead ii, iii and avf (figure 1). acute anterioseptal and lateral myocardial infarction (killip class ii) and hypertension were diagnosed and the patient was immediately transferred to the catheterization laboratory from the emergency unit. 300 mg aspirin, 600 mg loading dose of clopidogrel and 5000 u of intravenous heparin were administered. as soon as the coronary angiography was performed, the blood pressure of the patient had dropped to 80/50 mmhg with dyspnea. coronary angiography disclosed a left main occlusion in the middle portion of its body and a severe stenosis at the proximal posterior descending artery of right coronary artery (figure 2). intra - aortic balloon pump (iabp) was inserted and the blood pressure was increased to 95/50 mmhg, except for severe hypoxemia. so an extracorporeal membrane oxygenation machine (ecmo) was inserted via left femoral venoarterial access. after one coronary soft guide wire (0.014 inch run through ; teromo, japan) had been passed into the left anterior descending (lad) coronary arteries, the distal left main portion and lad was predilatated with 1.5 15 mm balloon inflated at 16 atm. subsequently, lad was dilatated with sprinter 2.0 12 mm balloon inflated at 14 atm and a fire star 2.5 15 mm balloon inflated at 14 atm. distally to this lesion with thrombolysis in myocardial infarction (timi) grade 3 flow, both lad and diagonal branch presented a significant diffuse lesion while circumflex (cx) ostia presenting with coronary aneurysm (figure 3). it demonstrated st segment elevation in leads i, avl, avr, v1 through v4 and st segment depression in lead ii, iii and avf. (a): right coronary artery angiograph shows a severe stenosis at the proximal posterior descending artery; (b): angiograph of left coronary artery viewed from spider position shows a left main occlusion in the middle portion of its body. the patient was then monitored in coronary heart disease care unit (ccu) with 3.4 l / min of extracorporeal membrane oxygenation (ecmo) and 1: 1 counterpulsation of iabp supporting on the first day after pci. the total 24 h urine volume increased from 1050 ml of the second day to 3170 ml of the third day after pci. so the iabp was withdrawn and ecmo volume was turned down to 2.4 l / min. however, the number of platelets in the whole blood of the patient dropped from 140 g / l on the first day to 76 g / l on the fifth day with normal liver, kidney and blood clot function, a successively cabg (ao - svg - lad and ao- svg - d1-pda) was then performed. the european society of cardiology for 2012 st - elevation myocardial infarction guideline recommends emergency revascularization with either pci or cabg in suitable patients with cardiogenic shock as class i and a level of evidence b. however, since lmss morbility is so low, we do not know exactly which is better for pci or cabg treatment and what we should do if iabp does not produce an positive effect in these patients. distally to the lesion of the left main, proximal left anterior descending, diagonal branch and proximal circumflex presented significant diffuse lesions while circumflex ostia presenting with coronary aneurysm and timi grade 3 flow. in this case, we selected pci and succedent cabg treatment strategy based on the following considerations. first, as common strategies of unprotected left main revascularization in acs, both pci and cabg were significantly associated with improved discharge and 6 month survival in comparison with no revascularization. pci became more feasible strategy of revascularization in this situation than cabg surgery, which will generally be delayed. third, it has been timi 3 grade flow after balloon inflation in lad, in which presented a significant diffuse and small vessel distal to coronary aneurysm lesion in lcx. as a previous study indicated, a small lumen diameter before the procedure is the independent predictor of death in patients with cardiogenic shock, we performed cabg surgery days later. guidelines also suggested iabp with a recommendation class iib and a level of evidence b as an effective measure in combination with balloon angioplasty in these patients. however, there is insufficient evidence endorsing the current guideline recommendation in the setting of lmss. on the contrary, in the meta - analysis included nine cohorts of st - elevation myocardial infarction (stemi) patients with cardiogenic shock (n = 10529) treated with pci, support by iabp was associated with a 6% (95%ci : 3%10% ; p < 0.0008) increase in 30 day mortality. whereas, a recent study indicated that patients who received primary pci and supported with iabp and ecmo had better 30-day and 1-year survival outcomes than those only with iabp. but the difficult decision to withdraw ecmo may need to be made if the patient is not eligible for conventional corrective surgery, or longer term ecmo. as observational data concerning iabp or ecmo therapy in the setting of lmss is importantly hampered by bias and confounding, therefore, randomized controlled trials in the future are needed to determine the use of iabp and ecmo in these patients treated with pci. all together , this case has shown that successful team treatments, including the prompt and suitable revascularization strategy, potent iabp and ecmo support are important to improve the clinical outcome in patients with lmss.

acute myocardial infarction complicated by cardiogenic shock and left main coronary artery disease is called left main shock syndrome. it is reported that the morbility and mortality of the syndrome is approximately 0.46% and 55%80%, respectively. however, the best treatment strategy in these cases is unknown. in this article , we present a patient with lmss who successively underwent emergency percutaneous coronary intervention and coronary artery bypass grafting with hemodynamic support within 5 days. the patient is now on his three month uneventful out - patient follow - up.

a simple method is described for growing rat embryos in vitro for 48 hr from head - fold to early limb - bud stages at rates of development and protein synthesis indistinguishable from those in vivo. culture of the embryos can be continued for longer periods but at a reduced growth rate. preheating the culture serum to 56 degrees c for 30 min improves embryonic development, but raising the culture temperature 2 - 3 degrees c or exposing the presomite embryos to 20% o2 (160 mm hg) causes malformations, particularly of the brain and spinal cord. the value of such culture methods for teratology is briefly discussed.imagesfigure 3.figure 6.figure 8.figure 9.figure 10.figure 11.

dna topoisomerases are ubiquitous enzymes found in all prokaryotic and eukaryotic cells and in some viruses. they are involved in all aspects of dna metabolism such as replication, transcription, recombination, and chromosome segregation. these reactions are based on sequential breakage and rejoining of the dna phosphodiester backbone. type i dna topoisomerases catalyze the cleavage of one strand of dna, whereas type ii dna topoisomerases catalyze the cleavage of a double - stranded dna, requiring atp as a cofactor. type i dna topoisomerases are further classified in two subfamilies, ia and ib, based on differences in amino acid sequence and reaction mechanisms. they are represented by bacterial topoisomerase i and iii and the eukaryotic topoisomerase iii enzymes. type ib topoisomerases, exemplified by eukaryotic topoisomerase i, in contrast, become attached to 3-phosphate end of the cleaved strand of the dna. while the function of topoisomerase ii and i are quite well established, the role of topoisomerase iii in dna metabolism is still being defined. genes encoding topoisomerase iii enzymes are highly conserved in evolution from bacteria to human, and the phenotypic consequences of loss of topoisomerase iii function are generally quite severe. it has been shown to possess a weak, atp - independent relaxation activity towards negatively supercoiled dna only and strict dependence on magnesium. the e. coli chromosome encodes two type ia topoisomerase, dna topoisomerase i and topoisomerase iii. loss of topoisomerase iii in e. coli in an increase in deletions arising from recombination events between direct repeats. yeast cells express a single type ia topoisomerase, topoisomerase iii encoded by the top3 gene. in s. pombe, top3 is essential for viability and plays a role in chromosome segregation. it has been shown that top3-ts mutant s. pombe cells are sensitive to the dna damaging agents uv and mms (methyl methanesulfonate) at the restrictive temperature revealing that topoisomerase iii is involved in dna damage survival. in s. cerevisiae, top3 mutants are viable, but very slow - growing and have defects in s phase responses to dna damage and in both mitotic and meiotic recombination. in vertebrates, there are two isoforms of topoisomerase iii enzymes termed and. deletion of mouse topoisomerase iii gene displayed shortened lifespan and infertility. dna topoisomerases of kinetoplastids represent a family of dna processing enzymes that essentially solve the topological problems not only in the nuclear dna but also in the kinetoplastid dna. the ib type of bi - subunit topoisomerase i and topoisomerase ii of the parasites which maintain vital cellular processes, are also proven target for clinically useful antitumor drugs. apart from this ib type of topoisomerase i, three type ia topoisomerases are there in the parasite genome, termed as topoisomerase ia, and two topoisomerase iii. topoisomerase ia of t. brucei has been reported and shown to be mitochondrial and essential for late theta structure resolution. very recently, a topoisomerase iii from t. brucei has been shown to play a critical role in antigenic switching. in the present study, for the first time , we have identified functionally active dna topoisomerase iii from kinetoplastid parasite l. donovani, which localized both inside the nucleus and kinetoplast of the parasite and rescued the topoisomerase iii mutant yeast from slow - growth phenotype. l. donovani strain ag 83 promastigotes were grown at 22c in m199 liquid media supplemented 10% heat inactivated fetal calf serum. transfected cells were maintained under the same conditions with 100 g / ml g418. if required, ampicillin and chloramphenicol were used at 100 and 34 g / ml final concentrations, respectively. the yeast strains used in the studies were w5909 - 3b (mat alpha trp1 - 1 his3 - 11, 15 leu2 - 3, 112 ura3 - 1 rad5 lys2 met15 ade2) and w2633 - 4c (a / alpha top3 : : trp1/+) (kindly gifted by dr . the yeast cells were grown at 25c on yepd medium containing 1% peptone, 2% yeast extract, 2% dextrose and 1.5% agar or synthetic minimal media as required . ldtopiii gene was pcr amplified from the genomic dna of l. donovani parasites using the sense primer 5-ggaaattccatatgggccgcaatgtgttgatg-3 and antisense primer 5-cgggatcctcacctgcgatcctcgcggttgcc-3 and was cloned in bacterial expression vector pet16b in nde1 and bamh1 restriction sites, termed as ldtopiii-pet16b . multiple sequence alignment of ldtopiii sequences from various species was carried out using clustal w ( http://expasy.org/tools). three - dimensional models of ldtopiii based on the crystal structure of e. coli topoisomerase iii were generated using swiss prot (http://expasy.org/sprot). the protein sequences were represented in ribbon format and the active site residues were represented in ball and stick format over the ribbon structure. ldtopiii genes was pcr amplified using ldtopiii-pet16b as templates and was subcloned using the sense primer 5-cgggatccatgggccgca atgtgttgatg-3 and antisense primer 5-gatatccctgcgatcctcgcggttgcc-3 in bamh1 and ecorv sites of leishmania transfection vector pxg - b2863 (a kind gift from dr . s. m. beverley), to produce c - terminal - gfp - tagged full - length ldtopiii protein and termed as ldtopiii-gfp. the constructs and empty vector pxg - b2863 were transfected into l. donovani promastigotes separately by electroporation as described earlier. briefly, late log - phase promastigotes were harvested and washed twice in opti - mem (gibco). cells were finally suspended at a density of 1 10/ml and 0.4 ml was taken into a 0.2 mm ice - chilled electroporation cuvette. thirty microgram of plasmid dna was taken in 100 l of electroporation buffer and added to the cells. after 10 min on ice, the cells were electroporated with a single pulse by bio - rad gene pulsar apparatus using 450 v and 550 f capacitance. the cells were incubated on ice for further 5 min and then added to10 ml of drug - free growth medium. after 24 h of survival 10 g / ml g418 was added and kept at 22c. finally the transfected cells were routinely maintained in medium containing 100 g / ml g418. localization of c - terminal gfp tagged chimeric ldtopiii-gfp protein was visualized by fluorescence microscopy (olympus ix81). differential visualization of the fluophores was achieved using a 488 nm excitation filters and 523 nm emission filter for gfp and 258 nm excitation and 361 nm emission filter for dapi. the full - length ldtopiii was subcloned in xbai and bamh1 sites into the yeast shuttle vector pvt100u, a kind gift from dr. rolf sternglanz and termed as ldtopiii-pvt using the sense primer 5-gctctagaatgggccgcaatgtgttgatg-3 and antisense primer 5-cgggatcctcacctgcgatcctcgcggtt-3. for construction of c - terminal deletion construct of ldtopiii, regions corresponding to amino acids 1 - 608 was pcr amplified using the primers 5-gctctagaatgggccgcaatgtgttgatg-3 (sense) and 5-cgggatccggcggcggagatggcggagaa-3 (antisense) and was cloned in xba1 and bamh1 sites of pvt100u vector. mutagenesis was performed by using the quikchangexl site - directed kit (stratagene, la jolla, ca) according to the manufacturer's protocol. to carry out the desired mutations, ldtopiii-pvt was used as templates for all mutagenesis experiments. for each mutation , the wild - type nucleotide was replaced using a specific pair of mutagenic primers. the following sense primer, along with the antisense counterparts (with codons in boldface and substitutions underlined), were used; for y327 of ldtopiii, sense primer was 5-ccgcggctatatttcgttccctcgtacccgaatcc-3 and antisense primer was 5-ggattcggtacgagggaacgaaatatagccgcgg-3. the top3 mutant yeast strain w2633 - 4c (a / alpha top3 : : trp1/+) (a kind gift from dr . r rothstein) was used for transformation with recombinant topo iii proteins from l. donovani by the lithium acetate and polyethylene glycol method. colonies were picked and cultured in tubes with 2 ml of synthetic minimal media at 30c overnight. in vitro transcription and translation of ldtopiii proteins ldtopiii-pet16b plasmids were used as dna templates for synthesis of the protein. after the reaction is over, the crude bacterial lysate containing the newly synthesized protein was tested for activity. the type ia dna topoisomerases were assayed by decreased mobility of the relaxed isomers of supercoiled pbs (sk+) dna in an agarose gel. relaxation assay was carried out with the crude lysates containing the in vitro transcribed and translated ldtopiii. supercoiled pbs dna (85%95% were negatively supercoiled with the remaining being nicked circles) was used as substrate in the relaxation buffer (25 mm tris - hcl, ph 7.5, 5% glycerol, 0.5 mm dtt, 2 mm mgcl2, 50 g / ml bsa). the amount of supercoiled monomer dna band fluorescence after etbr (0.5 g / ml) staining was visualized using gel doc 2000 under uv illumination (bio - rad quality one software). one is on chromosome 21, annotated as topoisomerase ia (lmjf21.0125) with an orf of 2453 bp. two other type ia topoisomerases are present on chromosome 28 and 36, respectively, both of which are annotated as topoisomerase iii (lmjf28.1780 and lmjf36.3200, resp .). one of the two topoisomerase iii genes present in l. major genedb is 2601 bp (lmjf28.1780) and encodes a 95 kda predicted protein. the other topoisomerase iii orf (lmjf36.3200) is 2844 bp, and encodes a 104 kda predicted protein. topoisomerase iii gene with 2601 bp was pcr amplified from the genomic dna of l. donovani, cloned and sequenced (genebank accession number gq499197). blast analysis of the sequence confirmed the topoisomerase iii lineage of the protein and henceforth referred as ldtopiii. the alignment of ldtopiii with s. cerevisiae and s. pombe topoisomerase iii and human topoisomerase iii is shown in figure 1. the active site tyrosine is located at the 327 position within a highly conserved gyisyprtes sequence. the protein has 46.22% identity and 76.09% similarity with human topoisomerase iii. it contains seven cxxc sequences instead of eight found in other topoisomerase iii proteins. glycine (g) and arginine (r) rich clusters at the c - terminus end, which is another hallmark of topoisomerase iii, are also present. it has a continuous stretch of 19 g and r residues in the c - terminus. three - dimensional structure generated by swiss prot has been shown in figure 2(a). the conserved amino acid residues are represented in ball and stick format and have been labeled. homology comparisons of ldtopiii with other ia type of topoisomerases have been provided in table 1, which strongly indicates its topoisomerase iii lineage. in silico search a 0.244 probability of mitochondrial transport was predicted by mitoprot (http://expasy.org/tools) analysis and 73.9% cytoplasmic and 17.4% nuclear distribution was revealed by psort ii analysis (http://expasy.org/tools). to determine the precise localization of the protein, full - length ldtopiii (865 aa) was cloned in leishmania expression vector as a c - terminal fusion protein with gfp, termed as ldtopiii-gfp, and the construct was transfected in l. donovani parasites. comparison of dapi and gfp fluorescence and merged images (figure 3(c) ) revealed that ldtopiii protein localized both inside the nucleus and kinetoplast of the parasites. figures 3(d) and 3(e) show cytoplasmic distribution of control gfp protein in l. donovani parasites. mutation of the s. cerevisiae top3 gene is known to in several phenotypes, including a growth rate which is only 50% that of wild - type. in order to assess whether the ldtopiii possesses functional similarity to the yeast topoisomerase iii, we have used a functional complementation assay of ldtopiii protein to rescue top3 mutant s. cerevisiae strain from slow - growing phenotype. we have cloned the ldtopiii gene in a shuttle vector pvt100u to generate ldtopiii-pvt and transformed in top3 yeast s. cerevisiae and ura+ colonies were selected. the ldtopiii-pvt partially complemented the slow - growth of top3 yeast (figure 4(a) ). the improved growth rate was not observed in case of the vector control (figure 4(a) ). this observation suggests that ldtopiii can be functionally expressed in yeast and shares functional similarity with s. cerevisiae top3 gene, which is consistent with earlier observations made with drosophila and human topisomerase iii proteins. to observe this complementation of ldtopiii in liquid medium equal amounts of the wild - type, topoisomerase 3 mutant yeast cells, topoisomerase 3 mutant yeast cells containing empty vector (pvt100u) and topoisomerase 3 mutant yeast cells containing ldtopiii (grown overnight at 30c) were inoculated in fresh minimal medium and grown at 30c. at every 2 hr interval up to 12 hrs, the growth was monitored and plotted. tyrosine 327 of ldtopiii was predicted to be the active site amino acid residue from sequence alignment analysis. in order to determine that ldtopiii functionally complements the top3 mutant yeast and the growth recovery was not due to any compensatory mechanism induced by ldtopiii we carried out site directed mutagenesis. we have mutated the active site residue of ldtopiii to phenylalanine (y327f) by site directed mutagenesis and transformed in top3 mutant yeast. it was observed that the active site mutant construct could not suppress the slow - growth of top3 mutant s. cerevisiae (figures 5(a) and 5(b) ) confirming role of active site tyrosine 327 in functional conservation of ldtopiii inside mutant yeast cells. the leishmania enzyme has a c - terminal segment of amino acids with no counterpart in yeast protein. the leishmanial protein contains zn - binding motif at its c - terminus, which is absent in the topoisomerase iii proteins of e. coli and yeast. the c - terminus residues of e. coli topoisomerase iii have been previously shown to be involved in dna binding. to determine the role of the c - terminal stretch of ldtopiii in functional complementation, we have made a c - terminal deletion construct (ldtopiiic258) removing the 258 amino acids and transformed in topoisomerase iii mutant yeast. the transformants were grown in plates and it was observed that the c - terminal deletion construct failed to rescue the mutant yeast from slow - growth (figure 5(a) ), suggesting essentiality of the c - terminal segment for functional complementation in vivo. to validate this observation in liquid medium we inoculated overnight grown cultures at 30c in fresh minimal medium and monitored their growth at 3 hr intervals. the growth curve (figure 5(b) ) clearly indicates that ldtopiiic258 could not functionally complement the slow - growing topoisomerase iii mutant yeast. this indicates that the conserved c - terminal region between amino acid residues 608866 contains important residues that are required for in vivo function of ldtopiiic258. to get a better insight into the functional characteristics of the enzyme, we next sought to obtain recombinant ldtopiii protein in vitro. ldtopiii was cloned in bacterial expression vector pet-16b and overexpressed in bl21 (de3)-plyss strain and induced with iptg. but the overexpressed protein went to inclusion body and were found in the pellet as insoluble protein which could not be recovered in the soluble fraction in active state. however, to test the activity of the recombinant protein, we have used in vitro transcription - translation kit, which is specially designed for in vitro transcription and translation of target dna to protein in a single reaction. the crude lysate containing the newly synthesized proteins were used for dna relaxation assay. figure 6(a) shows dna relaxation by increasing amount of recombinant ldtopiii (lanes 28). the clearly show that the recombinant protein containing lysates were able to relax the negatively supercoiled dna. to test that the activity was not coming from the lysate itself, we have carried out dna relaxation activity with the empty vector containing lysate which contained insignificant amount of activity, shown in figure 6(b) (lane 3). the type ia topoisomerases are among the most conserved proteins in nature, and their presence in all organisms is supported by extensive biochemical and genomic sequence data. this universal presence suggests that the type ia dna topoisomerases play an indispensable role in one or more fundamental processes involving dna, plausibly in the removal of double holliday junctions. topoisomerases iii and iii of kinetoplastid parasites seem to be orthologues of same kind of enzymes in other eukaryotes, notable for branching early within their respective groups. in the present study, for the first time we have identified functionally active dna topoisomerase iii from l. donovani. blast sequence alignments suggested topoisomerase iii from leishmania has high homology with human and drosophila topoisomerase iii. it shares many features, which are typical for other topoisomerase iii proteins including the cxxc type of motifs and a long stretch of g and r residues at its c - terminus. gfp - fused ldtopiii localized both inside the nucleus and the kinetoplast of l. donovani parasites indicating the involvement of ldtopiii in dna processing inside both the parasite organelle. our show for the first time the presence of an ia type of topoisomerase in the nucleus, as well as in the kinetoplast of leishmania parasites. previously, a ia type of topoisomerase from bacterial origin has been reported to be mitochondrial in t. brucei. ldtopiii could suppress the slow - growth phenotype of the mutant yeast indicating the functional conservation of topoisomerase iii activity. the is consistent with the earlier observations made with human and drosophila topoisomerase iii enzymes. the c - terminal deletion construct of ldtopiii lacking its zn binding domain was unable to rescue the topoisomerase iii mutant yeast from slow - growing phenotype revealing that the c - terminal 258 amino acids were indispensable for functional complementation of ldtopiii in vivo. previous report reveals the requirement of the c - terminus region of bacterial topoisomerase iii in substrate specificity. it is possible that c - terminal end of the leishmanial topoisomerase iii protein is essential for dna binding which requires further investigations. site directed mutagenesis study revealed that tyrosine at 327 position within the conserved amino acid stretch is the active site tyrosine of ldtopiii and when this tyrosine is mutated to phenylalanine, the protein failed to complement the slow - growing mutant yeasts. the indicates towards involvement of the functionally active ldtopiii in rescue of the mutant yeast from slow - growth. our attempts to purify recombinant ldtopiii enzymes in active state from bacteria were unsuccessful as the proteins consistently went to inclusion body. but we were able to study, for the first time, the in vitro dna relaxation activity the recombinant topoisomerase iii protein from the kinetoplastid parasite leishmania, when synthesized using cell free in vitro transcription - translation kit. altogether, this is the first report of functionally active topoisomerase iii protein from unicellular kinetoplastid parasite leishmania. important nonoverlapping function of the two isozymes of topoisomerse iii has been revealed by previous studies. the mouse - knockout experiments suggests, the form is essential for embryonic development, whereas the form is critical for life span. genetic experiments in yeast have demonstrated that top3 plays a role in suppressing mitotic recombination and in resolving recombined homologous chromosomes during meiosis i. preferential cleavage of plasmid - based r- and d - loops, has been reported by drosophila topoisomerase iii. furthermore, the combined action of either yeast or bacterial topoisomerse iii and the dna helicase recq can promote the formation of dna catenanes. the unwinding action of a recq type helicase appears to generate a dna structure that can be recognized by a topoisomerase iii. the only report of functionally significant topoisomerase iii from kinetoplastide parasite came very recently, which describes that topoisomerse iii from trypanosoma brucei influences antigenic variation by monitoring expression - site - associated vsg switching. existence of functionally active topoisomerase iii protein in leishmania indicates towards its role in dna metabolism in the parasites, which requires further studies and might emerge as a new therapeutic target that can be exploited against the deadly parasites. this work was supported by network project (nwp038) of council of scientific and industrial research (csir), government of india to h. k. majumder.

dna topoisomerases of kinetoplastids represent a family of dna processing enzymes that essentially solve the topological problems not only in nuclear dna but also in kinetoplast dna. we have, for the first time, identified a leishmania donovani homologue of bacterial and eukaryotic ia type of topoisomerase iii protein and termed as ldtopiii. complementation study of wild - type and mutant ldtopiii with slow - growing topoisomerase iii mutant yeast s. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase iii protein, the 327 tyrosine being the active site amino acid. a c - terminal deletion construct of ldtopiii could not suppress the slow - growth phenotype of mutant yeast, indicating the requirement of c - terminal region for the enzyme function in vivo.ldtopiii localized inside the nucleus and kinetoplast of the parasite. taken together, our study indicates functional conservation and possible role of ldtopiii in parasite dna processing.

a 76-year - old woman, gravida 3, para 2, presented with intermittent lower abdominal pain of increasing severity. the patient reported a 10 kg weight loss with associated anorexia and constipation for approximately six months. physical examination revealed abdominal tenderness and a fixed lower abdominal mass at roughly six months in gestational size. abdominal and pelvic ultrasonography showed a multilobulated solid and cystic mass in the pelvic cavity. a computerized tomography scan of the abdomen and pelvis demonstrated a 16108 cm lobulating - contoured and heterogeneously - enhanced mass occupying the lower abdomen and pelvic cavity. serum levels of tumor markers, with normal values in parentheses, were as follows: cancer antigen 125, 101.2 u / ml (< 35 u / ml); cancer antigen 19 - 9, 4.8 u / ml (< 30 u / ml). intraoperatively, a yellowish, hard, friable, 20157 cm mass had replaced the right lateral uterine wall and had adhered to the colon, rectum, and appendix. the right ovary was not recognized, but the left ovary and both fallopian tubes appeared normal. the tumor grossly occupied the entire right myometrium and was bulging into the endometrial cavity. the cut surface of the tumor had a variegated appearance, including components of greyish soft, whitish fish - flesh, and yellowish myxoid with small cystic changes (fig . microscopic findings exhibited highly cellular and necrotic tumor consisting of bundles or fascicles of spindle cells in a myxoid stroma ( fig . mitotic figures were frequently counted with more than 100 mitotic figures per 10 high power fields . right ovarian tissue was identified in the fibrous tumor capsule without direct invasion of tumor cells . 5), myo - d1, desmin, and vimentin. no stains for smooth muscle actin, pan - cytokeratin, epithelial membrane antigen, or estrogen receptor were postive. rms is a common soft tissue tumor seen in children, but pure uterine sarcomas arising in the female genital tract are rare, particularly in adults.1 in the pediatric population, the most commonly involved site is the vagina, whereas the uterine cervix and corpus are the most frequent locations of rms in adult women.2,3 in postmenopausal women, uterine rmss appear to be entirely the pleomorphic type with extremely poor clinical outcome.4,5 rmss can be morphologically classified into three categories: embryonal, alveolar, and pleomorphic. the spindle cell variant is a recently characterized and rare subtype of embryonal rms first described in 1992.6 spindle cell rms predominately occurs in the paratesticular region in children and young adults, and carries a relatively favorable prognosis. after rubin et al.7 reported the first two cases of spindle cell rms in adults, limited reports of adult spindle cell rms have been subsequently published.8 according to the largest review performed by nascimento and fletcher,9 the head and neck region were the most common sites for adult spindle cell rms followed by the retroperitoneum and lower extremity. these adult tumors seem to have a more aggressive clinical course compared to pediatric cases. only one case of uterine spindle cell rms has been previously reported in a 28-year - old woman who presented at international federation of gynecology and obstetrics (figo) stage i and was well after two years of follow - up. the case here, was figo stage iiia at presentation, and the patient died of disease three months after diagnosis. differential diagnosis for spindle cell rms of the uterus includes leiomyosarcoma, undifferentiated endometrial sarcoma, and rhabdomyosarcomatous components of a mullerian adenosarcoma or carcinosarcoma. immunohistochemical staining for skeletal muscle markers can be helpful in differentiating spindle cell rms from other spindle cell tumors. leiomyosarcoma is the most common malignant mesenchymal tumor of the uterine corpus and has similar histologic finding to spindle cell rms. tumor cells of leiomyosarcoma are spindle - shaped with characteristic cigar - shaped nuclei, or large pleomorphic resembling rhabdomyoblasts. markers such as myo - d1 and myoglobin are more specific than muscle - specific actin, smooth muscle actin, and desmin, because smooth muscle markers have limited value in differential diagnosis. undifferentiated endometrial sarcomas, highly aggressive neoplasms, do not histologically resemble entometrial stroma and may consist of spindle or markedly atypical tumor cells. although cd10 expression has been observed in both uterine rms4 and undifferentiated endometrial sarcoma, immunophenotypic evidence of skeletal muscle differentiation has only been seen with rms. mixed epithelial and mesenchymal tumors including adenosarcoma and carcinosarcoma consist of a mixture of benign or malignant epithelial and sarcomatous components. epithelial components are usually glandular or rarely non - glandular and can be constitute a small portion of the tumor, especially in carcinosarcoma. sarcomatous elements may be either homologous or heterologous with heterologous components frequently exhibiting rhabdomyoblastic differentiation. when sarcomatoid overgrowth predominates, epithelial components can be obscured. extensive tumor sampling with careful investigation for epithelial areas, and immunohistochemical confirmation may be required to differentiate rms from mixed epithelial and mesenchymal tumors. in , pure rms of the uterine corpus is uncommon, and the spindle cell variant is especially rare. here, we present a case of spindle cell rms of the uterine body in 76-year - old woman. uterine rms should be differentiated from leiomyosarcoma, undifferentiated sarcoma, and mixed epithelial and mesenchymal tumors. extensive tumor sampling, careful microscopic examination, and immunohistochemical staining may aid in a diagnosis of spindle cell rms.

uterine rhabdomyosarcoma (rms) typically presents as a mixed epithelial and mesenchymal tumors. pure rmss of the female genital tract are uncommon. spindle cell variant of rms is a rare morphologic subtype of embryonal rms and mostly occurs in the paratesticular region of children. here, we present a case of uterine spindle cell rms in a 76-year - old woman. the tumor, 20157 cm in size, was highly necrotic and adherent to the colon and rectum. tumor cells were mostly spindle - shaped, and isolated rhabdomyoblasts were scattered. immunohistochemical stains for myoglobin and myo - d1 showed diffuse positivity for tumor cells. the patient died only of disease three months after diagnosis.

focal motor status epilepticus (fmse) is characterized by repetitive and persistent myoclonic, clonic, or tonic contractions of the arm, face, or neck that can affect an entire side of the body, with or without alteration of mental functions. fmse can be associated with several medical conditions including brain tumors, metabolic disturbances such as hyponatremia or a hyperosmolar state, and focal cerebral lesions ing from stroke.1,2 fmse is often related to non - ketotic hyperglycemia (nkh) and accompanied by an underlying localized brain lesion.3,4 however, we are unaware of any report describing fmse with nkh that was accompanied by an acute cerebral infarction. furthermore, little is known as to whether it is epileptogenic or not. here , we report the case of a patient who presented with fmse associated with nkh and a magnetic resonance image (mri)-documented acute cerebral infarction, and later developed recurrent seizures. a 46-year - old woman presented with sudden and progressive erratic movements of the right hand and arm that had developed 3 days before visiting our hospital. on inspection, initial myoclonus of the right hand turned into a tonic contraction that spread to the wrist and forearm and persisted for approximately 30 seconds. the frequency of seizures gradually increased from once an hour at initial symptom onset, to once every 5 minutes 3 days later. she reported feeling tingling sensations in her right hand and arm prior to seizure onset. she was having diabetes mellitus for more than 15 years, but reported no previous stroke or seizure. no focal neurological abnormality, other than partial seizures, was observed. serum glucose level, osmolality, and sodium level were 690 mg / dl, 312 mosm / l, and 128 meq / l, respectively. mri demonstrated a small focal acute infarction in the left frontal subcortical area on a diffusion - weighted image (dwi). a focal cortical hyperperfusion in the left central area was observed on 99m - tc ecd single photon emission computed tomography (spect), which disappeared 2 weeks later. magnetic resonance angiogram (mra) and transfemoral cerebral angiogram (tfca) revealed atherosclerotic stenosis of the left internal carotid artery (ica) (fig . we administered antiepileptic drugs ( carbamazepine cr and valproate) for 2 weeks and maintained her glucose levels below 250 mg / dl with insulin. the clinical seizures were not observed after 2 days, and she was discharged without sequel. two years later, she visited the department of neurology because of recurrent generalized tonic - clonic seizures. during the previous month her fasting and 2-hour postprandial glucose levels were 125 mg / dl and 198 mg / dl, respectively. routine chemistry and assays for electrolytes and serum osmolality did not indicate any abnormality, except for mild elevation of serum creatinine level (1.4 mg / dl). a follow - up mri failed to indicate recurrent strokes or lesions other than a previous small infarction, and interictal eeg was normal. such seizures can be a presenting symptom of hyperglycemia without ketoacidosis.5 increased metabolism of gamma - aminobutyric acid triggered by hyperglycemia may lower seizure threshold.6 associated metabolic disturbances, such as mild hyperosmolality and mild hyponatremia, may contribute to the seizures.3,7 ketosis is known to have an anticonvulsant effect, and the direct stabilizing effect of ketone bodies and accompanying acidosis may play an important role in preventing seizures.8 therefore, seizures are more frequently developed in patients with nkh than in patients with diabetic ketoacidosis. even though the underlying mechanisms are not fully understood, seizures associated with nkh are not rare in clinical practice. clinical symptoms typically originate from a focal cerebral lesion, whereas hyperglycemia affects the entire brain. these symptoms may from previous underlying structural lesions that are more susceptible to the proconvulsant effects of nkh. a previous study reported that the majority of patients examined showed evidence of localized structural cerebral lesions.3,4 in contrast, pre - existing or acute structural lesions were not found in other case reports.7,912 our patient had a small focal subcortical infarct, as revealed by dwi, the location of which was relevant to the patient s symptoms. in previous reports , dwi was not used for evaluation, and small acute infarctions could have been missed on ct or routine mri. limb shaking ing from transient ischemic attacks (tias) should be differentiated from fmse in patients who have carotid artery disease. in our case, this differentiation can be made with several points. firstly, limb - shaking tias usually do not present with jacksonian march as the attack experienced by our patient did. secondly, tia symptoms persist less than 5 minutes and are often accompanied by hemiparesis, which was inconsistent with our patient s symptoms.13,14 moreover, brain spect showed hyperperfusion in the area relevant to the patient's seizures. based on the above findings, the abnormal arm movements observed in our patient appear unrelated to tia. however, conventional ictal scalp eeg often fails to detect seizure activities in fmse because of a limited discharges originated from central sulcus.2,15 in these cases, myoclonus - locked eeg back - averaging technique may be helpful in demonstrating the correlation of the myoclonus with paroxysmal discharges in the motor cortex.16 however, typical clinical manifestations with relevant spect finding of our patient are sufficient for the diagnosis. patients with histories of status epilepticus have a higher risk of recurrent unprovoked seizures, and the risk is much higher in patients with acute structural brain lesions than in patients with metabolic disturnbances.17,18 we usually administer antiepileptic drugs during the acute period of status epilepticus. however, long - term pro - phylactic antiepileptic treatment is not generally recommended because evidence for effectiveness of such long - term treatment in preventing epileptogenesis is still lacking.16,1719 in , our patient experienced acute symptomatic fmse and subsequent recurrent unprovoked seizures, that is, epilepsy. on the basis of our experience, we suggest that patients with nkh and fmse may have small cerebral infarctions, and dwi is crucial for detecting such lesions. furthermore, clinicians should pay careful attention to the emergence of epilepsy during follow - up.

focal motor status epilepticus (fmse) is often associated non - ketotic hyperglycemia (nkh). there are no previous reports describing fmse with nkh that was accompanied by an acute cerebral infarction and its long term follow - up . we describe the case of a patient having focal motor status epilepticus (fmse) associated with non - ketotic hyperglycemia (nkh) and acute cerebral infarction who later developed recurrent unprovoked seizures. a small acute infarct was observed in the left frontal subcortical area on diffusion - weighted images (dwi). fmse was initially controlled with short term antiepileptic drugs and strict glucose control. two years later, recurrent seizures occurred, and long - term antiepileptic drug treatment was administered. dwi should be considered for acute cerebral infarction in patients having fmse associated with nkh, and careful follow - up should be conducted for such patients.

one of the mysteries in prion research is the structure of the infectious form of mammalian prion protein, prpsc. we used ms analysis of h / d exchange to examine brain - derived prpsc. our data indicate that, contrary to popular models, prion protein conversion involves refolding of the entire region c - terminal to residue ~8090, and that this region in prpsc consists of - strands and relatively short turns / loops, with no native -helices present.

until recently, treatment for gynecologic malignancies has focused almost exclusively on prolongation of life, and few research studies have adequately addressed issues related to quality of life. quality of life (qol) typically involves the assessment of several dimensions: physical well - being, emotional well - being, social well - being, and functional well - being. as recently as 1993, andersen published an article acknowledging a grave lack of research on quality of life and challenging " institutions and study groups to support quality of life research for women with gynecologic cancer ". to date, few studies have utilized qol as a primary endpoint.. often, there are few, if any, symptoms until the tumor is in an advanced stage. further, these symptoms are often non - specific and may consist of things like abdominal distention, vaginal bleeding, abdominal or low back pain, often leading treating professionals to misinterpret early signs or defer further work - up. treatment for gynecologic malignancies is often quite morbid and may involve multiple modalities (surgery, radiation and chemotherapy). changes in bowel, bladder, hormonal, sexual and reproductive function are common. in addition, palliation is often difficult in the terminal stage, and death from a slow, obstructive, intra - abdominal process is not unusual. women frequently must adjust to physical changes after treatment including loss of ovarian function, hot flashes, vaginal dryness, hair and skin changes, and mood changes. surgical scarring may be another hurdle to adjustment, as are the need for urostomy or colostomy. sexual functioning may be impaired, and difficulties with desire and sexual response are common. infertility is an issue for many young women diagnosed with gynecologic cancer, and concerns may include adoption, egg donation, and surrogacy. the goals of the present article are to summarize what is known about qol in women treated for gynecologic malignancies, compare qol in women with gynecologic versus other malignancies, attempt to draw tentative about variables affecting quality of life and risk factors for maladjustment, and suggest directions for future research. bodurka - bevers and colleagues assessed the prevalence of depression and anxiety in 246 women diagnosed with ovarian cancer. these patients were at all stages of disease, and also were in various phases of active treatment or surveillance. suggested that 21% met criteria for depression and 29% scored above the 75percentile for anxiety. the authors conclude that the prevalence of depression and anxiety may be higher than expected in an ovarian cancer population, and this clearly highlights the need for further assessment of qol in this group of patients. miller, pittman and strong also highlighted the need for assessment of quality of life and emotional functioning. these researchers administered questionnaires to 95 patients with gynecologic cancer at least 6 months after completion of treatment. a majority of patients wanted their physicians to ask questions dealing with spirituality, death and dying, and emotional problems. studied 115 women between the ages of 21 and 83 years who were referred to a university hospital for ovarian, endometrial and cervical cancer. women completed the sf-36 questionnaire, a widely used and well - validated qol instrument. the authors then compared these scores with age - specific expected mean values in published data from a healthy population of women. of this research suggested that women with primary (as opposed to recurrent) gynecologic cancer had qol scores that were similar overall to healthy women. however, patients with recurrent disease scored an average of 10 points lower on each scale of the sf-36. also notable was the fact that women with primary gynecologic cancer scored lower than healthy women on scales measuring emotional and physical role functioning. patients undergoing palliative chemotherapy treatment had the lowest scores overall, as would be expected. the authors also used linear regression to adjust for age and primary vs. progressive / recurrent disease status. of this analysis showed that the poorest qol scores were reported by the youngest women with cervical cancer. this was in opposition to women with ovarian and endometrial cancer where age was negatively correlated with qol. studied the qol of long - term (over 5 years) survivors of ovarian cancer. 49 women were assessed and the indicated that this disease - free sample enjoyed a good qol compared to other cancer survivors and non - cancer cohorts. however, approximately 20% of survivors reported significant long - term treatment related side effects, including abdominal, gynecologic and neurologic toxicity. furthermore, greater than half of the women surveyed indicated that they would have attended a support group if one were available to them at the time of diagnosis and treatment. qol data (eortc-30, spitzer ql - i) was collected at six points from pre- to post - treatment. the first assessment of qol was conducted at one day prior to initiation of treatment. of the subjects, 26.2% were diagnosed with breast cancer, 31.9% with cervical cancer, 25.8% with ovarian cancer, and 16.1% with endometrial cancer. at pre - treatment , there were no statistically significant differences between breast cancer and gynecologic cancer patients on any qol domain. during active treatment, breast cancer patients had significantly higher qol scores, particularly in the areas of physical functioning and role functioning. at completion of treatment, breast cancer patients scored significantly lower on emotional functioning compared to patients with ovarian cancer. at six - month and one year post - treatment follow - up visits, there were no significant differences between breast cancer and gynecologic cancer patients on any of the qol domains assessed. overall, the researchers conclude that during active treatment patients with gynecologic cancer are significantly more physically impaired compared to breast cancer patients. however, qol is comparable between groups at one - year follow - up, suggesting that gynecologic cancer survivors experience significant improvement in qol following treatment. predictors of long - term qol included pre - treatment performance status and severity of surgery. not predictive was family support, number of treatments, age, stage or site of disease. miller, pittman, case & mcquellon compared qol in disease - free gynecologic cancer patients (n = 85) to that of 42 unmatched healthy women seen for standard gynecologic screening exams. their data showed no overall difference in fact - g scores between gynecologic cancer patients and normal women. in fact, cancer survivors scored slightly higher on the emotional well - being subscale. within the cancer survivors patients who had treatment over a longer period of time reported decreased functional well being and total fact - g scores. it should be noted that these patients tended to have an ovarian cancer diagnosis and most had been treated with surgery and approximately 6 months of combination chemotherapy. patients treated with surgery only had better overall qol, probably due to short treatment time and less advanced disease. the authors note that prior research has shown that acute treatment effects are resolved after 6 months, and there is only little change expected thereafter. the authors propose that lower levels of education may be predictive of a less supportive social environment, limited knowledge of health issues and poor general health. eisemann & lalos assessed well - being in women with endometrial and cervical cancer at pre - treatment and also at 6 month and 1 year post - treatment. furthermore, well - being before treatment was significantly predictive of post - treatment well - being. chan and colleagues performed a prospective, longitudinal study of 74 newly diagnosed gynecologic cancer patients. qol was measured at 4 points from pre - treatment to 18 months post - treatment. a structured interview was used to measure self - esteem, outlook on life, self - role and femininity. it should be noted that this study only included individuals who had no recurrence of their disease, so this may not be indicative of all patients diagnosed with gynecologic malignancies. also notable is the fact that all psychosocial variables were assessed at pre - treatment primarily by clinical interview (as opposed to more standardized assessment tools). the incidence of depression in this sample was twice that seen in a healthy population. subjects reported no change in relationship with spouse and sexual activity (though this may have been under - reported due to the fact that this variable was assessed by clinical interview). the authors found three high - risk groups for maladjustment; those with low religious belief, those who had received surgical treatment, and those with low educational level. lutgendorf and colleagues assessed 98 women with early stage or regionally advanced gynecologic cancer. prospective assessments were done measuring qol (fact - g), coping style (cope) and mood (poms) at pre - treatment and one year post - diagnosis. sleep disturbance was common throughout the study, and occurred in approximately 40% of the sample. surprisingly, medical factors such as disease extent and treatment intensity did not significantly predict physical well being at one year. however, coping strategies contributed significantly to the variance of physical well being, even when medical factors were controlled. over the course of the first year following diagnosis, emotional and functional the authors note that this improvement occurred even in the absence of significant increases in physical well being, suggesting possible adaptation to residual physical limitations. decreases in anxiety, depression and confusion were seen in both groups, but regionally advanced patients had poorer qol and mood compared to early stage patients. interestingly, coping strategies appeared to be very predictive of qol at one year post - treatment. greater disengagement (avoiding problems, giving up attempts to cope) was associated with poorer relationship with physician, poorer functional and physical well being, and greater mood distress. another study by chan and colleagues assessed 144 women with newly diagnosed gynecologic cancer. these subjects were assessed at pre - treatment, immediately post - treatment, 6 month, 12 month and 24 month post - treatment. they assessed the impact of age, symptoms, disease parameters, and treatment type on qol using the eortc-30. in this study as well, women with recurrent disease were dropped from final analysis, so this likely represents a sample of the most medically healthy patients. this is important, as this study may not accurately represent the qol of all newly diagnosed women, but may represent the qol of newly diagnosed women with the most favorable prognosis. in contrast to their earlier study, the suggested that patients treated with surgery alone reported the best qol (compared to those treated with multi - modality treatment). site and stage of disease had no significant effect on qol after treatment, and qol remained stable between 624 months following treatment. furthermore , overall qol appeared to improve after treatment, and this improvement was seen in global health status, functional scales and symptom report scales. there was a strong correlation between pre - treatment qol and that at 24 months post - treatment. in another compelling study, lutgendorf and colleagues assessed 48 women on qol (fact - g), mood (poms) and coping style (cope). 24 women had received one year of extensive chemotherapy, and 24 women had received no chemotherapy. overall, extensively treated women reported substantial, lasting decrements in physical, functional and emotional well being. avoidant coping again seemed to predict poorer qol, specifically in domains of physical and emotional well being. social well being appeared unimpaired in both groups. surprisingly, social support was not associated with any of the outcome variables, and the authors suggest that perhaps social support is not as important in maintaining qol post - treatment as it is during pre- and active treatment. bodurka - bevers and colleagues assessed the prevalence of depression and anxiety in 246 women diagnosed with ovarian cancer. these patients were at all stages of disease, and also were in various phases of active treatment or surveillance. suggested that 21% met criteria for depression and 29% scored above the 75percentile for anxiety. the authors conclude that the prevalence of depression and anxiety may be higher than expected in an ovarian cancer population, and this clearly highlights the need for further assessment of qol in this group of patients. miller, pittman and strong also highlighted the need for assessment of quality of life and emotional functioning. these researchers administered questionnaires to 95 patients with gynecologic cancer at least 6 months after completion of treatment. a majority of patients wanted their physicians to ask questions dealing with spirituality, death and dying, and emotional problems. studied 115 women between the ages of 21 and 83 years who were referred to a university hospital for ovarian, endometrial and cervical cancer. women completed the sf-36 questionnaire, a widely used and well - validated qol instrument. the authors then compared these scores with age - specific expected mean values in published data from a healthy population of women. of this research suggested that women with primary (as opposed to recurrent) gynecologic cancer had qol scores that were similar overall to healthy women. however, patients with recurrent disease scored an average of 10 points lower on each scale of the sf-36. also notable was the fact that women with primary gynecologic cancer scored lower than healthy women on scales measuring emotional and physical role functioning. patients undergoing palliative chemotherapy treatment had the lowest scores overall, as would be expected. the authors also used linear regression to adjust for age and primary vs. progressive / recurrent disease status. of this analysis showed that the poorest qol scores were reported by the youngest women with cervical cancer. this was in opposition to women with ovarian and endometrial cancer where age was negatively correlated with qol. studied the qol of long - term (over 5 years) survivors of ovarian cancer. 49 women were assessed and the indicated that this disease - free sample enjoyed a good qol compared to other cancer survivors and non - cancer cohorts. approximately 20% of survivors reported significant long - term treatment related side effects, including abdominal, gynecologic and neurologic toxicity. furthermore, greater than half of the women surveyed indicated that they would have attended a support group if one were available to them at the time of diagnosis and treatment. qol data (eortc-30, spitzer ql - i) was collected at six points from pre- to post - treatment. the first assessment of qol was conducted at one day prior to initiation of treatment. of the subjects, 26.2% were diagnosed with breast cancer, 31.9% with cervical cancer, 25.8% with ovarian cancer, and 16.1% with endometrial cancer. at pre - treatment , there were no statistically significant differences between breast cancer and gynecologic cancer patients on any qol domain. during active treatment, breast cancer patients had significantly higher qol scores, particularly in the areas of physical functioning and role functioning. at completion of treatment, breast cancer patients scored significantly lower on emotional functioning compared to patients with ovarian cancer. at six - month and one year post - treatment follow - up visits , there were no significant differences between breast cancer and gynecologic cancer patients on any of the qol domains assessed. overall, the researchers conclude that during active treatment patients with gynecologic cancer are significantly more physically impaired compared to breast cancer patients. however, qol is comparable between groups at one - year follow - up, suggesting that gynecologic cancer survivors experience significant improvement in qol following treatment. predictors of long - term qol included pre - treatment performance status and severity of surgery. not predictive was family support, number of treatments, age, stage or site of disease. miller, pittman, case & mcquellon compared qol in disease - free gynecologic cancer patients (n = 85) to that of 42 unmatched healthy women seen for standard gynecologic screening exams. their data showed no overall difference in fact - g scores between gynecologic cancer patients and normal women. in fact, cancer survivors scored slightly higher on the emotional well - being subscale. within the cancer survivors patients who had treatment over a longer period of time reported decreased functional well being and total fact - g scores. it should be noted that these patients tended to have an ovarian cancer diagnosis and most had been treated with surgery and approximately 6 months of combination chemotherapy. patients treated with surgery only had better overall qol, probably due to short treatment time and less advanced disease. the authors note that prior research has shown that acute treatment effects are resolved after 6 months, and there is only little change expected thereafter. the authors propose that lower levels of education may be predictive of a less supportive social environment, limited knowledge of health issues and poor general health. eisemann & lalos assessed well - being in women with endometrial and cervical cancer at pre - treatment and also at 6 month and 1 year post - treatment. furthermore, well - being before treatment was significantly predictive of post - treatment well - being. chan and colleagues performed a prospective, longitudinal study of 74 newly diagnosed gynecologic cancer patients. qol was measured at 4 points from pre - treatment to 18 months post - treatment. a structured interview was used to measure self - esteem, outlook on life, self - role and femininity. it should be noted that this study only included individuals who had no recurrence of their disease, so this may not be indicative of all patients diagnosed with gynecologic malignancies. also notable is the fact that all psychosocial variables were assessed at pre - treatment primarily by clinical interview (as opposed to more standardized assessment tools). the incidence of depression in this sample was twice that seen in a healthy population. subjects reported no change in relationship with spouse and sexual activity (though this may have been under - reported due to the fact that this variable was assessed by clinical interview). the authors found three high - risk groups for maladjustment; those with low religious belief, those who had received surgical treatment, and those with low educational level. lutgendorf and colleagues assessed 98 women with early stage or regionally advanced gynecologic cancer. prospective assessments were done measuring qol (fact - g), coping style (cope) and mood (poms) at pre - treatment and one year post - diagnosis. sleep disturbance was common throughout the study, and occurred in approximately 40% of the sample. surprisingly, medical factors such as disease extent and treatment intensity did not significantly predict physical well being at one year. however, coping strategies contributed significantly to the variance of physical well being, even when medical factors were controlled. over the course of the first year following diagnosis, emotional and functional the authors note that this improvement occurred even in the absence of significant increases in physical well being, suggesting possible adaptation to residual physical limitations. decreases in anxiety, depression and confusion were seen in both groups, but regionally advanced patients had poorer qol and mood compared to early stage patients. interestingly, coping strategies appeared to be very predictive of qol at one year post - treatment. greater disengagement (avoiding problems, giving up attempts to cope) was associated with poorer relationship with physician, poorer functional and physical well being, and greater mood distress. another study by chan and colleagues assessed 144 women with newly diagnosed gynecologic cancer. these subjects were assessed at pre - treatment, immediately post - treatment, 6 month, 12 month and 24 month post - treatment. they assessed the impact of age, symptoms, disease parameters, and treatment type on qol using the eortc-30. in this study as well, women with recurrent disease were dropped from final analysis, so this likely represents a sample of the most medically healthy patients. this is important, as this study may not accurately represent the qol of all newly diagnosed women, but may represent the qol of newly diagnosed women with the most favorable prognosis. in contrast to their earlier study, the suggested that patients treated with surgery alone reported the best qol (compared to those treated with multi - modality treatment). younger patients reported poorer physical health compared to older patients. site and stage of disease had no significant effect on qol after treatment, and qol remained stable between 624 months following treatment. furthermore, overall qol appeared to improve after treatment, and this improvement was seen in global health status, functional scales and symptom report scales. there was a strong correlation between pre - treatment qol and that at 24 months post - treatment. in another compelling study, lutgendorf and colleagues assessed 48 women on qol (fact - g), mood (poms) and coping style (cope). 24 women had received one year of extensive chemotherapy, and 24 women had received no chemotherapy. overall, extensively treated women reported substantial, lasting decrements in physical, functional and emotional well being. avoidant coping again seemed to predict poorer qol, specifically in domains of physical and emotional well being. social well being appeared unimpaired in both groups. surprisingly, social support was not associated with any of the outcome variables, and the authors suggest that perhaps social support is not as important in maintaining qol post - treatment as it is during pre- and active treatment. gynecologic malignancies pose special risks for quality of life. despite the fact that gynecologic malignancies occur in approximately 1 in 20 women in the united states, qol has not been widely researched, with the bulk of research devoted to prolongation of life. reports in the literature have been conflicting, with some finding deterioration in qol and some finding stability or improvement over time. little has been known about the impact of various treatments, diagnoses, stages of illness, and other risk factors on qol in these patients. given the challenges and changes that women must face after a diagnosis of gynecologic cancer, qol is an especially pertinent issue on which to focus. before concluding anything regarding qol, however, several caveats are important to note. first, it is important to utilize a well - validated measure of qol in order to compare qol in patients with gynecologic malignancies to any other group of patients or healthy subjects. the studies reviewed in the current paper have generally done so, with the exception of three. second, due to the relatively small number of gynecologic cancer patients seen at any one cancer center, most of the research studies above have grouped gynecologic cancer patients into one group, as opposed to separating out by diagnosis. because of this, any interpretation of the impact of diagnosis on qol must be tentative. related to this is the fact that type of treatment may be reflective of stage of disease (i.e. patients with advanced disease will be more likely to have multi - modality treatment, while patients with early stage disease may have surgery alone). as such, any interpretation of the impact of treatment type on qol must also be done cautiously and in the context of disease stage. there have been no prospective studies in which gynecologic cancer patients have been randomly assigned to any psychological or psychiatric treatment before, during or after treatment. the author of this review is currently planning such a study to investigate how a structured , psychological support group will impact levels of a growth factor (vegf) which has been linked to cancer progression in women with ovarian cancer. despite the limitations of the current research , there appears to be a higher than expected incidence of depression and anxiety in patients undergoing treatment for gynecologic malignancies. this is likely related to the high treatment toxicity and often poor prognosis of many of these illnesses. it appears that qol is most negatively affected from the time of diagnosis through the completion of treatment. it also appears that qol is more substantially impaired during treatment of gynecologic malignancies than during treatment of other cancers. 12 years after treatment, disease free patients report qol that is generally equivalent to other cancer survivors and healthy women. further, emotional and functional well - being increase over the first year following treatment, even in the absence of corresponding increases in physical well - being, suggesting adaptation to residual physical limitations. despite these positive , a significant minority of patients continue to report lasting emotional problems and treatment related toxicities. risk factors for maladjustment include treatment with radiotherapy or multi - modality treatment, increased length of treatment, younger age, and coping using a disengaged style. lower levels of education and spiritual / religious belief, as well as lack of help at home, are also risk factors for poor qol. future research should include large, multicenter studies, which would allow comparative analysis of qol by diagnosis. also, studies measuring the impact of various chemotherapeutic agents on qol should be undertaken, as there appear to be long - lasting toxicities from many of the chemotherapeutic regimens, which are only beginning to be understood. due to the difficulty in palliating terminal illness, any studies focusing on improvement of qol in end - stage disease patients would be welcome and clinically relevant. finally, prospective studies of the impact of psychological treatments on qol and prognostic factors should be undertaken. future studies will hopefully assist women in coping with the challenges and rigors of treatment and post - treatment toxicities. ideally, these studies would determine risk factors not only for psychological morbidity, but also medical mortality and morbidity, and attempt to modify psychosocial variables to improve survival time and quality of life.

gynecologic malignancies occur in approximately 1 in 20 women in the united states. until recently, clinical management of these cancers has focused almost exclusively on prolonging the survival of patients. a recent literature search using medline revealed relatively few research studies that reported data on quality of life (qol) in a gynecologic cancer population. reports in the literature have been conflicting, with some studies finding deterioration in qol and some finding stability or improvement in qol over time. until recently, the impact of various treatments (surgery, radiation, chemotherapy) on qol in this population was unknown. recently, the qol of women with gynecologic cancer has been compared to that of women with other types of cancer. also , risk factors for poor adjustment in gynecologic cancer are beginning to be investigated. this presentation will attempt to 1 ) summarize the relevant literature on qol in a gynecologic cancer population, 2 ) compare qol in this population to other types of cancer, 3 ) examine risk factors for poor adjustment and 4 ) describe the limitations of the literature and future research directions.overall, it appears that qol is most negatively affected from time of diagnosis through completion of treatment. following treatment , qol appears to improve over the course of 612 months, but then appears to remain stable from that time through two years post - treatment. compared to breast cancer patients, it appears that gynecologic cancer patients experience poorer qol on several domains during active treatment, but that after completion of treatment, overall qol is similar between groups. risk factors for maladjustment include treatment with radiotherapy or multi - modality treatment, increased length of treatment, younger age, and coping using a disengaged style. other risk factors include lower education, poor social support and lower levels of religious belief. the significance of these findings and future research directions will be discussed.

the success ratio of root canal treatment is reportedly around 90% for pulpectomy and approximately 80% for infected root canal. however, success ratios display large fluctuations according to periapical lesion, condition of infection in the root canal, periodontal disease, and pre- or postoperative occlusive condition. some reports have described no difference in depending on the tooth kind. however, swartz reported low success rates for the mandibular first molar. likewise, yamaki and yamamoto reported significantly inferior treatment for the maxillary first molar. the reason for inferior treatment on the maxillary first molar are the high rates of occurrence of curved canal and advanced curved canal. krithkadatta et al. using the cone - beam - computed tomography, reported unusual root canal morphology in the mandibular first molar. while employing the cone - beam - computed tomography, kottoor et al. furthermore, treatment of the secondary mesio - buccal canal (mbii) is often insufficient or overlooked. research into mbii has mainly examined the rate of occurrence and morphological classifications. weine defined four types according to the divergence state of the canal in the mesio - buccal root: type i, single canal from the pulp chamber to the root apex; type ii, two canals leaving the chamber and merging to form a single canal short of the root apex; type iii, two separate and distinct canals from the pulp chamber to the root apex; and type iv, one canal leaving the chamber and dividing into two separate and distinct canals. however, few reports have examined detection and effective treatment methods. radiographic techniques offer effective, nondestructive examination. the observation by dental radiography does not always agree with that by naked eye or some other anatomical assessments, because the two canals of mesio - buccal root are overlapped in the direction of irradiation. computed tomography for dentistry (dental ct) has recently been developed, allowing an anatomical view of the microstructural organization of the tooth and periodontal hard tissues with three - dimensional images. dental ct offers high picture resolution compared with conventional high - speed spiral - type ct, but the imaging range is restricted. dental ct is used to examine lesions inside bone, tumors, implants, and tooth transplantation, providing effective imaging information. however the purpose of this study was to identify detection characteristics of the mbii for maxillary first molars according to four test methods: dental ct; digital dental radiography; magnifier; and the naked eye. the root canal systems of 86 extracted human maxillary first molars without root canal treatment were completely inspected using micro - focus computed tomography (micro ct) (mct100-mfz ; hitachi medical, tokyo, japan) to accurately assess the number of canals. two researchers experienced in diagnostic imaging observed that the number of orifices, root canals, and apical foramina of all experimental teeth on micro - ct, using these as the gold standard. furthermore, canal systems in mesio - buccal roots were classified as types i - iv using the anatomical classification defined by weine. for all samples, radiographs or floors of the pulp chamber were observed by 10 dentists using the four methods after accessing the cavity preparation. sensitivity, specificity, positive, and negative predictive values and diagnostic accuracy for the four test methods were investigated using the of micro - ct as the gold standard. specifically, the four methods comprised dental ct (mean effective does per once : 0.03 msv . ; psr9000 ; asahi roentgen industry, kyoto, japan); conventional digital dental radiography (mean effective does per once : 0.02 msv . ; dfw-20 ; asahi roentgen industry, or denooptix ; dentsply - sankin, tokyo, japan); magnification telescope with 2-times magnification (surgitel gl275n ; gsc, michigan, usa); and the naked eye. imaging conditions for dental ct were dental ct mode; peak voltage, 60 kv; peak current, 2 ma; and radiation time, 20.48 seconds. conditions for digital dental radiography were peak voltage, 65 kv; peak current, 20 ma; radiation time, 0.1 seconds. the radiation direction was set from the buccal side at right angles to the longitudinal axis of the tooth. with the magnifier or naked eye, orifices of the root canal of all samples were searched using # 08 root canal instruments on the floors of the pulp chambers. imaging conditions for micro - ct when establishing gold standard were peak voltage, 65 kv; peak current, 100 a. the time intervals of observation between conditions for a sample were set up long enough to avoid the examiner's preconception. sensitivity, specificity, positive, and negative predictive values and diagnostic accuracy were calculated from measured values of various observations. using these , the test was used to compare analyze differences between the various conditions. mbii (arrows) in typical images from the micro - focus - computed tomography (micro ct) (a) mbii (arrows) in typical images with the x - ray - computed tomography for dentistry (dental ct); (b) mbii (arrow) in typical images of digital dental radiography. mbii was not verified on the image of photography from buccal - palatal direction; (c) mbii (arrow) in the image of the floor of the pulp chamber as the gold standard, micro ct - revealed mbii (types ii, iii, or iv) in 52 of 86 teeth (60.5%) in all samples. anatomical classification showed type i in 34 teeth (39.5%), type ii in 13 teeth (15.1%), type iii in 24 teeth (27.9%), and type iv in 15 teeth (17.5%). dental ct showed 56.4% sensitivity, 76.4% specificity, 78.9% positive predictive value, 47.1% negative predictive value, and 64.2% diagnostic accuracy. digital dental radiography images showed 13.4% sensitivity, 84.6% specificity, 58.9% positive predictive value, 36.0% negative predictive value, and 40.3% diagnostic accuracy. magnification telescope observation showed 26.1% sensitivity, 72.0% specificity, 59.3% positive predictive value, 38.5% negative predictive value, and 44.1% diagnostic accuracy. naked eye observation showed 20.3% sensitivity, 76.0% specificity, 56.8% positive predictive value, 37.9% negative predictive value, and 42.0% diagnostic accuracy. dental ct offered superior compared with the other three methods for all inspection ratios, and significant differences were seen between the of dental ct and the other three methods (p<0.01). digital dental radiography was significantly superior to the naked eye in terms of sensitivity, specificity, and positive predictive value, but the naked eye was superior for negative predictive value and diagnostic accuracy in detecting mbii (p<0.01). in this study, a classification of the mesio - buccal root canal by the micro - focus - computed tomography (micro ct) inspection n=86 sensitivity rate in each observation method as the standard in micro - ct (n=86) specificity rate in each observation method as the standard in micro - ct (n=86) positive predictive value rate in each observation method as the standard in micro - ct (n=86) negative predictive value rate in each observation method as the standard in micro - ct (n=86) diagnostic accuracy rate in each observation method as the standard in micro - ct (n=86) micro - ct as used for setting the gold standard was developed using industrial equipment for the purpose of nondestructive inspection of various materials. very clear images can be obtained by irradiation with extremely small doses of radiation to the target. however, clinical application of micro - ct is impossible, as an extended period of radiation is required for imaging. one is the dental ct mode that revolves around the circumference of the subject and photographs the region at 42.7 mm height and 30 mm width. the other method is the panorama mode, in which the x - ray tube rotates five times in a spiral orbit. furthermore, these images can be reconstructed to form multiplanar reconstructions and three - dimensional images. the radiation dose involved in dental ct investigation is basically comparable with conventional panoramic (mean effective does per once ; 0.025 msv .) however, depending on the anatomical location and the setting of ct device, the radiation dose exposed to the patient could be kept to a minimum. the cone beam ct, which is a variant of conventional ct, is even more useful in dentistry as its radiation can be restricted only to the anatomical area being investigated. in addition, the radiation dose required by the cone beam ct is only one - fifth of the conventional ct and time needed is as short as 1015 seconds. as for cone beam ct application, although there are many advantages than risks, it is important to have a clear objective for using this technique and then to define the anatomical area. surgetel is a magnifier that was developed for dentistry, offering superior resolution with a three - layer lens coating, in addition to enlargement power. determining the anatomical condition of the mesio - buccal root of maxillary first molars before root canal treatment is difficult. weine and kulilid et al. reported that the second mesio - buccal canal is located 1 - 3 mm toward the palatal canal from the larger mesio - buccal canal. however, discovery has been difficult in many cases with searches using thin reamers, since the root canal orifice of mbii is quite small and of variable position. in terms of diagnostic accuracy and negative predictive value, in digital dental radiography, the two root canals of the mesio - buccal root can not be distinguished in many cases, since these canals overlap with the direction of irradiation. the detection rate considerably decreased due to the interviewing of maxilla or periodontium in clinical situations. good diagnostic accuracy was obtained under observation by sergitel or the naked eye because the orifice of the root canal was searched for using reamers on the basis of anatomical knowledge. reported mbii in 54% of maxillary first molars, while seidoberg et al. reported an mbii detection rate of 62% for extracted teeth and 33% in clinical situations the detection rate by the naked eye was 51.4% of a of micro - ct, which was 30% in all experimental teeth. detection by magnification telescope or the naked eye is impossible for type iv. in other words the root canal treatment has often been finished without discovery of mbii, including types ii and iii. even if mbii is detected however, success rate does seem to improve with detailed knowledge of the anatomical features. radiographic and visual information generally can not be directly compared, because type iv mbii can not be absolutely detected by magnification telescope or the naked eye. the existence of type iv has a same adverse effect with mbii when its not discovered. the present clearly show that detection rate of mbii is higher for dental ct than for digital dental radiography. few differences in the detection rate of mbii were seen between surgitel and the naked eye, although sergitel was superior in terms of the time required and ease of detection. detectability of mbii in the maxillary first molar was superior using dental - computed tomography compared to digital dental radiography, magnification telescope, and the naked eye.

aim: the purpose of this study was to clarify detection characteristics of the secondary mesio - buccal canal in maxillary first molars using various methods.materials and methods: the root canal system of 86 extracted human maxillary first molars was inspected using micro - focus - computed tomography to accurately determine the number of canals. radiographs or floors of the pulp chamber for all samples were observed for the secondary mesio - buccal canal with computed tomography for dentistry, digital dental radiography, magnifier, or the naked eye. sensitivity, specificity, positive, and negative predictive values and diagnostic accuracy for these four methods were investigated using the from the micro - focus - computed tomography inspection as the gold standard. all samples of each method were observed by 10 endodontists. using these , the 2 test was used to compare and analyze differences between the various conditions (p<0.05).: the secondary mesio - buccal canal could be recognized in 60.9% of samples with the micro - focus - computed tomography. no significant difference was seen between efficiencies of the computed tomography for dentistry and the micro - focus - computed tomography. the computed tomography for dentistry was superior to the other three methods.:detectability of the secondary mesio - buccal canal in the maxillary first molar was superior using dental - computed tomography compared to digital dental radiography, magnification telescope, and the naked eye.

since the first elucidation of three - dimensional models of protein structures and polynucleotides, a wealth of structural information has become available. for proteins, different secondary structure elements have been described, and also for dna, two different helices were proposed very early on. while proteins are readily classified in terms of helices, sheets, and various kinds of turns, the full structural diversity of dna and rna is only recently becoming clear. on the basis of our previous work on the classification of protein structures using an algorithm called disicl (dihedral - based segment identification and classification) , we here propose a definition of polynucleotide structural elements based on two dihedral angles of the nucleotide backbone complemented by the dihedral angle linking the sugar and the base. while studies of backbone dihedral angles are available for polynucleotides, secondary structure prediction and classification of dna and rna models are more often based on sequence- or knowledge - based approaches such as sequence alignments, context free grammar, and machine learning or empirical energy functions and dynamic programming to determine the most stable secondary structure or an ensemble of structures with a central member. there has been significant effort to combine these methods to predict and construct both the secondary and tertiary structure of rna molecules. structure - based analysis and classification methods on the other hand rely on three - dimensional models to evaluate the shape and intramolecular and intermolecular interactions between dna, rna, and other molecules such as proteins. established structure - based analysis methods for polynucleotides are commonly based on complex helical parameters, such as in the program schnaap (structure and conformation of helical nucleic acids : analysis program). the x3dna analysis tool also relies on helical parameters but performs a local dna classification based on phosphate coordinates of dinucleotide base pair steps. another very useful and effective package, curves, can analyze global helical curvature and local base pair parameters, as well as groove dimensions. while the recently reimplemented curves+ program can effectively analyze molecular dynamics trajectories, its intrabase and interbase pair parameters, which can be used to assign local structure information, are almost identical to those reported by x3dna. almost all of the structure - based approaches for dna or rna classification require double helical structures (originating from a single or from multiple strands) and seem relatively limited in terms of the diversity of the structural classes that are being considered. in the current work, we define an extensive library for the classification of nucleotide sequences and apply this classification on databases containing 260,000 trinucleotide segments. after a description of the data sets to be analyzed the suggested classifications are discussed in more detail and demonstrated by selected examples of dna and rna structures obtained from the brookhaven protein databank. the performance of disicl is compared to that of x3dna, and finally, some are drawn. for the purpose of testing and comparing different classification algorithms, two large scale polynucleotide data sets were obtained from the brookhaven protein databank (pdb, www.rscb.org). both data sets were selected from all pdb entries available on october 23, 2012, using the following criteria entries show at most 30% sequence identity. entries obtained from x - ray crystallography have a resolution of 0.82.0. separate dna and rna data sets were defined (dna_comb and rna_comb, respectively), containing structural models determined by both x - ray crystallography and nuclear magnetic resonance (nmr) spectroscopy, because the number of entries for polynucleotides was considered too small for further partitioning. the resolution range for x - ray structures was chosen such that the relevant dihedral angles can be reliably determined, but the number of alternative locations for groups of atoms in the data set is kept low. prior to the analysis, multiple chains and multimeric structures were retained, but residues were renumbered to avoid identical residue numbers from different chains. if any of the classification algorithms failed on a particular entry, it was completely discarded from the full analysis to ease the comparison of methods. this approach yielded approximately 80,000 and 180,000 segments for dna and rna models, respectively. further details are provided in the database summary section of table 1, where the number of downloaded pdb entries (file number), number of extracted individual structures (model number), and total number of classified nucleotides / residues (total data set) is provided, along with the average number of structures per pdb entry (ave . model length), and amount of base - paired nucleotides (base pairing). x3dna is a freely available analysis, reconstruction, and visualization tool for dna modeling (www.x3dna.org). it has many smaller modules that can be used to produce idealized dna models based on their sequence and the required helix type, as well as to analyze existing dna and rna structures. the analysis package can determine base pairing and obtain helical parameters (such as roll, twist, displacement, and groove dimensions) based on simple geometric calculations, and it also features a dinucleotide segment - based local helix classification algorithm. this classification takes the mean phosphorus atom z coordinates and helix inclinations (zp and zph, respectively) of a - dna to distinguish a - dna, b - dna, and transitory ta - dna forms (often found in tata boxes). this particular algorithm does not recognize z - dna forms (unless the full helix is left handed), and the classification should still be verified by other helical parameters also printed by the analysis program. other structure - based programs focus on full helical descriptions of dna sequences and global hydrogen bonding, while the x3dna analysis program performs more localized dinucleotide segment classification, which is better suited to capture the structural diversity of, for example, molecular simulations. the disicl algorithm for protein structure classification first, relevant (backbone) dihedral angles are calculated and attributed to regions in the dihedral angle space. the pair of regions occupied by the two central residues of the segment determines the structural class to which the segment is assigned. while the disicl algorithm was originally designed for protein analysis, the purely dihedral - based classification can be applied to other biopolymers as well. using a study of dihedral angles in selected dna backbones published one- and two - dimensional distributions on eight backbone dihedrals of dna oligomers crystallized in different helical structures (a, b, z). this work provided an excellent base for our classifications, while other papers confirm that helical structures and their subconformations are important factors when dna interacts with proteins and drug - like molecules. finally, rna molecules, which often fold into complex structures, also have a tendency to form dna - like helical segments. on the basis of the two - dimensional distributions, we chose three dihedral angles that can characterize helical structures of nucleotides: backbone dihedrals and and base torsion angle. while some helical parameters (like groove dimensions) can be more easily measured for full helix turns (45 base pair segments), a pair of the triplets (, ,) provided by a base triplet has a sufficiently high resolution to separate the polynucleotide helices. the backbone dihedral angle definitions and the 14 region definitions of the dihedral angle space are shown in figure 1. similar to the first and last amino acid of the protein segments of disicl, the third nucleotide only provides one atom for the calculations, and as such, the first two nucleotides were used as central residues for the comparison studies. on the basis of the (, ,) definitions, 17 detailed (table 3) and eight simplified (table 4) classes were defined for dna and rna structures. their region mapping and precise region definitions are shown in table 2 and table s1 of the supporting information, respectively. representation of region definitions used for polynucleotide classification (on the left) based on subsequent (, ,) values within a trinucleotide segment (on the right). atoms and bonds that define 1, 2, and 2 are marked in red. segments are assigned to a class if their central residues fall into regions separated by a dot in the segment definitions. the same data is given for the x3dna classes at the bottom of the table. the region definitions of the dna classes are not as straightforward as the protein region definitions, so a summary is provided here. the 14 region definitions can be divided into five groups marked by greek letters. regions have high density in rna structures, with 1 containing the most densely populated area associated with the a - helix. regions are dominant in the dna data set and are associated with the different forms of the b - helix. the experimentally derived subconformations of b - dna, and bi and bii, fall in the 1 and 2 regions, respectively. three regions contain local density peaks normally found in z - dna, although 1 residues are also regularly found in dna quadruplexes, and 2 residues regularly appear in sharp backbone turns. regions have populations comparable to the regions and are separated from the , , and regions by their low value. the 1 region appears in distorted a - helices, and the 2 region regularly appears in backbone turns, while the 3 region is almost exclusive found in dna quadruplexes. the fifth group represents a / b transitions and contains the regions ab1 and ab2. the ab1 region represents the intersect volume of the 1 and 1 regions (which contain the maximal peak densities in the rna and dna data sets, respectively) and is densely populated for both rna and dna structures. region ab2 is a moderate density volume surrounded by the regions 1, 3, 1, 2, and 3. 1, 2, 1, 2, and 3 are based on the angular distributions of schneider et al. but were modified to better fit the density distribution of both dna and rna data sets. this procedure was based on the classification of a data set containing 150,000 nucleotides consisting of approximately equal amounts of dna and rna. the rectangular regions were adjusted to include 75% of the data points including the nearest local density maxima. afterward, selected subsets of structures with common structural elements (dna quadruplexes, junctions, rna tetraloops, riboswitches, etc .) were analyzed. if structurally important nucleotides were repeatedly observed near unassigned peaks in the dihedral angle space, additional regions were assigned to those areas (ing in regions 2, 3, 3, 1, 2, and 3). on the basis of chemical intuition, visual checks concerning the shape of the backbone, directionality of the bases, and the annotation provided in the pdb entries, segment definitions (pairs of regions) were associated with structural classes. associated segment definitions were assigned to a class after a careful visual analysis of 20100 randomly picked structures. segment definitions were assigned if at least 50% of the examples were of one particular class. finally, the borders of neighboring regions were fine - tuned in an iterative process, where the effect of shifting the border was determined by performing structural analyses of structures containing the segments that were reassigned to a different class. all structural models were analyzed separately by both classification algorithms. as the different programs produced output in different formats, all were ordered into identically formatted data series. the data series contained the name of the class along with all the segments in the model that belonged to that class. second, the data series of all models were collected and combined into a single data set for each of the individual algorithms containing elements anj, which was assigned the value 1 if nucleotide n was classified to belong to class j. tables 3 and 4 show the abundance (occj) and average length (lj) of each structural element (in nucleotides), which were calculated based on the number of residues in the class (nj), number of interruptions (nj), and total number of residues (nsum) according to eqs 13. the number of interruptions was increased by one whenever a gap was found in a continuous chain of dinucleotide segments of class j.123to compare the classification algorithms, the correlation matrices of algorithms were calculated containing the correlation scores cij where i and j mark the i class of the first algorithm and the j class of the second algorithm, respectively. three types of correlation scores were used: pearson correlation (rij), match score (mij), and scaled match score (mij). the pearson correlation (rij) is calculated from eq 4, where ani is the average occurrence of the class i (ani = ni / nsum).4while the r - score drops quickly with the amount of mismatches (or different occurrences of classes i and j), a large positive r - score is still a good measure to determine agreement between algorithm classes. the unscaled match score (mij) is calculated using eq 5 and represents the absolute number of residues assigned to class i in one algorithm and to class j in the other algorithm.5the m - score is additive, which makes it possible to group classes or track distributions of correlations for one class. the scaled match score (mij) provides a better comparison between algorithms and is calculated by eq 6.6 in words, the scaled match score is obtained by dividing the observed match (mij) between two classes with the maximal theoretical match (mmax). here, mmax is equal to size of the smaller data set.7 to summarize comparisons, the weighted average of the scaled match scores were calculated for a - helical, b - helical, and transitory dna or rna forms for nucleotides (table 1, methods agreement). additionally, the weighted average of all these superclasses and the scaled match score for unclassified residues were calculated to obtain an overall match between methods. the grouping for superclasses is provided in table s2 of the supporting information. for dna classifications, dna groove dimensions were measured with a simple algorithm using a similar basic idea as used in x3dna (see figure 2 for a schematic representation of relevant nucleotides for this calculation). because the full turn of the b - dna structure consists of approximately five (base - paired) nucleotides on each of the two strands, helical fragments of a given classification with five consecutive base pairs identified were used to determine the groove dimensions. sj-1, paired with central residues i j and (i + 1)(j - 1), such that base pair i as a rough estimate for major and minor groove widths, the distance between phosphorus atoms p(i-2) and p(j-2) yields the major groove width, while the distance between atoms p(i+2) and p(j+2) provides the minor groove width. groove depths were estimated by the distance between the midpoint of the vector defining the width and the midpoint of the vector pi pj. schematic representation of the calculation of groove dimensions in double - stranded dna helices. the classification of polynucleotides was performed on two data sets containing models of dna molecules (dna_comb) and rna molecules (rna_comb) using two segment - based algorithms, namely, x3dna and disicl. disicl defines 17 detailed classes, which can be grouped into classical helical structures (a-, bi-, bii-, biii-, and z - helices), special loops and turns (a - loop, tetraloop bulge, b - loop, sharp turns, and quadruplex loops), and transitory classes (ab, ab2, ad, az, bd, bz, and zd) (see table 3 for the average occurrences and lengths of these structural elements). in the simplified version of the nucleotide disicl library (table 4) , this is reduced to eight classes (a-, b-, z - helices, irregular a and irregular b structures, quadruplex loops, ab transitions, and other transitory segments). the majority of dna and rna molecules in the databases assume double helical structures. dna under physiological conditions assumes a right - handed double helical form usually referred to as b - dna. the nucleotides in the b - dna form have two identified subconformations (bi and bii) mostly differing in their and angle. under certain salt concentrations, rna and dna can form a different helix, normally referred to as the a - form, while some dna structures can assume left - handed helices, normally referred to as the z - form. the bi class (bi) contains the dna (, ,) density maximum associated with continuous repeats of the bi subconformation (located in the region 1). occurrence of longer stretches of the bi class on both strands forms the classical b - helix, which makes up 35% of all dna nucleotides. the bii class (bii) contains definitions for alternating segments, which are an alternate form of the b - helix (3.5% of dna segments). while the bii class rarely appears in longer stretches in both strands, bii - rich areas of dna form helices that are more varied in their groove dimensions and on average have wider and more shallow grooves (table 5), which might be important for dna the bii class in longer stretches also appears in single strands for dna loops and three - way junctions. the biii class (biii) was defined for pure 3 segments, which occur in 2.5% of the analyzed nucleotides. biii segments (often accompanied by b - loop segments) distort the b - dna helix leading to a wider major groove but narrower minor groove. examples of bi-, bii-, and biii - rich dna models are depicted in panel a of figure 3. examples of dna structures and structure classification by disicl. for each model, the pdb identification code is given followed by the abbreviation of classes according to table 3, which are color coded to match the structures they mark. helices are sorted based on the assigned disicl classification for the central segment of the helix turn on both strands. groove dimensions are given as averages (mean) and root - mean - square fluctuation (rmsf) in. the a - helix class (ah) relates to the bent a - form helix of dna (2%), and it is the predominant form of ribonucleic acids (50%). the class is defined by segments with pure 1 conformation, which usually appear in fully a - helical models or prior to turns in more complex dna structures. the z - helix class (zh) appears predominantly in z - helical dna structures and consists of definitions with an alternating pattern of either 12 or 13. the z - helix class appears consecutively only in z - helical dna models (see, for example, panel b of figure 3), but it is observed isolated in segments of dna loops and quadruplexes. in rna structures, the predominant class by far is the a - helix, containing over 50% of rna residues, building up the helices and stem loops that form the majority of the more complex structures. segments which are classified as b - helix appear with less than 2% occurrence and mostly at isolated positions. these segments sometimes have a backbone shape different from the normal helical forms appearing in dna, resembling more the sharp turn class (this is especially common for segments of the b2 class). z - helical segments also appear at isolated positions in rna structures, mostly at the end of stem - loops with receptor functions, suggesting an important functional role (as shown in figure 4 panel a). examples of rna structures and structure classification by disicl. for each model, the pdb identification code is given followed by the abbreviation of classes according to table 3, which are color coded to match the structures they mark. apart from the classes associated with the classical dna helices, a number of special classes were defined for functionally important segments mostly found in more complex rna and dna structures. while the classes defined here help to monitor possible structurally important parts of polynucleotide structures, these structures do not separate sharply in the (, ,) space, leading to a lower (4070%) selectivity for individual definitions. the quadruplex loop class contains definitions highly specific for dna quadruplexes, which typically appear at the ends of chromosomes. the quadruplex loops rarely appear in longer stretches than three residues and instead are connected by sharp turns and transitory structures to form repeats. as quadruplexes are mainly formed in dna structures, the occurrence of this class is significantly higher in the dna data set (3.5%) than in the rna set (0.5%) while the quadruplex loop class is highly selective for quadruplex structures (especially for quadruplexes made from one or two strands), quadruplexes formed by multiple strands of dna can exist with one, two, or all four parts built from b - helical segments (see examples in panel c of figure 3). the tetraloop bulge class (tl) was defined for a special bulged loop structure, which appears often in rna loops. the model structure of this class derived from tetraloop receptors, where the loop contains at least one 90 turn in the backbone, with a base facing outward from the loop to interact with bases further away in the rna sequence, possibly playing an important structural role. however, based on visual checks, it is only moderately selective for the required shape, and many segments belong to the more general a - loop class. a bulged loop from a tetraloop receptor is shown in panel b of figure 4. the sharp turn class (st) collects definitions, which are enriched in segments with a more than 90 turn in the backbone and/or the torsion of their bases (defined by the atoms c1i, c1i, c1i+1, c1i+1). sharp turn segments typically appear where the bases of the stem loops are connected, at the end of certain riboswitch and aptamer rna loops and in dna and rna knot structures. the occurrence of the sharp turn class is less than 2% in both rna and dna data sets, and sharp turns typically appear as isolated segments. for examples of the sharp turn, see panels d and e of figure 4. the a - loop class (al) contains definitions of the region, which were not found to be highly selective for any of the special classes, and they are typically not forming a perfect a - helix either. a - loop structures appear often in and between rna - stem loops, connecting the classical a - helical segments with each other or with tl and st segments. the a - loop class takes up about 10% of all rna structures, but it is rarely found in dna. the al residues can form longer stretches as well, but these stretches are often single stranded or have significant distortions compared to a - helix structures. examples of a - loop segments in different rna structures are shown in panels b, c, and d of figure 4. the b - loop class (bl) contains the atypical definitions of -regions. similar to the al class, b - loop segments usually connect the b - helical parts of dna models and are often found in different junctions (holliday junctions, kissing complexes, etc .), dna - loop structures, and at sites where small molecules are intercalated into a dna helix. longer helical stretches of b - loop structures also appear in single strands, typically complemented by pure biii segments on the other strand. the average occurrence of b - loop segments in dna is 16% and around 1% in rna models. the ab class collects definitions for segments with a transition from the density maxima of rna and dna structures (1 and 1 region, respectively). the volume bridging these two peaks is also highly populated in both data sets (around 10%) and was suggested to have a functional structure of its own. we found that the ab class is often observed in helical structures in three functional roles: isolated or short ab segments often serve as junctions for a - helical and b - helical parts of both dna and rna (as shown on the left side of panel d in figure 3). short stretches of ab segments temper the bending of a - helices in rna stem - loops allowing for less strained loop structures (panel a in figure 4). longer stretches of ab segments (especially pure ab1 stretches) are often found in three stranded structures, like dna triplexes (right side of panel b in figure 3) and rna pseudoknots (panel e in figure 4). the ab2 class collects definitions typically transiting between the 3 and 2 regions. unlike the ab class that mostly looks helical, ab2 segments typically appear more linear as the backbone dihedrals are close to 180 with bases looking well aligned or pointing away from each other this nonideal position for stacking interactions agrees well with the observation of the ab2 class near unpaired or mismatched nucleotides and interaction sites of more bulky drug molecules. the remaining five transitory classes, namely, the ad, az, bd, bz, and zd, were defined based on the major areas that their segments connect. no particular selectivity for any of the previous classes was detected for the definitions of which they are comprised. these classes contain 3% of nucleotides in both data sets and have a similar role as the different turn definitions in the protein classification libraries. the simplified disicl library for nucleotides is designed to provide an easier comparison to cd spectroscopy, where a-, b-, z-, and quadruplex forms of dna can be distinguished from each other. for this reason, the detailed disicl classes bi - helix (renamed to b - helix or bh), a - helix, z - helix, and quadruplex loop remain as separate classes in the simplified library. as the bii - helix , biii - helix, and b - loop classes relate to distorted but mostly b - helical forms of the dna, they were grouped together into the irregular b (ib) class. the a - loop and tetraloop bulge classes usually appear in rna - stem loops and are not sharply separated in the (, ,) dihedral angle space. they are grouped together to form the irregular a (ia) class in the simplified classification. although the ab class is a transitory class, we decided to keep it as a separate class in the simplified classification because of its high abundance, enrichment in special helical segments, and definition through its own region (ab1). the remaining seven classes in the detailed classification (st, ab2, az, ad, bd, zb, and zd) typically stand for nonhelical segments, which connect helical parts in stem loops and other complex polynucleotide structures grouped together in the transitory (tr) class in the simplified classification. the average structure element length and occurrence of the simplified disicl classes for nucleotides is shown in table 4, along with the codes of the detailed classes grouped together in each simplified class. full correlation matrices (pearson scores and scaled match scores) for the comparison of disicl and x3dna are given in tables s3s6 of the supporting information. here, we provide an overview of the overall correlation analysis in table 6. the abundance and average lengths of a- and b - helix structures are similar for the two algorithms (table 3). the average helix length of disicl is shorter due to the more detailed classification and the fact that disicl can classify one residue less for fully base - paired chains (as x3dna requires a base - paired dinucleotide step for classification, while disicl uses a segment of three nucleotides in one strand). for both algorithms , the occurrence of each class is displayed in the first row or column, respectively. correlation analysis reveals that the assigned helical structures show only a partial overlap between the algorithms. a - helix classes of disicl and x3dna show the best agreement both in the dna and the rna data sets amounting to 65% of disicl residues and set, the b - helical classes as determined by disicl, show a comparable amount of correlation with the b - helix in x3dna. highest correlations are observed for the b - helix class or bi - helix (m score 48% and r - score 0.4), closely followed by the irregular b class, for which 43% of the residues are also classified as b - form by x3dna (with similar values for b2, b3, and bl classes), but its lower abundance decreases the r - score to 0.3. for the rna data set, the amount of segments assigned by x3dna as b - helix is extremely low (0.04%) and shows little or no correlations with the disicl b - helical classes (or any other class), leading to a combined agreement amounting to 6% the x3dna class. the abundance of b - helical segments in rna according to disicl is 1.5% (mostly due to the b - loop class). visual checks reveal that x3dna b - helix segments do not show the shape normally associated with disicl b - helical segments, while disicl b - helix segments appear in rna mostly as bulges in a - helices or at the end of stem - loops (an example is shown in panel c of figure 4). we found no models in the rna data set, with hydrogen - bonded base pairs for which disicl classified both strands as b - helix, which partially explains the low correlation with x3dna as this program monitors paired bases only. while the z - helix class in disicl has a low abundance (1% and 0.4% in dna and rna, respectively), it has no overlap with any of the x3dna classes in the dna data set and a minimal overlap with the a - helix class in rna (due to the isolated z - helix segments in rna stem loops). this shows that x3dna very rarely mistakes z - helical segments for a- and b - form segments, even though not explicitly making the classification (except for full z - helices in dna). considering transitory and special classes, the ta - transitory class of x3dna shows moderate correlations (m scores) with the bi - helix (32%), ab (13%), and b - loop (12%) classes of disicl in dna and with the ab (38%) and a - helix (26%) classes in rna, showing that the peak of its density distribution falls in the ab1 region. the correlation might be low for dna because the ta class was based on special dna segments meant for interacting with polymerase enzymes, and protein nucleotide complexes were filtered out from our data sets. about one - third of the ab class in disicl was considered as b - helix in dna (34%) and a - helix in rna (33%) by x3dna. additionally the bd class shows a moderate correlation (30%) with x3dna b - helix in dna, while ad (15%) and ab2 (14%) classes correlate weakly. in rna models, moderate agreement with the x3dna a - helix is also observed for the a - loop (39%) and ad (38%) classes (often found in distorted a - helices) and the tetraloop bulge class (25%). the rest of the disicl classes remained mainly unclassified by x3dna with no significant correlations. a summary of the correlation analysis is shown in table 1 (methods agreement), which reveals an overall agreement between x3dna and disicl slightly below 60% for both the rna and dna data sets, which is slightly lower than the agreement between protein classification algorithms. the analysis of the dna and rna data sets provides a solid basis to define and characterize the disicl nucleotide structure classes, and their correlations with the classification of x3dna. the higher level of detail in disicl allows us to monitor the structural effects of interactions of nucleotides with small molecules and proteins as well. two examples for rna protein and dna protein complexes are shown in figure 5. panels a and b show a model of an aaug tetraloop hairpin in complex with a yeast rnase binding domain (pdb code 2lbs). the bulk of the interactions take place between a short -helix of the protein at the end of the tetraloop hairpin. while x3dna steadily recognizes the a - helical conformation at the base of the hairpin, the interaction site remains unclassified disicl assigns a classification for 70% of the nucleotides over the nmr solution models, mainly to the tetraloop bulge or ab2 class. another interesting example is the ternary complex of double - stranded dna and a protein fragment of the polymerase i from t. aquaticus (pdb code 2ktq). in this case shown in panels c and d of figure 5the longer template strand of the mainly b - form dna is bent by the protein, recognized as an a - helical stretch in disicl. as in the previous example, x3dna readily recognizes the b - helical nature of the double - stranded part but leaves the dna at the interaction site unclassified. the examples suggest that fine structural changes might be revealed by disicl, yielding additional information on interactions between nucleotides, proteins, and small molecules. examples of dna / rna protein complexes classified by disicl and x3dna. for each model, the pdb identification code is given, followed by the method of classification and the abbreviation of structural classes according to table 3. the disicl algorithm for dihedral - based structure classification was extended to allow for the classification of nucleotide structures. starting from previously published distributions of dihedral angles, three dihedral angles (, ,) were selected to perform the classifications. fourteen distinct regions were defined in the ing three - dimensional dihedral angle space. a classification is performed based on the assignment of the two central nucleotides in a trinucleotide segment, first to their regions and as a pair to one of 17 structural classes. apart from helical structures, we define loop regions, turns, and transitory structural elements, and examples of these were given with dna and rna models from the brookhaven pdb. newly suggested structural classes include the quadruplex loop, sharp turn, and tetraloop bulge, as well as a number of transitory elements. the detailed classification was simplified into eight more general classes and were compared to the classification in x3dna. overall, disicl seems a very powerful tool for the detailed structural analysis of both proteins and polynucleotides. studies of practical applications for the disicl algorithm are currently the focus of our attention. additionally,

in an accompanying paper (nagy, g. ; oostenbrink, c. dihedral - based segment identification and classification of biopolymers i : proteins . j. chem . inf . model . 2013, doi : 10.1021/ci400541d), we introduce a new algorithm for structure classification of biopolymeric structures based on main - chain dihedral angles. the disicl algorithm (short for dihedral - based segment identification and classification) classifies segments of structures containing two central residues. here, we introduce the disicl library for polynucleotides, which is based on the dihedral angles , , and for the two central residues of a three - nucleotide segment of a single strand. seventeen distinct structural classes are defined for nucleotide structures, some of which to our knowledge were not described previously in other structure classification algorithms. in particular, disicl also classifies noncanonical single - stranded structural elements. disicl is applied to databases of dna and rna structures containing 80,000 and 180,000 segments, respectively. the classifications according to disicl are compared to those of another popular classification scheme in terms of the amount of classified nucleotides, average occurrence and length of structural elements, and pairwise matches of the classifications. while the detailed classification of disicl adds sensitivity to a structure analysis, it can be readily reduced to eight simplified classes providing a more general overview of the secondary structure in polynucleotides.

diabetes mellitus, a metabolic disease with manifestation of hyperglycemia and dyslipidemia, is still one of the most leading causes of death and disability. over time , diabetes leads to serious complications such as nephropathy, retinopathy, neuropathy, stroke, and peripheral vascular diseases. currently, beside insulin, the most widely used medications for diabetes are insulin and the oral hypoglycemic drugs. although early onset manifestations of diabetes can be controlled by current antidiabetic drugs, late onset complications appear in many patients. in addition, the clinical uses of the current drugs are usually accompanied with some adverse effects including abdominal discomfort, severe hypoglycemia, lactic acidosis, and peripheral edema. therefore, the search for new antidiabetic agents with more effectiveness and lesser side effects has continued. today, antidiabetic effects of several plants have been supported by from animal studies or clinical trials. among them, allium sativum, cinnamomum zeylanicum, citrullus colocynthis, juglans regia, nigella sativa, olea europaea, punica granatum, salvia officinalis, teucrium polium, trigonella foenum, urtica dioica, and vaccinium arctostaphylos are widely used as medicinal plants for management of diabetes. several studies have shown that each one of these plants is effective in the decrease of plasma glucose and serum lipids in diabetes. we hypothesized that a combination of them may have more beneficial effect on improving metabolic indexes. the present study is a part of a research effort to develop the best polyherbal mixture from these antidiabetic plants for management of glucose and lipid in diabetes. in our previous work , we observed that administration of a combination of hydroalcoholic extracts of six of them inhibited the progression of hyperglycemia and decreased serum lipids and hepatic enzyme activity in diabetic rats. since nonprocessed herbs are usually more tolerated than extracted or formulated products and patients prefer to consume plants as salad, spice, and so forth, in this work, we aimed to investigate the effect of a mixture of powders of the above mentioned twelve plants on glycemic and lipidemic status of diabetic rats. the air - dried a. sativum (cloves), c. zeylanicum (bark), c. colocynthis (fruit), j. regia (leaf), n. sativa (seeds), o. europaea (leaf), p. granatum (fruit), s. officinalis (areal parts), t. polium (areal parts), t. foenum (seeds), u. dioica (areal parts), and v. arctostaphylos (fruit) were powdered and mixed with ratio of 5%, 5%, 5%, 10%, 5%, 5%, 5%, 5%, 5%, 20%, 15%, and 15%, respectively. this polyherbal mixture (phm) was added (15% w / w) to the standard pellets diet of animals in treatment group. male albino wistar rats (280330 g) were obtained from laboratory animals house, mashhad medical university (iran) and housed in a room with controlled lighting (12 h light/12 h darkness) and temperature (22 2c). all animal procedures were done according to the ethical guidelines of the animal care of shiraz university of medical sciences (iran). to generate diabetes, some rats received a single dose (55 mg / kg, ip) of streptozotocin (stz) (enzo life, usa). induction of diabetes was confirmed by measuring fasting blood glucose (fbg) two days after stz injection. rats with fbg level of 250 mg / kg or higher were considered to be diabetic. the animals were randomized into three groups: normal control rats which were fed standard diet (n = 6), diabetic control animals which were fed standard diet (n = 6), and diabetic rats which received diet containing 15% (w / w) of phm (n = 8). the treatment was initiated two days after stz injection and continued for 4 weeks. at the end of the 30th day, the rats fasted 16 h and blood samples were collected from retroorbital sinus for biochemical measurements. at the end of the experiment, animals were placed in individual metabolic cages for urinary collection. after acclimatization (1 day), the 24 h urinary samples were collected from diabetic animals. serum triglyceride and total cholesterol were evaluated with standard enzymatic colorimetric kits from pars azmun (iran). blood glucose was measured using glucose oxidase reagent (ziest chem diagnostics, iran). serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) activities were evaluated with colorimetric methods by commercially available kits (pars azmun, iran). as shown in figure 1, before injection of stz, fbg levels of both groups were statistically not different from each other. at day 2 the diabetic rats in control group showed further increase of fbg at the end of experiment (374 17 mg / dl in day 30 versus 272 14 mg / dl in day 2, p < 0.01). however, administration of phm to diabetic rats blocked the increase of blood glucose. at day 30, the level of fbg in this group was 263 29 mg / dl which was significantly lower than that in diabetic control rats (p < 0.01). during experiment, both diabetic control and diabetic phm treated groups exhibited a significant reduction in their body weight. at day 30 , the weight reduction reached to 27% (p < 0.001 versus day 0) and 25% (p < 0.001 versus day 0) for control and phm treated animals, respectively (table 1). prior to diabetes induction, the level of water intake was not significantly different between three groups. however, after stz administration, there was a significant increase in the levels of water intake in both groups of diabetic rats. although the polydipsia condition was evident during the treatment period, the level of water intake in phm group was significantly (p < 0.001) lower than that in diabetic control group. also, at the end of the experiment, the phm - fed diabetic rats showed an improvement in polyurea state and the excretion of urine was significantly lower than that of diabetic control group (30 3 ml/24 h versus 72 9 ml/24 h, p < 0.01). there was a significant elevation in the level of triglyceride (129 27 mg / dl versus 59 11 mg / dl, p < 0.05) and total cholesterol (105 9 mg / dl versus 55 3 mg / dl, p < 0.01) in diabetic control rats as compared with normal group. the levels of triglyceride and total cholesterol in phm treated group were 66 27 mg / dl (p < 0.05 versus diabetic control group) and 83 4 mg / dl (p < 0.05 versus diabetic control group), respectively. at day 30, diabetic control rats showed higher ast (110 18 versus 71 8 u / l) and alt (77 4 versus 28 5 u / l, p < 0.05) activity than that of normal group, suggesting hepatic dysfunction in these animals. the level of ast and alt activity in phm treated groups was 107 15 u / l and 58 10 u / l, respectively, which was not significantly different from that of diabetic control rats (figure 3). although numerous herbs have been suggested for the treatment of diabetes, but at present no one could completely treat diabetic patients. one approach for the development of an effective phytochemical compound can be mixing a number of hypoglycemic and hypolipidemic herbs to produce more potent antidiabetic agent. the present work was a part of a research effort to find the best mixture from some medicinal plants which their hypoglycemic and hypolipidemic effects were supported by several studies. here, we investigated the effect of a diet containing mixture of twelve medicinal plants on glycemic and lipidemic status of diabetic rats. although this mixture failed to completely restore stz - induced hyperglycemia and had no effect on weight reduction, it significantly prevented further elevation of blood sugar and improved the polydipsia and polyurea. this beneficial effect on glycemic status is expected to happen as antihyperglycemic effect of all twelve plants forming phm has been confirmed with repeated studies. antihyperglycemic effect of medicinal plants is achieved by different mechanisms including decreasing glucose absorption from intestine, enhancing insulin secretion from beta cells, increasing glucose uptake by tissues, inhibiting glucose production in liver, and increasing pancreatic tissue regeneration and/or presence of insulin - like agents in plants. regarding phm, inhibitory effect of t. foenum on intestinal glucose absorption and the beneficial effects of j. regia and t. polium on regeneration of pancreatic islets also, it has been demonstrated that n. sativa and s. officinalis decrease hepatic glucose production through inhibition of gluconeogenic enzymes. furthermore, the insulin secretory effects of c. colocynthis, o. europaea, t. foenum, v. arctostaphylos, and u. dioica were shown in vitro in the isolated pancreas or islets. therefore, the levels of serum triglyceride and cholesterol are usually elevated in diabetic patients. in our study the hypolipidemic action of phm is in agreement with earlier studies that reported that a. sativum, c. colocynthis, c. zeylanicum, j. regia, n. sativa, s. officinalis, p. granatum, t. foenum, and t. polium decrease the levels of serum triglyceride and cholesterol in diabetic subjects. also, in our previous work, we showed that t. foenum led to a significant reduction in lipid droplet accumulation in adipocytes. elevation of alt and ast enzyme activities is considered as an evidence for hepatic damage. an increase of these enzyme activities is also associated with fatty liver disease and decreased hepatic insulin sensitivity in type 2 diabetes. according to the reports of previous studies, a. sativum, j. regia, s. officinalis, o. europaea, t. foenum, and t. polium could decrease the alt and ast levels in diabetic rats. on the other hand, there are some reports that chronic administration of t. polium in elevation in plasma levels of liver enzymes. in our study therefore, consumption of this polyherbal mixture accompanied with no hepatoprotective or hepatotoxic activity. taken together , our demonstrated that phm has beneficial effects on blood glucose and lipid profile of diabetic rats. in our previous work , we observed approximately the same level of antihyperglycemic and hypolipidemic effect with administration of a polyherbal mixture made from the combination of macerated and soxhlet (hydroalcoholic) extracts of a. sativum, c. zeylanicum, n. sativa, p. granatum, s. officinalis, and t. polium. therefore, we did not find any favorable effects on diabetes when the powders of twelve plants were combined instead of giving a combination of hydroalcoholic extracts of six of these plants. also, some studies reported more antihyperglycemic effect when the extracted or formulated products of herbs especially t. foenum were given individually. however, it should be considered that when they are administrated individually, the antidiabetic effect was achieved usually with high doses of the plant extract which may be accompanied with unpleasant effects in the body. therefore, we still believed that the phm, which was constructed simply from mixing plant powders without the need for extraction procedure, has the potential to be used as a dietary supplement for the management of diabetes, particularly diabetic dyslipidemia. obviously, more study is needed to find a more potent polyherbal mixture from antidiabetic plants. further, combination of active ingredients isolated from these plants may be a subject if interest in future perspective related to diabetes management.

the effects of a polyherbal mixture containing allium sativum, cinnamomum zeylanicum, citrullus colocynthis, juglans regia, nigella sativa, olea europaea, punica granatum, salvia officinalis, teucrium polium, trigonella foenum, urtica dioica, and vaccinium arctostaphylos were tested on biochemical parameters in diabetic rats. the animals were randomized into three groups: normal control, diabetic control, and diabetic rats which received diet containing 15% (w / w) of this mixture for 4 weeks. diabetes was induced by intraperitoneal injection of streptozotocin (55 mg / kg). at the end of experiment, the mixture had no significant effect on serum hepatic enzymes, aspartate aminotransferase, and alanine aminotransferase activities. however, the level of fasting blood glucose, water intake, and urine output in treated group was lower than that in diabetic control rats (p < 0.01). also, the levels of triglyceride and total cholesterol in polyherbal mixture treated rats were significantly lower than those in diabetic control group (p < 0.05). our demonstrated that this polyherbal mixture has beneficial effects on blood glucose and lipid profile and it has the potential to be used as a dietary supplement for the management of diabetes.

the central hallmark of vaccination is to prime the adaptive immune system to develop immune responses that will protect the host organism upon a second encounter with the same pathogen. however, priming the adaptive immune system requires activation of nave b- and t - lymphocytes into effector cells that translate into protective immunity. while studies on the immunological basis of vaccine protection have for a long time focused on humoral and cellular responses as measures of protective immunity, growing evidence shows that the mode by which antigens are presented to b- or t - lymphocytes has a significant influence on the outcome of adaptive immune responses induced by vaccination which is also influenced by the mode in which antigens are administered to host cells. put together, these elements drive vaccine development into a cross - talk between vaccinology and immunology in which vaccine design and its delivery (vaccinology) on one hand have to be optimized in order to gain an effective immune response (immunology) on the other. hence, optimization of antigen design and its delivery into host cells is a prerequisite to inducing an optimal protective immune response. unlike b - lymphocytes, which are precursors of antibody secreting cells that can recognize antigens through primed antigen presenting cells (apcs)/activated b - cells, t - cell receptors (tcrs) endogenous peptides derived from intracellular sources such as replicating virus are synthesized and processed for presentation to nave cd8 t - cells by mhc - i molecules while exogenous peptides derived from extracellular sources are processed and presented to nave cd4 t - cells by mhc - ii molecules. an alternative mechanism that permits some extracellular antigens to activate nave cd8 t - cells called cross presentation exists which occurs via the mhc - i pathway. for antigens delivered via the endosomal route, proteosomes degrade soluble antigens after ubiquitination which have been synthesized in the cytosol or escaped to the endoplasmic reticulum (er) by cross presentation. thereafter, the processed antigens are released after proteosomal degradation to generate peptides that are transported into the er by the transporter - associated antigen processing (taps). once in the er, the antigenic peptides are loaded onto mhc - i molecules for presentation on the cell surface where they initiate the activation of nave cd8 t - cells into effector cytotoxic t - lymphocytes (ctls). in the case of antigens delivered by the exogenous route, lysosomes degrade endocytosed antigens after endosomal fusion with lysosomes. in general, lysosomes can degrade complex structures such as whole viral particles that are delivered to them via endocytosis by the extracellular route. presentation of processed peptides by endosomal degradation leads to maturation of apcs into professional apcs which is characterized by expressing mhc - ii molecules and antigen specific signaling molecules such cd40l, cd80, and cd86. the ing professional apcs are the prime initiators of adaptive immune responses that activate nave t - cells into effector cells through the mhc - peptide complexes and immune modulation molecules. therefore, it follows that, for a vaccine antigen to turn nave b- or t - lymphocytes into protective cell, there has to be an efficient antigen delivery system that stimulates the activation of cell of the adaptive immune system. although studies on antigen presentation in fish immunology have gained prominence in recent years , there is still limited research on activation of cells of the adaptive immune system by apcs, which precludes our understanding of the role of innate immunity in optimizing vaccine performance. despite that, several studies have been carried out trying to deliver viral antigens into different compartments of fish cells. hence, in this review we provide an overview of these delivery systems and based on this approach we highlight the different immune responses induced by antigens delivered by the intracellular route compared to antigens delivered by the extracellular route. in addition, we also highlight the differences in vaccine efficacy from fish immunized using antigens delivered by the exogenous route compared to fish vaccinated using the endogenous route. overall, we anticipate that the synopsis of different antigen delivery systems put together in this review will shed new insights into limitations and successes of the current vaccination strategies used in fish vaccinology. in mammals, antigen presentation is carried out by different cell types that include monocytes, macrophages, and dendritic cells. these cells possess pattern recognition receptors (prrs) that recognize and bind to pathogen associated molecular patterns (pamps) known as danger signals on pathogens. upon binding to pamps using prrs, monocytes mature into macrophages while immature dendritic cells (dcs) also transform into mature dendritic ones to become professional apcs, which induce the expression of proinflammatory cytokines that attract more apcs to the sites of antigen deposition. upon encounter with the apcs, nave b- and t - cells undergo maturation to become memory cells capable of recognizing the antigens in subsequent encounters thereby creating the basis acquired immunity. in teleosts fish, apcs known to possess prrs having the capacity to bind to different pamps on pathogens have been described in different species and these include monocytes, macrophages, and dendritic - like cells. in addition fish b - cells have been shown to carry out antigen presentation apart from their role as antibody secreting cells. as a , in vitro methods for culturing fish monocytes, macrophages, and dendritic - like cells have been developed which makes it easy to study antigen presentation using cell cultures. it is interesting to note that tap genes comparable to those seen in mammals have been identified and mapped to mhc regions in different cartilaginous and bony fish species suggesting that similar mechanisms of endogenous antigen processing seen in higher vertebrates also exist in teleosts fish. unlike in mammals where apcs carrying processed antigens migrate to the lymph nodes, in fish apcs carrying antigens migrate to the head, kidney, and spleen, which are the major lymphoid organs. apart from lymphoid organs, apcs have been detected in other organs such as the gills, skin, and intestines in fish. in addition, different phagocytic cell types have been characterized in fish although their antigen presentation capabilities have not been investigated. similar to their mammalian counterparts, fish apcs possess a wide range of surface markers that include cd80/cd86, cd83, cd209, mhc - i, and mhc - ii proteins. in fish , cd83 has been shown to be an activation marker for macrophages and dendritic - like cells. apart from cd83, other surface markers identified for fish dendritic - like cells include cd208/lysosomal associated membrane protein (lamp3). recently, zhu et al. showed that fish b - cells act as pivotal apcs in priming the adaptive immune system using cd80/cd86 molecules. in another study, abs et al. showed upregulation of mhc - ii genes that coincided with upregulation of cd80/cd86 genes in a nonlethal infection (no cytopathic effects observed) of viral hemorrhagic septicemia virus (vhsv) in igm+ cells which consolidates the notion that fish b - cells use cd80/cd86 molecules to activate the adaptive immune system using the mhc - ii pathway. as shown in table 1, all four t - cell receptor chains (, , , and) required for binding to apcs together with the four chains (-, -, -, and -chain) of the cd3 coreceptor complex required for t - cell activation in mammals have been reported in fish. in addition , the t - cell costimulatory marker cd28 and the negative regulatory marker ctla-4, which bind to cd80 and cd86 receptors on apcs, have also been characterized in fish. as for cd8 t - cells, two subsets have been characterized, namely, cd8 and cd8, from different fish species of which cd8 has been the most widely used marker for t - cell activation in different studies. moreover, cell mediated cytotoxicity against allogeneic targets and virus infected cells has been reported by different scientists. put together these observations suggest that fish t - cells possess surface receptors essential for the binding to apcs comparable to those found in mammals and that activation of t - cells into effector cytotoxic t - lymphocytes (ctls) could be based on similar mechanisms to those seen in mammals. there are three immunoglobulin isotypes characterized in fish, this far, and these include igm and igd also present in mammals while the recently identified igt is only found in fish where it exists as membrane bound and secreted form in serum. igm is the most abundant isotype in serum where it is estimated to be > 1000-fold higher than igt. in addition, igm has been detected in the mucus of the skin, gills, and intestines although its levels in these organs are far much lower than levels detected in serum. on the contrary, igt is predominantly found in the mucus of the skin, gut, and intestines where it is > 100-fold higher than levels detected in sera. it is interesting to note that the costimulatory marker cd40l mostly expressed in activated cd4 + t - cells, which binds to the cd40 receptors on apcs in order to activate b - cell proliferation, has been characterized in fish. in addition, transcription factors involved in specification of cd4 t - cells into different t - helper (th) subtypes have also been characterized, which include t - bet , gata-3 , and ror for the differentiation of nave cd4 t - cells into th1, th2, and th17 subtypes, respectively. in addition, several cytokines linked to specification of cd4 t - cell into different subtypes have been characterized and these include il-2, il-4, il-6, il-10, il-12, ifn, il-15, il-21, il-22, and tgf. overall, the characterization of different apcs and adaptive immune cells together with their receptors and regulatory cytokine presented here suggests that teleosts fish antigen presentation mechanisms could be comparable to those used by mammals suggesting that antigen presentation mechanisms have been conserved across the vertebrate taxa. intracellular antigen delivery systems involve immunization strategies that administer the vaccine antigens into the cytoplasm (figure 1) and these include the following. live vaccines use attenuated viruses or recombinant antigens encoded by live virus vectors that have the capacity to replicate in host cells with attenuated pathogenicity lacking the ability to cause disease. as analogues of pathogenic viruses, they engage with the cell membrane by binding to surface receptors using epitopes similar to their native virus, thereby gaining entry into endosomal structures and the cytosol where they use the host cell machinery to replicate. consequently, the processed antigens are presented on the cell surface by mhc - i molecules while soluble antigens expressed by replicating virus are engulfed by apcs to induce humoral immune responses (figure 1). hence, live vaccines induce both cellular and humoral immune responses. in general, different scientists have reported the induction of ctl responses in fish.. showed activation of the ctls by viral hemorrhagic septicemia virus (vhsv) infection in rainbow trout, while we recently showed activation of eomesodermin, a transcription factor involved in activation of cd8 cells in atlantic salmon exposed to infectious pancreatic necrosis virus (ipnv). similarly, chang et al. showed activation of cd8 cells after exposing orange spotted grouper (epinephelus coioides) to nervous necrosis virus (nnv). flow cytometry analysis of the spleen cells from fish exposed to nnv showed increased mean fluorescent intensity of the cd8 cells and peripheral blood leukocytes (pbls) which were linked to increased cytotoxicity and mhc - i restriction of the sorted lymphocytes by recombinant cd8 antibodies. several fish species have shown upregulation of mhc - i and -ii molecules as well as expression of high antibody levels after exposure to viral infections suggesting that attenuated viruses administered as live vaccines could evoke both cellular and humoral immunity. based on these observations, several attempts have been made to develop live viral vaccines for fish (table 2) and some of the strategies explored this far are outlined below. roberti et al. discovered a naturally attenuated mutant of infectious hematopoietic necrosis virus (ihnv) that conferred protection against ihnv in rainbow trout although the vaccine ed in causing low level mortality after challenge. used an oral vaccine against vhsv obtained from a naturally attenuated live virus selected using monoclonal antibodies. in their study, they showed high expression levels of mhc - ii and cd4 mrnas. in addition, they detected antibody responses that were linked to significant protection in rainbow trout after challenge. an attenuated ihnv vaccine was developed at oregon state university by multiple passages using a rainbow trout isolate propagated using the steelhead trout cell culture. the vaccine showed high protection (95%) in vaccinated chinook salmon while mortality in control fish reached 90%. although the vaccine was highly protective in chinook salmon, when used in rainbow trout it showed significant mortality and as such it was stopped. since the ab strain of infectious pancreatic necrosis virus (ipnv) was found to be less virulent than the west buxton, sp, or jasper strain, dorson et al. attempted to develop an attenuated strain of ipnv from the ab strain after several passages on rtg cells. reverse genetics has been used in fish vaccinology to generate avirulent strains for use as live vaccines. for example, recombinant ihnv having a deletion of the nv gene ed in irreversible attenuation of the wild type virulent strain ing in the induction of high protection levels in rainbow trout. in another study, recombinant ihnv generated by replacing the nv gene with green fluorescent protein (gfp) or substituting the ihnv g - gene with the g - gene of vhsv induced heterologous protection in rainbow trout. for ipnv, reverse genetics was used to generate an avirulent strain for use as a live vaccine against the wild type strain by substituting amino acids on positions 217 and 221 of the vp2 capsid. these studies showed that the strain encoding the t217a221 motif caused high mortality in atlantic salmon while the strain encoding the p217t221 motif was avirulent and linked to subclinical infections. infecting atlantic salmon with high and low virulent strains at a nonpermissive physiological state (presmoltification stage) did not in mortality. when the vaccinated fish were challenged at smolt stage (permissive) the avirulent strain was less immunogenic than the virulent vaccine strain. in addition, we observed that the avirulent strain reverted to virulence under stress conditions. in general, the fear of reversion to virulence has been the major hindrance for the licensure of live vaccines in aquaculture. the strategy of dna vaccination is based on the principle that the encoded immunogenic protein is injected into the muscle or other tissues where it enters the host cells and directs the synthesis of its polypeptide antigen from the plasmid vector. once transfected into host cells, transcribed antigens replicate in the cytosol using the endogenous pathway while soluble or secreted antigens are phagocytized by apc and gain access into the exogenous pathway (figure 1). in principle, dna vaccines in the in vivo synthesis of antigenic proteins using the host cell machinery in a manner identical to natural virus infection in the case of dna vaccines made for viral diseases. this culminates into antigenic proteins expressed by plasmid dna gaining access to both the exogenous and endogenous pathways in the activation of both humoral and cellular mediated immune responses. boudinot et al. demonstrated the intracellular delivery of the plasmid dna encoding the recombinant g protein of vhsv inside the muscle cells of vaccinated rainbow trout. intracellular detection of the g - protein was shown up to 45 days at the injection sites. transcription of the g - protein was demonstrated by detection of mrna in muscle tissue extracts, which was linked to expression of high antibody and mhc - ii mrnas levels. in another study, utke et al. showed activation of the ctls following immunization using the g - protein of vhsv in rainbow trout. in their study, they used pbls collected from fish immunized with a dna vaccine encoding the recombinant g -protein of vhsv and showed that pbls from vaccinated fish killed the vhsv mhc - i matched rtg-2 cells indicating that the g - proteins had the capacity to induce ctl responses in vaccinated fish. they also showed the homing of leukocytes to the injection site suggesting that cells expressing the recombinant g - protein had a chemoattractant effect. this observation was recently supported by castro et al. who showed that b - lymphocytes, both igm and igt cells, represent one of the major cell types infiltrating the injection sites expressing the g - protein of vhsv. in their study, they showed upregulation of cxcr3b, a receptor for cxcl11, together with ck5b and ck6 chemokines, which could play chemotactic roles in the early recruitment of b - cells at the injection sites. put together, these studies show that the intracellular expression of proteins transcribed from dna vaccines in fish cells leads to homing of leukocytes and b - cells to injection sites with possible involvements of chemoattractant chemokines. further, these studies suggest that antigens delivered by this endogenous route evoke both the humoral and cellular mediated immune responses in vaccinated fish. finally, it is important to point out that immunization using dna vaccines exhibits many advantages over the live and inactivated vaccines. intracellular synthesis of the antigenic proteins poses no danger of reversion to virulence and does not require inactivation of viruses using toxic substances. high expression levels of humoral and cellular responses can be achieved at low doses as shown by corbeil et al. that nanogram quantities of a dna vaccine protected rainbow trout against ihnv infection after challenge. in addition, intracellular synthesized antigens tend to fold in their native conformation and correctly glycosylated displaying the neutralizing epitopes in a similar pattern to the native virus. in terms of genetic engineering for example, the use of molecularly encoded cytokine adjuvants like il-2 in dna engineered vaccines has shown the ability to enhance dna delivery and increase the duration and magnitude of plasmid dna expression in vivo. jimenez et al. coinjected recombinant il-8 with plasmid dna encoding the g - protein of vhsv in rainbow trout and showed massive infiltration of neutrophils at the injection site linked to upregulation of proinflammatory cytokines. table 3 shows the dna vaccines explored for use in fish, this far, of which only the dna vaccine for ihnv has been licensed in canada (novartis ltd .). this mode of antigen delivery which has been referred to as the first class ticket to induction of mhc - i responses relies on receptor mediated internalization of viral antigens to the er followed by retrograde translocation into the cytosol. only a few studies have explored this delivery system in fish vaccinology, this far. constructed a fusion protein vaccine made by fusing the vp2-vp3 polyprotein of ipnv with the exotoxin of lactobacillus casei, which ed in reduced viral loads in vaccinated rainbow trout after challenge while in our group we constructed a fusion protein vaccine made by fusing the vp2 of ipnv with the pseudomonas aeruginosa exotoxin a (ep). the exact mechanisms in which viral antigens are translocated into the cytosol are dependent on three bacterial proteins as illustrated from the pe fusion protein in figure 2. the pe protein has three functional domains, namely, the receptor binding domain - i, transmembrane targeting domain - ii, and the toxic moiety domain - iii. domain - i binds to the 2-macroglobulin receptor on the cell surface. after binding to domain - i after enzymatic cleavage by the protease furin in the endosome, the protein fragment encoding domains - ii and -iii is delivered into the golgi by the er retrograde transport and further into the cytosol using domain - ii, which is responsible for transmembrane translocation of the toxin proteins into the cytoplasm. as shown in figure 2, domain - iii, which is toxic to cells, is eliminated and is replaced with the immunogenic protein (vp2) of ipnv bound to the kdel signaling peptide. the purpose of including the kdel signaling peptide in the final construct (pe - vp2-kdel) is that it enables the binding of the whole construct to the golgi membrane kdel - receptor. once bound to the golgi membrane receptor, the ligand - receptor complex is packaged into vesicles for retrograde transport back to the er where processed peptides are packaged on mhc - i molecules for presentation on the cell surface. in our studies, we employed the pe - vp2-kdel fusion protein to deliver the vp2 immunogenic protein of ipnv intracellularly as a vaccine. although we did not assess the ctl responses induced by this antigen delivery system, our findings show that these vaccines were able to induce a low level antibody response suggesting that antigens delivered using this method could gain access to induction of humoral responses in vaccinated fish. however, there is a need for detailed investigation to determine the role of ctl responses induced by this mode of antigen delivery in vaccinated fish. polymeric nanoparticles formulated from biodegradable polymers have been widely explored as carriers for controlled delivery of vaccine antigens. this system can potentially deliver antigens to the desired location at predetermined rates and durations to generate an optimal immune response. for example, tian et al. and zheng et al. showed that lymphocytic disease virus (lcdv) encapsulated in particles sustained a much longer release of the dna antigen than naked dna injected in japanese flounder. in addition, carriers protect the antigen from degradation until release as shown by rajeshkumar et al. that encapsulated dna antigens were protected from degradation by dnaase for vaccines used in the asian sea bass (lates calcarifer). to deliver the antigens into host cells , nanoparticle materials are internalized by endocytosis. to deliver the antigens into the cytosol, the release of antigens from the acidic endosomes requires membrane disruptive agents, which release the internalized proteins into the cytosol. therefore , encapsulation carriers should include membrane penetrating peptides and polymers that disrupt the membranes when the ph declines in the endosomes. made acid degradable nanoparticles, which were designed to release encapsulated proteins in a ph - dependent manner. in their study, they made nanoparticles that were stable at ph 7.4 but quickly degradable at ph 5.0 in the acidic endosomal environment enabling the release of antigens into the cytosol, ultimately ing in upregulation of mhc - i. another method explored is the use of amphiphilic polymers , which also have ph - dependent membrane disruptive properties protonated at the endosomal ph range. upon reduction of the endosomal ph, these particles increase their hydrophobicity to facilitate the disruption and penetration of the endosomal membranes culminating in the release of antigens in the cytosol. these amphiphilic polymers have been shown to increase cd8 + responses and to improve vaccine potency. in summary, these studies show that nanoparticle antigen delivery systems can be designed to deliver antigens through the intra- or extracellular routes to evoke immune responses linked to the mhc - i or -ii pathways. studies in higher vertebrates have shown that apcs easily carry out phagocytosis of nanoparticles and microparticles between 150 nm and 4.5 m with the optimal size for phagocytosis being 500 nm while monocytes have been shown to easily phagocytose nanoparticles > 100 nm. and, as pointed out by gutierro et al. , nanoparticles that encapsulate antigens resemble pathogens in terms of their uptake into host cells by mirroring the route of pathogen uptake and the immune response triggered after nanoparticle uptake. have also pointed out that nanoparticles can also be used to carry antigens on their surface which would serve as a good stimulant for the induction of b - cell responses. although antigen delivery using nanoparticle vaccines has been well studied in higher vertebrates, indications are that fish cells use similar mechanisms of antigen uptake from nanoparticle based vaccines. for example, ruyra et al. showed entry of liposome - based nanoparticles in zebrafish hepatocytes and trout macrophages by endocytosis. upon entry, the nanoparticle laden cells initiated specific proinflammatory responses while fredriksen and grip showed intracellular cytoplasmic localization of polylactic - coglycolic acid (plga) nanoparticles in to - cells. these findings support earlier observations, which showed that because plga particles are less hydrophilic than alginates, they are easily incorporated into host cells, which makes them suitable vehicles for delivering antigens into intracellular compartments. recently, we used plga nanoparticles to deliver inactivated whole viral particles of ipnv as a vaccine, which expressed low antibody levels comparable to those induced by inactivated whole virus (iwv) vaccines suggesting that delivery of antigens using plga nanoparticles has the ability to induce humoral immune responses in vaccinated fish. although there are limited studies that categorically demonstrate the intracellular delivery of nanoparticle based vaccines in fish cells, rajeshkumar et al. were able to induce low level cytotoxicity (tested in vitro) using chitosan nanoparticle vaccines in asian sea bass vaccinated against vibriosis (listonella anguillarum). table 4 shows that only a few studies have been carried out using nanoparticle based technologies to administer viral antigens in fish. in general, indications show that nanoparticle based vaccines have the potential to deliver antigens into different host cell compartments and thus that they can induce cellular and humoral immune responses in vaccinated fish. this approach involves antigen delivery systems that administer viral antigens into the extracellular compartments using the exogenous pathway (figure 1). this mode of antigen delivery ensures that the antigenic protein is preserved in its native structure while the virus is rendered nonreplicative using chemical or physical methods. given that inactivated whole virus (iwv) vaccines are nonreplicative, it follows that their antigens enter the host cells by the exogenous route and their processed peptides are presented to cd4 cells via the mhc - ii pathway. and, as such, several studies have shown upregulation of mhc - ii genes in response to vaccination using iwv vaccines. in terms of cd4 t - cell differentiation, iwv vaccines have been shown to predominantly activate genes linked to the t - helper 2 (th2) responses. for example, we showed upregulation of gata-3, a transcription factor linked to activation of nave cd4 cells into th2 responses, when genes linked to activation of th1 and cd8 t - cell responses were downregulated. in this study, we showed a high correlation between gata-3 and antibody levels expressed against ipnv. in terms of antibody responses, iwv vaccines have been linked to high expression levels of igm and igt in vaccinated fish. in our studies , we showed a high correlation between postchallenge reduction of mortality and systemic igm levels, suggesting igm levels could serve as a correlate of protection for iwv vaccines. overall, these studies strongly suggest that iwv vaccines are to be considered as exogenous antigens, mainly inducing humoral immune responses. the basic principle for this vaccination strategy is that the gene encoding the antigenic proteins is isolated from the native virus and transferred into a heterologous vector that is nonpathogenic for propagation. table 6 shows the antigenic proteins identified for the major fish viral diseases and the different expression vectors used for propagation. in the case of vhsv and ihnv, production of subunit vaccines has focused on cloning the g - protein into heterologous vectors while, for viruses such as ipnv, the strategy has been to clone the entire outer capsid encoding the protective epitopes, instead of protein segments coding the neutralizing epitopes in heterologous vectors. subunit vaccines are nonreplicative and are delivered exogenously to host cells by the extracellular route (figure 1). similar to iwv vaccines, subunit vaccines induce humoral immune responses and upregulation of mhc - ii genes , which is consistent with observations in higher vertebrates in which it has been shown that immune responses induced by subunit vaccines are mainly dependent on the mhc - ii pathway and that they elicit antibody responses. showed a high correlation between reduction of viral rna and activation of cd4 markers in atlantic halibut (hippoglossus hippoglossus l.) immunized using a subunit vaccine for nodavirus. although cross presentation of exogenously processed peptides from endosomes into the cytosol has been reported in higher vertebrates , there is no study demonstrating cross presentation of peptides processed from endosomes into the cytosol for subunit vaccines in fish. structural proteins of most viruses self - assemble to forms capsids in different expression systems that resemble the native virus structure in size and morphology and, hence, they are referred to as virus - like particles (vlps). made vlps of nodavirus expressed in e. coli or spodoptera frugiperda (sf21) insect cells that formed small, nonenveloped t = 3 quasi - symmetric particles. they showed that the capsid of malabaricus grouper nervous necrosis virus (mgnnv) spontaneously self - assembled into vlps when expressed in sf21 cells infected with a recombinant baculovirus. these vlps were indistinguishable from the native virus particles by electron microscopy and the 3d structure of the vlps was resolved at 2.3 nm by cryomicroscopy. produced vlps that were devoid of the nucleoprotein but resembled the outer capsid of the native grass carp reovirus (gcrv). however, in some cases vlps are formed from replication of surface particulate components that do not form the entire capsid, but they contain elements of the outer capsid that are immunogenic. these protein structures are called subviral particles (svps). both vlps and svps table 7 shows the vlps, svps, and immature virus particles (ivps) made from different fish viruses. as shown in table 7, different expression systems were used to make vlps, svps, and ivps for ipnv. given the similarity to their native viral capsids, this property was demonstrated by lai et al. who produced vlps for nnv expressed in e. coli and showed that nnv failed to infect the asian sea bass cells that were exposed to the vlps prior to infection, suggesting that the cell surface receptors were occupied by the vlp - epitopes blocking the wild type virus from entering the cells and thereby protected the cells from developing cytopathic effect (cpe) while control cells not exposed to vlps developed full cpe. liu et al. showed that vlps generated from nnv induced high antibody responses that lasted for more than five months, similar to those produced by the native wild type virus, which were correlated with long - term protection in vaccinated orange spotted grouper. produced vlps in e. coli for nnv that expressed high antibody levels, which were correlated with igm, mhc - ii, and cd4 levels in vaccinated fish. these observations suggest vlps induce the expression of cd4 responses through the mhc - ii pathways in a similar pattern to those induced by subunit vaccines in mammalia. the most explored strategies for the delivery of antigens using the intracellular route in fish vaccinology involve the use of live and dna vaccines. the use of dna vaccines in fish has undergone intense investigation in the last decades as a substitute of replicative antigens for live vaccines. although factors leading to higher performance of dna vaccines for rhabdoviruses compared to other fish viral families have not been elucidated, similar observations seen in higher vertebrates show that dna vaccines for rhabdoviruses are more protective than some of the dna vaccines for other viral families. in general, replicative vaccines delivered via the intracellular route have been linked to activation of cellular and humoral immune responses in vaccinated fish, which makes these vaccines be more protective than nonreplicative vaccines delivered by the extracellular route. for antigens administered by the extracellular route, several antigen delivery strategies have been explored in fish vaccinology, which include the use of iwv, subunit, svp, vlp, and imp vaccines. in general, all exogenous antigens induce humoral immune responses. in terms of cellular immunity, exogenous antigens were linked to expression of mhc - ii and cd4 genes. however, new innovations such as the use of fusion protein and nanoparticles vaccines having the potential to deliver nonreplicative antigens into the cytosol are likely to induce ctl responses in vaccinated fish. the use of nanoparticle vaccines has attracted a lot of interest in the delivery of oral vaccines for fish production systems that require a boost vaccination when fish have been transferred in cages to the sea after prime immunization using parenteral vaccines at the freshwater stage. in general, iwv vaccines are superior to subunit vaccines given that they produce high antibody levels, which correlate with protection in vaccinated fish. in addition, these vaccines have been shown to activate the expression of cd4 and th2 genes that correlate with high antibody levels consolidating the common notion that exogenous antigens stimulate humoral immune responses orchestrated by th2 cytokines. although we did not review the role of apc prime / activated b - cells in antigen uptake and presentation to cells of the adaptive immune system in detail given the limited studies carried out on this topic in fish vaccinology, we can conclude that the different antigen delivery systems explored in fish this far deliver their antigens into the intra- and extracellular compartments and that they activate either the cellular or humoral immune response or both depending on the route of antigen delivery. as pointed out by howarth and elliot, the most protective vaccines are those that stimulate both the cd4 + and cd8 + t - cells responses and, as such, replicative vaccines such as the live and dna vaccines that stimulate both the mhc - i and -ii pathways are likely to produce better protection in fish (table 8). so far only the ihnv - dna vaccine for use in atlantic salmon in canada is the only one licensed while live viral vaccines are feared to revert to virulence. hence, the use of iwv vaccines which accounts for the largest proportion of licensed vaccines is likely to continue dominating the vaccine industry in aquaculture. therefore, the search for better antigen delivery systems that stimulate both cd4 + and cd8 + responses that have the potential to induce long - lasting protective immunity has to continue. overall, we anticipate that the synopsis of different antigen delivery systems presented here will shed new insights into the limitations and successes of the current immunization strategies used in fish vaccinology.

vaccine efficacy in aquaculture has for a long time depended on evaluating relative percent survival and antibody responses after vaccination. however, current advances in vaccine immunology show that the route in which antigens are delivered into cells is deterministic of the type of adaptive immune response evoked by vaccination. antigens delivered by the intracellular route induce mhc - i restricted cd8 + responses while antigens presented through the extracellular route activate mhc - ii restricted cd4 + responses implying that the route of antigen delivery is a conduit to induction of b- or t - cell immune responses. in finfish, different antigen delivery systems have been explored that include live, dna, inactivated whole virus, fusion protein, virus - like particles, and subunit vaccines although mechanisms linking these delivery systems to protective immunity have not been studied in detail. hence, in this review we provide a synopsis of different strategies used to administer viral antigens via the intra- or extracellular compartments. further, we highlight the differences in immune responses induced by antigens processed by the endogenous route compared to exogenously processed antigens. overall, we anticipate that the synopsis put together in this review will shed insights into limitations and successes of the current vaccination strategies used in finfish vaccinology.

pancreaticobiliary cancer remains a lethal disease where treatment continues to be a major oncologic challenge. most patients present with advanced disease in which survival is dismal. for patients diagnosed early with small tumors, surgery offers a chance of cure. in an attempt to diagnose these tumors at an early stage, many studies have evaluated the use of tumor markers, but so far, due to low sensitivity and specificity, the have not been promising. the high metastatic potential of pancreatic cancer has led researchers to investigate its ability to invade the basement membrane and extracellular matrix (ecm) allowing it access to capillaries and lymphatics. one process that has been shown to be involved in invasion of most types of cancer is the urokinase - type plasminogen activator system. upa is a serine proteinase that is best known for catalyzing the conversion of inactive plasminogen to the active proteinase plasmin. upa and its receptor upar have been demonstrated to be involved in tumor invasion, growth, and metastasis. independently or through the activation of plasmin, upa can degrade ecm, activate matrix metalloproteinases, mediate the release of growth factors (including transforming growth factor , fibroblast growth factor, vascular endothelial growth factor, insulin growth factor, tumor necrosis factor , and hepatocyte growth factor), stimulate cellular migration, induce chemotaxis, and promote angiogenesis. since the 1980s when upa was postulated to have a role in tumor invasion, there have been multiple studies evaluating it as a prognostic marker of cancer. it was first studied in breast cancer where it has been shown as an independent prognostic marker for predicting survival second only to lymph node status. following the initial in breast cancer, upa overexpression was shown to confer a worse prognosis in many other cancers including colorectal, esophageal, gastric, hepatocellular, prostate, sarcoma, and head and neck squamous cells among others. the importance of the upa activator system has also been demonstrated in pancreatic cancer. the first study to show overexpression of upa in pancreatic cancer was by takeuchi et al. in 1993. he demonstrated by immunohistochemical staining that 78% of pancreatic cancers overexpressed upa and this overexpression correlated with decreased survival. another study by cantero et al. in 1997 showed that concomitant overexpression of upa and its receptor upar correlated with shorter survival times. we detected upa overexpression in pancreatic intraepithelial neoplasia (panin) and histologically normal ducts / acini that are in the immediate vicinity of the tumor. upa was also found in the vessels of tumor stroma suggesting that upa is in circulation, and therefore should be detectable by serum analysis. it allows further evaluation through imaging of the ductal system and allows to sample pancreatic juice and brush biopsy cytology for further analysis. ercp has also been studied in small numbers to screen high - risk patients with a family history of pancreatic cancer, and it demonstrated that cytologic brushings showing dysplasia correlate with the pathologic specimen after resection. despite combination of sampling with brush cytology, fine needle aspiration, and biopsy, the sensitivity of all three combined is only 62%. most practitioners perform only brush cytology which by itself has a sensitivity of only 30% in the diagnosis of pancreaticobiliary malignancies. we hypothesized that molecular markers such as upa may improve our ability to differentiate malignant from benign pancreaticobiliary strictures and assist us in planning therapeutic strategies. we performed a feasibility study of patients undergoing ercp for confirmed or suspected pancreaticobiliary cancers to determine whether upa overexpression could be identified from cytologic brushings. serum levels of upa were measured in this patient group and compared to healthy subjects. eleven patients with known or suspected pancreaticobiliary malignant strictures secondary to either pancreatic cancer or cholangiocarcinoma as determined clinically by radiologic studies were consented to have cytologic brushings obtained during ercp for evaluation of expression of upa. at ercp, one slide was routinely stained for h&e to identify the presence of epithelial cells and the other was stained for upa in a blinded fashion. presence and type of cancer were determined by biopsy and reviewed by a pathologist. reagents used in the immunohistochemical procedure including blocking solution, secondary antibody, and vectastain elite abc reagent were purchased as the r.t.u vectastain universal elite abc kit (vector laboratories, burlingame, ca). color - development reagents were purchased as part of a dab substrate kit for peroxidase (vector laboratories, burlingame, ca). the dab substrate kit contained the reagents required to make a working solution of 3,3-diaminobenzidine (dab) for staining tissue sections. upa expression in cytologic brushings obtained during ercp was evaluated by immunohistochemistry (ihc). the upa1 primary antibody was used at a concentration of 20 g / ml. cells were blocked with normal horse serum (2.5%) for one hour and incubated overnight at 4 celsius with the primary antibody. after gently rinsing the cells with triton - tbs, they were treated with the secondary antibody (r.t.u . biotinylated universal antibody and anti - rabbit / anti - mouse igg made in horse), gently washed and treated with vectastain elite abc reagent. a slide of cytologic brushings was also stained by the same procedure, except that an irrelevant isotype igg was substituted for the primary antibody as a negative control. staining intensity for upa was classified as 03 + (0 absent ; 1 +, weak ; 2 +, moderate ; 3 +, strong). serum levels of upa were analyzed using an enzyme - linked immunosorbent assay (elisa). correlation between ihc grade and serum levels of upa was estimated with the spearman rank correlation coefficient. all tests were carried forth using sas 9.2, sas institute inc., cary, nc. given n = 11 subjects per group and assuming exchangeability under the null hypothesis, the wilcoxon rank - sum test would be able to detect approximate shift alternatives of roughly 0.6 ng / ml, 1.3 ng / ml, and 1.7 ng / ml given 0.80 power and common standard deviations under the null hypothesis of no difference of 0.5 ng / ml, 1.0 ng / ml, and 1.9 ng / ml, respectively. 8 patients had pancreatic adenocarcinoma and 3 had cholangiocarcinoma. when immunohistochemistry for upa was performed on the cytology specimens, six of the eight patients (75%) with pancreatic cancer (figure 1) and two of the 3 patients (67%) with cholangiocarcinoma overexpressed upa (figure 2). the two patients with pancreatic cancer who did not overexpress upa were being treated with chemotherapy and underwent ercp for biliary stent exchange. the mean serum upa in the 11 patients with cancer was 1.27 ng / ml (sd = 1.54). the mean serum upa in the 11 normal healthy individuals was 0.56 ng / ml (sd = 0.16). serum analysis demonstrated a 2-fold higher concentration of upa in the pancreaticobiliary cancer patients compared to the healthy controls (p = .0182). table 1 compares the level of upa staining in cytologic specimens to serum levels of upa for all 11 patients. the ihc grade for upa staining correlated with serum levels for upa (r = 0.72 ; p < .0001). a definitive diagnosis of cancer can be difficult in patients with pancreaticobiliary strictures with no obvious mass on imaging studies. we have previously shown that upa is an early event in the malignant transformation of pancreatic cancer. therefore we sought to determine the feasibility of determining upa expression in cytologic brushings of established malignant strictures to evaluate its potential as an adjunct to ercp in diagnosis of pancreaticobiliary malignancies. in our small sample, we found it is possible to identify overexpression of upa in cells obtained from brushings during ercp. upa overexpression (72.7%) appears to be a marker for pancreatic or biliary cancers. although there is no current literature to show that chemotherapy directly inhibits upa synthesis, we have postulated that this treatment impedes growth, and thus decreases upa expression. the relationship between upa overexpression and cholangiocarcinoma has not been studied, but in two of the three (66%) samples, the immunohistochemical analysis of the cytologic brushings showed overexpression. as ercp is an invasive technique with potential morbidity, we also looked at serum upa in our patient population, and it was elevated in patients with pancreaticobiliary cancer. the serum levels correlated with the ihc grade of upa in tissue (p < .001). serum upa levels have been evaluated in breast cancer by rha et al., who showed blood upa levels correlated with those of tissue. because our study contains only a small number of patients, the value of serum upa as a tumor marker needs to be further evaluated in large numbers of patients including patients with chronic pancreatitis before it can be compared to the standard tumor markers such as ca19 - 9. currently there are no established screening tests for pancreatic cancer. the united states preventive services task force (uspstf) has recommended against routine screening for pancreatic cancer based upon the lack of data demonstrating its clinical value in the general population. familial pancreatic cancer (fpc) accounts for up to 10% of patients with pancreatic cancer. iii, and patients with panin iii have been considered for pancreatectomy by some groups. surveillance by ercp and endoscopic ultrasound is performed in an attempt to identify patients with fpc who have developed panin, but the diagnosis is difficult. evaluating upa overexpression from ercp obtained cytologic brushings may have potential application in screening high - risk patients by identifying either panin or invasive pancreatic cancer. although ercp arguably remains the golden standard for pancreaticobiliary evaluation, there is a risk of complications associated with ercp including pancreatitis (5.4%). endoscopic ultrasound (eus) with eus - guided fna has emerged as an extremely sensitive and specific method of diagnosing pancreatic cancer and can detect small lesions more effectively than conventional imaging. for the diagnosis of pancreatic cancer, as we have shown that upa staining can be performed on brush cytology, it can be done on fna specimens as well and may improve diagnostic accuracy. as targeted therapy has stepped to the forefront of cancer research, molecular markers have moved from their traditional roles of diagnosis and staged to possible aims of treatment. already upa has been evaluated as a potential target for treatment to decrease the invasive and metastatic activity of pancreatic tumor cells. evaluated an anti - upar monoclonal antibody in mice and found that it significantly decreased tumor growth, hepatic metastases, and retroperitoneal invasion. small molecule inhibitors of upa have been evaluated in a fibrosarcoma model in mice ing in decreased metastases and prolonged survival. these suggest that detecting overexpression of upa in pancreatic cancers may not only be prognostic and diagnostic but may also direct treatment in the future. a serious limitation of the current study is its small sample size and lack of an adequate control group precluding meaningful analysis. in this study, a control group of healthy subjects for the analysis of upa in the serum was obtained. there are obvious ethical issues in obtaining brushing from healthy volunteers and it would appear more suitable to utilize patients with benign pancreatic and biliary strictures. we elected to exclude potential control groups such as those presenting with obstructive jaundice from benign pancreaticobiliary strictures. the possibility that some of these patients harbored occult malignancy would be difficult to discern without long term follow up. even in patients with choledocholithiasis , there may be a potential for the associated inflammatory process to elevate upa levels. thus, we performed this feasibility study in patients with known or suspected pancreaticobiliary malignancies by brush cytology to corroborate our prior findings in resected pancreatic cancer specimens. we performed a feasibility study to determine whether upa overexpression could be identified from cytologic brushings of patients undergoing ercp for confirmed or suspected pancreaticobiliary cancers. serum levels of upa were measured in this patient group and correlated with upa overexpression. larger studies involving all forms of pancreaticobiliary pathology need to be performed before upa overexpression can be used either as a serum or a cytological tumor marker, especially those with no identifiable masses. also comparison with other tumor markers, that is, ca 19 - 9 and cea will be required to substantiate the role of upa as a diagnostic or prognostic tumor marker.

we have previously demonstrated that upa is overexpressed in pancreatic tumors. in an attempt to diagnose these tumors earlier , we sought to determine whether upa could be identified in endoscopic retrograde cholangiopancreatography obtained brushings in patients with malignant pancreatic and biliary strictures. secondarily, upa was measured in the serum of this patient population. upa overexpression was identified in the cytologic tissue in 8 of 11 patients (72.7%). serum analysis demonstrated a 2-fold higher concentration of upa in the pancreaticobiliary cancer patients (1.27 versus 0.56 ng / ml ; p = .0182). also, upa overexpression correlated with serum levels (p < .0001). this study confirms that upa can be detected in the ercp cytologically obtained tissue and is frequently present in a higher concentration in the serum of pancreaticobiliary cancer patients. a larger sample size will be required to address its value as a sensitive marker for the diagnosis of pancreatic or biliary cancers.

new york city has one of the highest reported human immunodeficiency virus (hiv) and acquired immunodeficiency syndrome (aids) rates in the united states, more than triple the national average. approximately one quarter of these hiv - positive individuals live in bronx county (known as the bronx), with areas reaching an hiv / aids prevalence of greater than 2%. relative to the united states, new york state, and the other counties in new york city, the bronx also has a disproportionately higher prevalence of sexually transmitted infections (stis) and teen pregnancy. in 2005, the teen pregnancy rate in the bronx was 140% higher than that of new york city, and the chlamydia case rate was more than double the national rate. therefore, condom access, as a means of hiv / aids, sti, and teen pregnancy prevention, is a high priority in this community. although the determinants of condom use are complex and multifactorial, prior work has established that preparedness by possession of condoms is one strong predictor of use. however, it has been well documented that embarrassment during the purchase of condoms remains a barrier to acquisition and use among young adults and women. condoms have been described as a socially sensitive product; a real or imagined social presence around such products creates embarrassment, which in turn affects purchasing behavior. by the same token, research has shown that adolescents prefer to purchase condoms from places where they are clearly visible and quickly purchased, even when given a cheaper option. in 2004, brackett reported that one of several strategies employed by young adults to reduce the embarrassment of condom purchase is to avoid asking for help or for location of condoms within a store. yet the popular press has reported that condoms are often stored in locked cases in drug store aisles. in 2006, the washington post reported that one national pharmacy sold condoms in locked cases in 22 of 50 of their washington, dc stores. requiring assistance from store personnel to purchase condoms makes the sale more public, lengthy, complicated, and potentially embarrassing, thereby creating a barrier to access. the theory of planned behavior proposes that intentions and perceived control over behavior predict behavioral performance. part of this model suggests that one's intentions and behavior in performing a given act are related to the control and difficulty one perceives in performing that act. therefore, among individuals intending to acquire condoms, a setting in which those vulnerable to embarrassment are obliged to ask for assistance creates a structural disincentive with a potentially negative outcome. while condom purchase preferences and behavior have been explored, condom placement within physical reach has received narrow attention. in 2006, scott - sheldon et al. they examined condom placement relative to the proximity of other embarrassing products, and the positive / negative associations such placement fosters. in this study, two thirds of condoms were positioned behind or next to the checkout counter, though the proportion behind the counter and requiring assistance is not specified. klein et al. investigated several aspects of condom availability, including condom visibility and placement within store aisles. this paper found that the majority of drug stores displayed condoms in the aisles, while the majority of private grocery and convenience stores kept them behind the counter. however, the limited data on this barrier have not been validated in a community of such significant risk as the bronx, nor has condom accessibility through the lens of mandatory interaction been researched. the goal of this paper was to describe the prevalence of structural barriers to condom access in the bronx, specifically physical inaccessibility, defined as the sale from locked cases or behind store counters requiring interaction with store personnel. we hypothesized that physical inaccessibility of condoms would be found in the majority of the sites selling condoms in the bronx. in addition, we sought to determine whether or not the prevalence of these structural barriers was associated with the socioeconomic status (ses) of the health districts within the bronx. the bronx is a densely populated urban community of approximately 1.4 million people, with commercial areas in close proximity to or interspersed within residential neighborhoods. one - third of bronx residents live below the federal poverty line, though socioeconomic status varies widely between the seven designated health districts of the county. health data for the bronx are listed in table 1. the bronx yellow pages lists 320 pharmacies (80 of them are chain pharmacies) and approximately 900 grocery stores in the 20 zip codes in the county. as pharmacies are largely represented in commercial areas, they were used as focus points for sampling of the two nearest grocery stores or supermarkets. if the pharmacies were so closely clustered that two different grocery stores per pharmacy were not encountered, we would survey the maximum number of stores within walking distance of those pharmacies, operationalized as within 5 blocks. staff entered each selected location in the manner of an ordinary customer and observed the location of condom placement. if condoms were not visible, the representative asked the store clerk whether condoms were sold and if so, where in the store they were located. on leaving the store, the representative provided the following data on a coding form developed by the investigators: the type of store, zip code, availability of condoms, specific condom location within the store, whether personnel assistance was needed to obtain condoms prior to purchase, and the number of interactions required prior to purchase. information regarding ses, hiv, teen pregnancy, and sti prevalence was obtained from the new york city department of health (doh), which provides data for the seven health districts and the 20 zip codes in the bronx. each site was assigned to one of these districts based on its zip code, to determine whether the manner of condom sales correlated with the health statistics of that region. we determined that 75 pharmacies and 90 grocery stores needed to be sampled based on a presumed point estimate of 50% of sites keeping condoms physically out of reach, in order to achieve a 95% confidence interval of 10% around this point estimate. to even the distribution of grocery stores and achieve the minimum sample size, we surveyed the two closest grocery stores to each pharmacy, aiming for 150 stores and 225 total sites. data organization and analysis were performed using epi info version 2000 (epiinformatics, doraville, ga). we sampled a total of 215 sites, 75 pharmacies and 140 stores. because of the close proximity of some of the pharmacies, condoms were sold at 195 (91%) of these sites (table 2). condom purchase required assistance from site personnel at 160 of the 195 sites selling condoms (82% ; 95% ci : 76%87%). condoms were more likely to be kept out of reach in the 115 grocery stores (96%) compared to the 75 pharmacies (60%) selling them (or = 15, 95% ci : 548). condoms were also kept out of reach in more independent pharmacies (78%) compared to chain pharmacies (10%) (or = 32, 95% ci, 6235). four sites required assistance from two or more personnel prior to condom purchase whereas the remainder required assistance from one person prior to purchase. we stratified for the ses / hiv prevalence of each bronx health district relative to the manner of condom distribution. in health districts 1&2, with the greatest hiv, sti, and teen pregnancy prevalence and the greatest number living below the federal poverty level, condoms were kept out of reach in 90% out of 55 sites sampled (table 4). in health districts 6&7, with the lowest hiv, sti, and teen pregnancy prevalence, and the lowest number living below the federal poverty level, condoms were kept out of reach in 70% out of 30 sites (table 4). condoms were more likely to be kept out of reach in the lowest ses / highest infection and teen pregnancy prevalence districts compared to the highest ses / lowest infection and teen prevalence districts (or = 4.3, 95% ci : 1.117). although 91% of stores surveyed in the bronx sold condoms, the vast majority (82%) sold them in locked cases or behind sales counters. in almost all of the convenience stores and in 78% of independent pharmacies, consumers required assistance from site personnel in order to purchase condoms. thus, condom accessibility was poor in the sites most commonly encountered; most bronx stores are convenience stores and 3 out of every 4 pharmacies are small and privately owned. since the low - ses districts also have the highest rates of hiv, stis, and teen pregnancy, barriers to condom access are of particular concern. the study conducted by klein et al. in 2001 found that almost two - thirds of adolescents who purchased their condoms did so only from pharmacies. the purchasing preferences reported in klein's work highlight the potential relevance of our finding that the majority of bronx pharmacies sold condoms from behind the counter. our findings also differ from those of scott - sheldon et al., who report on 66% of condoms being sold from behind the counter or while we do not know what proportion of these condoms was sold exclusively from behind the counter, we found that significantly more condoms were sold in this manner, and this practice was more common in a higher - risk community. this reinforces the negative implication of selling the vast majority of condoms in a manner that obligates assistance from a stranger. a limitation of our study is that condoms can be acquired from sources other than stores, such as high schools or community health centers. there were no data available on the number or rate of condom distribution in local high schools. however, students who have previously reported on condom availability in schools have found them to be inadequate in supply. moreover, distribution in schools does not benefit those who are older or adolescents who are not in school. a further limitation was inadequate information about where individuals in our region actually acquire their condoms. we were unable to quantify whether or not individuals seek condoms from free sources and the number of such sites in the region. though we do not know whether young adults usually purchase condoms from chain pharmacies, where condoms are usually sold with open access, those pharmacies represent the minority of the total in our community and may be difficult for some to access. finally, although the small sample size led to wide confidence intervals around the odds ratios, the are nonetheless significant as the lower limit of the confidence interval was greater than one. although not confirmed, it has been suggested that the fear of theft is one reason why condoms are sold from locked cases or behind store counters. therefore, efforts to change the sales practices in smaller stores with less financial stability may be unrealistic. however, this does not change the fact that access to condoms is critical, especially in areas which had the highest sti rates; that there may be a structural barrier to condom purchase in these communities represents an incongruity between what is most needed and what is available. to this end the vast majority of sites surveyed in the bronx sold condoms in locked cases or behind sales counters. failure to make condoms readily accessible for unmediated purchase may disproportionately deter adolescents and women from acquiring condoms and ultimately from adopting recommendations for condom use. this information can prompt similar investigations in other high - risk communities and inform public health efforts to increase condom accessibility and the distribution of free condoms in public and practice settings.

as embarrassment is a known obstacle to condom acquisition, selling condoms from physically inaccessible places that require personnel assistance constitutes a barrier to access. this study investigates the extent of this barrier in the bronx, a high hiv / sti prevalence county of new york. 75 of 320 listed bronx pharmacies were sampled via computer randomization. investigators coded condom placement and physical accessibility within these pharmacies and 140 surrounding stores. 91% of sites sold condoms. in 82%, condoms could not be accessed without assistance. condoms were physically inaccessible in venues most encountered in the community: grocery stores versus pharmacies (or=15 ; 95% ci, 548), independent versus chain pharmacies (or=32 ; 95% ci, 6235). they were physically inaccessible more in the lowest ses / highest hiv prevalence areas versus the highest ses / lowest hiv prevalence areas (or = 4.3, 95% ci, 1.117). findings can inform efforts to increase accessibility of condoms, distribute condoms in alternative settings, and prompt similar investigations in other high - risk communities.

the deposition of coloring matter, coloration, or discoloration by a pigment pertaining to the gingiva is gingival pigmentation. our knowledge about gingival pigmentation (gp) and their etiologies has increased enormously over the past decade. the racial - physiological pigmentation is not of medical concern, but may at times be of esthetic concern. light brown to black pigmentation may be physiologic or racial in healthy colored - skinned individuals, whereas the same oral pigmentation in caucasians may be abnormal. the intensity of pigmentation is frequently altered by physical, chemical, and hormonal factors. the normal physiologic color of gingiva is coral pink or salmon pink, with physiological variations of melanin pigmentation. it is a nonhemoglobin - derived brown pigment produced by melanocytes and is also a powerful cationchelator. they work independent of the surrounding epithelial cells and behave as unicellular exocrine gland, convert tyrosine to melanoprotein (melanin), which is transferred to keratinocytes by way of melanosomes. benign and malignant lesions, cultural intentional tattooing, drugs, heavy metal ingestions - poisonings, iatrogenic, smoking, and systemic problems can all cause gp. we propose a new improved classification for gp and pigmented lesions, based on our experience gained from previous classification systems. we also propose a new index for gp to assess the treatment needs for such patients. classification is a mode to arrange the disease(s) and or condition(s) in cohorts, to help communicate among the professionals and to study them. classification systems are necessary in order to provide a rational and scientific framework to study the etiology, pathogenesis, and treatment of diseases and to plan the treatment in an orderly fashion. in addition, such systems help us prioritize and organize the health care needs of the patients. they should have clear categories that make it easy to make a decision as to which category a condition should fit into. an index should be valid possessing, in statistical terms, good sensitivity, and specificity. furthermore, the ideal index should also be reliable and reproducible with no variations as a of internal flaws within the index and give the same if the condition being assessed has not changed. it should also be able to detect small changes and should be able to measure changes in either direction, that is, whether the condition being measured improves or deteriorates. finally, it should be acceptable and free of discomfort for patients or subjects, with the length of time to complete any assessment and examination taken into consideration. oral pigmentation index (dopi):this index of oral pigmentation is the commonly used index due to its simplicity and ease of use. the scores are as follows: no clinical pigmentation (pink - colored gingiva)mild clinical pigmentation (mild light brown color)moderate clinical pigmentation (medium brown or mixed pink and brown color)heavy clinical pigmentation (deep brown or bluish black color) melanin index: this index has classified pigmentation as follows: no pigmentationone or two solitary unit(s) of pigmentation in papillary gingiva without the formation of a continuous ribbon between solitary unitsmore than three units of pigmentation in papillary gingiva without the formation of a continuous ribbonone or more short continuous ribbons of pigmentationone continuous ribbon including the entire area between canines melanin pigmentation index: takashi et al. have proposed another index to measure gingival melanin pigmentation. the index is as follows: score 0: no pigmentationscore 1: solitary unit(s) of pigmentation in papillary gingiva without extension between neighboring solitary unitsscore 2: formation of continuous ribbon extending from neighboring solitary units this index is not equipped to describe the degree of melanin pigmentation.gingival pigmentation index: score 0: absence of pigmentationscore 1: spots of brown to black color or pigments.score 2: brown to black patches but not diffuse pigmentationscore 3: diffuse brown to black pigmentation, marginal, and attached oral pigmentation index (dopi): this index of oral pigmentation is the commonly used index due to its simplicity and ease of use. the scores are as follows: no clinical pigmentation (pink - colored gingiva)mild clinical pigmentation (mild light brown color)moderate clinical pigmentation (medium brown or mixed pink and brown color)heavy clinical pigmentation (deep brown or bluish black color) no clinical pigmentation (pink - colored gingiva) mild clinical pigmentation (mild light brown color) moderate clinical pigmentation (medium brown or mixed pink and brown color) heavy clinical pigmentation (deep brown or bluish black color) this index has classified pigmentation as follows: no pigmentationone or two solitary unit(s) of pigmentation in papillary gingiva without the formation of a continuous ribbon between solitary unitsmore than three units of pigmentation in papillary gingiva without the formation of a continuous ribbonone or more short continuous ribbons of pigmentationone continuous ribbon including the entire area between canines one or two solitary unit(s) of pigmentation in papillary gingiva without the formation of a continuous ribbon between solitary units more than three units of pigmentation in papillary gingiva without the formation of a continuous ribbon one or more short continuous ribbons of pigmentation one continuous ribbon including the entire area between canines melanin pigmentation index: takashi et al. the index is as follows: score 0: no pigmentationscore 1: solitary unit(s) of pigmentation in papillary gingiva without extension between neighboring solitary unitsscore 2: formation of continuous ribbon extending from neighboring solitary units score 0: no pigmentation score 1: solitary unit(s) of pigmentation in papillary gingiva without extension between neighboring solitary units score 2: formation of continuous ribbon extending from neighboring solitary units this index is not equipped to describe the degree of melanin pigmentation. gingival pigmentation index: score 0: absence of pigmentationscore 1: spots of brown to black color or pigments.score 2: brown to black patches but not diffuse pigmentationscore 3: diffuse brown to black pigmentation, marginal, and attached score 0: absence of pigmentation score 1: spots of brown to black color or pigments. score 2: brown to black patches but not diffuse pigmentation score 3: diffuse brown to black pigmentation, marginal, and attached in our view, all the aforementioned indices seem to lack the capacity to relate various aspects of gp. moreover, other gingival - pigmented lesions are beyond their scope, as they were intended only for racial pigmentation. an index quantitatively reflects the clinical state or conditions using set of criteria on a graduated numerical scale whereby one can easily define, describe, distinguish, compare, and analyze the status of an individual or a group with reference to that state and/or conditions. dummett in 1944 noted that some dark complexioned individuals possessed perfectly pink gums devoid of any melanogenous pigmentation. in our view, mild shades of gp may require esthetic treatment in light - skinned individuals but may not have esthetic concern in dark - skinned subjects. in our proposed index, 0 - 3 is the range available to record the gingival color and its variation within physiological limits. a clinician may recommend a depigmentation procedure when the patient scores 1 - 2 of our index and has up to class 2 of liebart and deruelle. smile line classification, which is as follows: class 1: very high smile line - more than 2 mm of the marginal gingiva visibleclass 2: high smile line - between 0 and 2 mm of the marginal gingiva visibleclass 3: average smile line - only gingival embrasures visibleclass 4: low smile line - gingival embrasures and cemento - enamel junction not visible. class 1: very high smile line - more than 2 mm of the marginal gingiva visible class 2: high smile line - between 0 and 2 mm of the marginal gingiva visible class 3: average smile line - only gingival embrasures visible class 4: low smile line - gingival embrasures and cemento - enamel junction not visible. the classification of gp and the proposed new index should provide a workable framework upon which future studies can be performed to develop effective managements for this complex group of diseases. it is expected that as our knowledge progresses; future revisions to the classification system will be needed. , the present proposed index represents the most comprehensive of all the indices that evaluate the gp and other pigmented lesions. it takes into account the esthetic aspects, that is, the presence of melanin pigmentation in the esthetic anterior smile region and the severity of gp; it also considers the etiology of various other pigmented lesions in the gingiva. hence, we believe that it will be a useful tool for both clinicians as well as periodontal epidemiologists. due to clarity and simplicity of the proposed index, this classification can be applied even by nave professionals. the broad implementation of this classification and index may facilitate the comparison of gp across the globe and help esthetic management of such presentations. the baseline data obtained by using the parameters of our index can help in studying the prevalence of gp and pigmented lesions.

cosmetic expectations have increased with time and current trends speak volumes about gingival esthetics and smile designing. gingival pigmentation especially on the labial aspect of anterior teeth has become an important component of general esthetics. various physiologic and pathologic factors cause gingival pigmentation. the existing indices do not deal with the etiology, extent and severity of gingival pigmentation. hence, we propose a new classification and index for gingival pigmentation to assess the treatment needs for the patient.

a 48-year - old woman visited our pain clinic for 2-year episodes of the right hand and wrist pain with a pain score of 9 - 10/10 by vnrs (verbal numeric rating scale ; 0 with no pain, 10 with maximal pain). each time she had been diagnosed with different conditions, such as rheumatic arthritis, raynaud's phenomenon, and other musculoskeletal diseases. they prescribed painkillers such as acetaminophen, tramadol, pregabalin and even alprazolam along with various physical therapies, but her pain became worse so that she could not even do her house work. the pain started with a sudden onset of " burning " as she described it. her vital signs including blood pressure were normal and she had no co - existing diseases such as hypertension or diabetes. electromyography and nerve conduction studies of both upper extremities were normal and showed no evidence of peripheral neuropathy. infrared thermography studies were performed to assist in understanding the patient's disorder. in the ing images, the affected areas of her upper extremities appeared red, indicating a higher temperature, while other areas appeared yellow or green, indicating a lower temperature; this visualization also revealed vascular markings in the affected hand. the difference in temperature for the region of interest (roi) was 2.35 (35.37 vs 33.02) at the most differentiable area in the third and fourth fingers (fig . laboratory tests were performed including cbc ( complete blood count) with differential and platelet counts. initial red blood cell count was 4.84 10/l; white blood cell (wbc) count was 8.3 10/l; hemoglobin level was 13.8 g / dl; hematocrit was 41.7%, and platelet count was 1,131,000/mm. prothrombin time (pt) and partial thromboplastin time (aptt) were 11.3 seconds and 31.2 seconds, respectively. erythrocyte sedimentation rate (esr), rheumatoid factor (rf), and antinuclear antibody (ana) screening data were within the normal range. we treated her with 500 mg of aspirin per day, and transferred her to a hematologist for further evaluation. she was diagnosed with essential thrombocytosis after a bone marrow biopsy and aspiration study, and chemotherapy was started with anagrelide while continuing to administer a low dose of aspirin (100 mg per day). a month later, her platelet count measured 340,000/mm, and her symptoms dramatically disappeared with a pain score of 1/10 (vnrs). the temperature had decreased in the affected areas, where the color was now yellow; the color boundary between the affected area and the remaining areas was negligible, and the temperature was similar to that of the unaffected arm. primary erythromelalgia, also known as weir mitchell's disease, is an autosomal dominant disorder, and it has recently been accepted as a channelopathy caused by mutations in the voltage - gated sodium channel -subunit nav 1.7 encoding gene (scn9a), hich is selectively expressed within the nociceptive dorsal root ganglion and sympathetic ganglion neurons. primary erythromelalgia is the first human disorder that may serve as a model of the association between an ion channelopathy and chronic neuropathic pain. secondary erythromelalgia can from a number of diseases such as myeloproliferative disorders (i.e. pv, et), hypercholesterolemia, autoimmune disorder, small fiber peripheral neuropathy, fabry's disease, mercury poisoning, mushroom poisoning, sciatica and some medications including bromocriptine, verapamil and ticlopidine. diagnosis is mostly based on the clinical picture because there is no confirmatory diagnostic test. the classic description of erythromelalgia is red, painful, warm hands or feet brought on by warming or hanging the limb downward, and relieved with cooling and elevation of the affected area. secondary erythromelalgia often predate myeloproliferative disorders by a median of 2.5 years, thus a blood test should be serially performed once erythromelalgia is diagnosed. abnormal cbc data such as elevated platelets confirms secondary erythromelalgia. most patients are subjected to polypharmacy in an attempt to manage pain if it is so severe that it interferes with daily living. medications such as opioids, gabapentin, lidocaine patches, benzodiazepines and nonsteroidal anti - inflammatory drugs (nsaids) are used frequently; however, in most cases, these treatments either have no proven efficacy or provide only limited relief. various nerve blocks including epidural blocks and sympathetic blockades have been introduced to treat erythromelalgia unresponsive to non - invasive therapies, ing in considerable efficacy, but no treatment is consistently effective in the management of erythromelalgic patients. for secondary erythromelalgia, treatment of the underlying disorder secondary erythromelelgia is prevalent in 3% to 65% of patients with myeloproliferative disorders, especially polycythemia vera and essential thrombocytosis.. pathological signs presenting with secondary erythromelalgia linked to thrombocythemia include arteriolar intimal proliferation with thrombotic occlusions secondary to platelet aggravation. some researchers suggest that both hypoxia and hyperemia occur in the disease process with high levels of arteriovenous shunting in the extremities. secondary erythromelalgia associated with myeloproliferative disease can be dramatically alleviated with high - dose aspirin therapy. aspirin prevents platelet aggravation through an irreversible inhibition of cyclooxygenase; the effect of a single dose of 500 mg aspirin usually lasts for about three days. this specific long - lasting effect of a single dose of aspirin is pathognomonic and can be used as a diagnostic test in secondary erythromelalgia linked to myeloproliferative disease. a regular low dose of aspirin can also treat erythromelalgia and be a safe method to prevent erythromelalgia. infrared thermography can be used in the diagnosis and assessment of therapeutic for erythromelalgia. thermography of an erythromelalgic patient may reveal increased temperature in the affected area and the thermal changes can be linked to the relief of symptoms as in our present case. thompson et al. had reported thermographic findings for a case of primary erythromelalgia with increased skin temperature in the affected area. primary erythromelalgia often presents as bilateral and symmetric erythro - hyperthermic congestion in the extremities, while secondary aspirin - responsive erythromelalgia linked to myeloproliferative disease has unilateral or bilateral effects but in an asymmetric area. in our case, the thermography showed a hyperthermal area specifying vascular markings in the unilateral upper extremity, which may suggest an infective or inflammatory disease like vasculitis. however, we could rule out vasculitis or phlebitis due to her normal range in inflammation markers such as her wbc counts and esr. erythromelalgia can be confused with complex regional pain syndrome (crps) because crps may also present severe burning pain and/or erythema with thermal change which can be present in thermography. however, these symptoms in patients with crps are often unilateral and can be proximal while those in erythromelalgic patients are primarily distal and symmetric if not associated with myeloproliferative diseases. in thermography , crps can have increased or decreased thermal areas while the affected area of erythromelalgia is usually increased in temperature. in addition, symptoms triggered by warming of the affected area and relieved by cooling and elevation are less common with crps. in this case , we saw a typical case of secondary erythromelalgia associated with thrombocythemia that was treated successfully with aspirin. in , pain clinicians should suspect erythromelalgia when seeing a patient with a triad of redness, pain and elevated temperature in the extremities. infrared thermography can help in diagnosing and understanding the disease course of erythromelalgia by presenting the affected area. the symptom of secondary erythromelalgia with myeloproliferative disorders, such as essential thrombocytosis can be alleviated with aspirin as in this case.

erythromelalgia is a rare neurovascular pain syndrome characterized by a triad of redness, increased temperature, and burning pain primarily in the extremities. erythromelalgia can present as a primary or secondary form, and secondary erythromelalgia associated with a myeloproliferative disease such as essential thrombocythemia often responds dramatically to aspirin therapy, as in the present case. herein, we describe a typical case of a 48-year - old woman with secondary erythromelalgia linked to essential thrombocythemia in the unilateral hand. as this case demonstrates, detecting and visualizing the hyperthermal area through infrared thermography of an erythromelalgic patient can assist in diagnosing the patient, assessing the therapeutic , and understanding the disease course of erythromelalgia.

possible causes of mechanical prosthetic valve dysfunctions include obstruction due to valvular thrombosis or pannus formation or regurgitation of valvular or paravalvular origin. in case of regurgitation, major concern is paravalvular leakage, because mechanical prosthetic valves are proven to be durable and valvular regurgitation due to structural valve deterioration is extremely rare. we report a sudden leaflet dislocation of a edward - duromedics mitral valve (baxter healthcare corp ., cleveland, ms, usa) corrected by the prompt diagnosis followed by an emergency operation. a 72-year - old woman visited emergency department as she suddenly experienced chest pain with resting dyspnea while washing dishes at home. she underwent mitral valve replacement 27 years ago, at the age of 45, due to mitral stenosis. mitral valve replacement was done with edwards - duromedics 29 mm mitral valve (baxter healthcare corp ., apart from valvular heart disease, the patient was on medication for diabetes mellitus and hypertension as well as paroxysmal atrial fibrillation . she remained to be the status of nyha functional class ii and anticoagulation with coumadin was appropriately done ( international normalized ratio 2 - 2.5). the recent transthoracic echocardiography (tte) done 4 month ago confirmed normal left ventricular systolic function and well functioning of prosthetic mitral valve with mild pulmonary hypertension and mild tricuspid regurgitation. on physical examination, her vital signs were as follows; blood pressure 69/51 mmhg, pulse rate 130/min, body temperature 36.7 with respiratory rate 35 breath / min. 1 ). complete blood count showed white blood cell count of 9820/l, hemoglobin of 9.8 g / dl, and platelet of 386000/l. other labs showed elevated creatinine (1.12 mg / dl, estimated glomerular filtration rate 50 ml / min/1.73) and slightly elevated cardiac enzymes (creatine kinase 555 iu / l, creatine kinase - mb 22.29 iu / l, troponin t 0.016 ng / ml) with elevated n - terminal prohormone of brain natriuretic peptide level of 832 pg / ml. arterial blood gas analysis showed oxygen pressure of 97 mmhg, carbon dioxide pressure of 27.4 mmhg, with ph of 7.353 with oxygen mask of 10 l. after central line through right jugular vein was inserted, central venous pressure was 1 mmhg. the patient was intubated as she became intolerably tachypneic and progressively hypoxic. to evaluate the cause of the shock and acute decompensated heart failure, bed side tte was performed and revealed normal sized left ventricle (end diastolic dimension : 44 mm) and hyperdynamic left ventricular systolic function (ef : 80%) without regional wall motion abnormality. moderate tricuspid regurgitation (grade iii) and severe pulmonary hypertension (right ventricular systolic pressure 75 mmhg) with plethora of inferior vena cava were demonstrated. on two - dimensional (2d) echocardiogram , there was suspicious finding of single prosthetic mitral leaflet, but details of the mitral leaflet morphology and color doppler were not sufficient for evaluating the prosthetic mitral valve function due to tachycardia and poor echo window. however, elevated mean diastolic pressure gradient (10 mmhg) across the prosthetic mitral valve without prolongation of pressure half time (54 ms) and low velocity of mitral regurgitation (mr, 4 m / s), and rapid declined in mr velocity suggested existence of severe mr (fig . the diagnosis of acute severe mr due to escape of prosthetic valve leaflet with embolization was made and the patient immediately went through emergency operation to surgically correct dysfunctions of previously replaced mitral valve . when mitral valve was exposed, there was one leaflet missing without evidence of paravalvular dehiscence and pannus or thrombus formation ( fig . tte after the surgery demonstrated well functioning bioprosthetic mitral valve with decreased tricuspid regurgitation ( grade i) and resolution of pulmonary hypertension (rvsp 39 mmhg). in an attempt to localize the missing leaflet, computed tomography (ct) was done, and a plate like metallic density in infra - renal abdominal aorta was noted (fig . the remaining fragment of leaflet in infra renal abdominal artery was removed 11 days after bioprosthetic valve replacement . the abdominal aorta was vertically dissected 7 cm, and the fragment was safely removed ( fig . the patient developed mild fever after the surgery but recovered well and was discharged 37 days after the surgery . leaflet escape of bi - leaflet mechanical prosthesis has been reported ( tekna and duromedics) to be extremely rare.1 - 5 ) the leaflet escape is reported to happen more frequently in mitral than aortic positions. a few factors have been reported to account for material deterioration.6 ) the cavitation, which is the rapid formation of vaporous microbubbles in a fluid by a local reduction of pressure below the vapor pressure7 ) is recognized as the most contributing factor to the series of valve failure in edward - duromedics prosthesis. other factors identified are asymmetric closure with local stresses, inadequate compliance of calcified sewing ring, clustered microporosity of the pyrolytic carbon and surgical mishandling.6 ) the time of leaflet escape varies from 19 days6 ) to 12 years8 ) after the implantation of the mitral valve. the clinical presentation is usually acute pulmonary edema with cardiogenic shock as the of acute valvular incompetence.1 - 5 ) other causes of the clinical symptoms, such as myocardiac infarction, para - valvular leak, thrombosis of the prosthetic valves, malignant arrhythmia and pulmonary embolism should be considered. tte is usually not helpful for the diagnosed as it may be mis - interpretated as obstructed closure of the prosthetic valve, paravalvular leak or thrombosis.9 ) transesophageal echocardiography (tee) is diagnostic most of times. cineflouroscopy may role as non invasive diagnostic tool to determine leaflet escape from valve thrombosis.9)10 ) it is acknowledged that timely diagnosis and emergent surgical replacement of the prosthetic valve is most important.11 ) there are a few cases of mitral leaflet escape reported in korea.12 - 14 ) as a patient deteriorates with symptoms of acute heart failure with unstable vital signs, in most of the reported cases, an emergent operation is performed with tte finding of acute valvular dysfunction. therefore, an exact diagnosis of leaflet escape is made during the surgery, except there was a case reported by kim et al.13 ) which the diagnosis of a leaflet escape was made before an emergency operation by using fluoroscopy. in our case, although the images of tte were not sufficient for evaluating the exact mitral valve morphology and function, a single mitral leaflet was suspicious on 2d echocardiogram. in addition to the ambigious 2d images of single mitral leaflet, elevated mean diastolic pressure gradient with low velocity of mitral regurgitation, we could make diagnosis of acute severe mr by comprehensive interpretation of tte without performing tee. the location of the missing leaflet can be difficult to identify in case the leaflet embolized to distal aorta or its branches. plain x - rays are not helpful because of lack of radio - opacity of the prosthetic valves. removing dislocated leaflet is recommended as it may cause arterial wall damage leading to erosions, infections, and further migrations. this case is notable that the patient who presented with severe cardiogenic shock after the prosthetic valve implanted 27 years ago suddenly dislodged, recovered from the debilitating condition owing to the prompt diagnosis based on tte and immediate surgical correction. although rare, when a patient with previous history of prosthetic valve replacement presents with symptoms of acute decompensated heart failure, possibility of leaflet and escape of the valve leaflet should be contemplated. in cases of the leaflet escape, the urgent diagnosis and emergent surgical replacement is mandatory to prevent the mortality.

leaflet escape of prosthetic valve is rare but potentially life threatening. it is essential to make timely diagnosis in order to avoid mortality. transesophageal echocardiography and cinefluoroscopy is usually diagnostic and the location of the missing leaflet can be identified by computed tomography (ct). emergent surgical correction is mandatory. we report a case of fractured escape of edward - duromedics mitral valve 27 years after the surgery. the patient presented with symptoms of acute decompensated heart failure and cardiogenic shock. she was instantly intubated and mechanically ventilated. after prompt evaluation including transthoracic echocardiography and ct, the escape of the leaflet was confirmed. the patient underwent emergent surgery for replacement of the damaged prosthetic valves immediately. eleven days after the surgery, the dislodged leaflet in iliac artery was removed safely and the patient recovered well.

dorzolamide hydrochloride (drz) was synthesized in 1980 and it was the first carbonic anhydrase inhibitor approved for the topical treatment of glaucoma in humans in 1995. the drug reduces the production of bicarbonate ions, thereby reduces secretions and lowers intraocular pressure. elevated iop may lead to glaucoma in patients with ocular hypertension. nowadays due to their higher efficiency and lower systemic adverse reactions, ophthalmic preparations are considered to be the first choice in glaucoma treatment. one of the most important problems of the ophthalmic drops is their short ocular residence time. the drug is quickly diluted by tears and is removed through nasolachrymal ducts. therefore, it is beneficial to design a biocompatible ocular drug delivery system that can increase the residence time of the drug in the eye. liposomes are self - assembled colloidal particles; consist of lipid bilayers surrounding an aqueous compartment. due to their cell - like nature , liposomes are able to attach to cellular tissues of the body. today, nanotechnology provides smaller dimension of liposomes; under the name of nanoliposomes. drug - loaded nanoliposomes can maintain a relatively constant drug concentration, increase the drug residence time in the eye and consequently enhance therapeutic efficiency; therefore, lower doses of the drug with less frequency can be applied. the objective of this study is to develop ocular drz nanoliposomes and evaluate their potential use for the treatment of ocular hypertension. dorzolamide hcl supplied by baselux (s.a - switzerland), soy phosphatidylcholine (phospholipon 85 g) purchased from lipoid (germany) and cholesterol supplied by sigma (germany) were used for the preparation of formulations. nanoliposomes reverse - phase evaporation vesicle (rev) method: the lipid components, with 7:3 and 7:4 molar ratio of soy phosphatidylcholine (spc): cholesterol (ch), were dissolved in chloroform - methanol solution (2:1 v / v). the organic solvent was evaporated at 65 c under reduced pressure at 150 rpm using a rotary evaporator (ev311, lab - tech, germany) and the thin lipid film obtained was maintained at 4 c for 24 hours to ensure complete removal of solvents. the film was dissolved in diethyl ether, followed by addition of 10 ml phosphate buffer (ph 5.8) containing 2% (w / v) drz. the organic solvent was removed at 37 c and the dispersion was sonicated for 30 min using a bath sonicator (t710, elma, germany). to ensure full lipid hydration and maturation,. thin layer hydration (tlh) method: in this method the thin lipid film obtained by previously described conditions was directly hydrated with phosphate buffer (ph 5.8) containing drz (2% w / v), sonicated and kept at 4 c overnight for maturation. nanoliposomes in order to determine the encapsulation efficiency (ee%) of drz nanoliposomes, the formulation was transferred into centrifugal filter device (centritrep-50 kda), and centrifuged at 3000 rpm and 25 c1 for 30 min (clements 2000, germany). then, the supernatant was analyzed for free drz using uv spectrophotometer (biochorm, england) at 254 nm. ee% was calculated as follows: ee%=total drug - free drugtotal drug100 the following parameters were measured: the mean particle size, polydispersity index (pdi) and zeta potential of the nanoliposomes by nanosizer (nano zs, malvern, uk), viscosity by brookfield viscometer (model dv - ii+pro, middleboro, usa) at 100 rpm, surface tension by the de nouy ring method (csc scientific company, usa), ph value, and refractive index (ri) at 251 c. in - vitro drug release study nanoliposomal samples enclosed in dialysis bags (cellulose membrane mw cut- off 12 kda, sigma) were immersed in 250 ml phosphate buffer (ph 7.4) at 251 c and were stirred at 100 rpm. at predetermined time intervals, samples were withdrawn and analyzed for drz spectrophotometrically at 254 nm. drz solution (2% w / v) ex - vivo drug permeation study the experiment was carried out using franz diffusion cells designed for ocular permeation studies with receiver medium of 10.5 ml phosphate buffer (ph 7.4) at 351 c. albino rabbits were sacrificed by iv injection of sodium phenobarbital and the whole eyes were enucleated. samples (1 ml) were withdrawn at specific time intervals and assayed spectrophotometrically at 254 nm. the permeation behavior of free drug as a control experiment was determined using drz solution (2% w / v). the steady - state flux (jss) of different formulations was determined by the slope of the linear portion of the plots of the amount of drug in the receiving chamber versus time, divided by exposed corneal surface area and the lag time that was estimated from the x - intercept of the linear portion of the graph. apparent corneal permeability coefficient (papp) was calculated according to the following equation: cd represents the drug concentration in the donor compartment. in - vivo iop measurement the animal experiments were conducted in full compliance with regulatory principles of ethics committee of ahvaz jundishapur university of medical sciences. albino rabbits (2.53 kg) were housed at controlled temperature (252 c), and humidity (605%), with a 12/12-h light - dark cycle. nanoliposome, group ii was treated with drz solution and group iii received biosopt ophthalmic drop (bakhtar biochemie co, iran). one drop (0.05 ml) each of drz nanoliposome, drz solution or biosopt drop was instilled into the right eye, while the non - treated eye was considered as control. iop of both eyes was determined using iopen tonometer for 8 h. the mean of three consecutive tonometric measurements was calculated for each animal. rev and tlh nanoliposomes with lipid molar ratio of 7:4 were stored at 4 c; their particle size and ee% were monitored after 1 and 3 months. statistical analysis all studies were carried out in triplicate and data were reported as a mean sd. one - way anova and multiple comparison tukey s test were used to assess the significance of the differences between the various groups, p<0.05 was considered statistically significant. nanoliposomes the data showed that nanoliposomal formulations with lipid ratio of 7:4 (spc : ch) produced significantly higher ee% than those with lipid ratio of 7:3 (spc : ch) (p<0.05) (table 1). particle size showed that higher proportion of cholesterol in lipid ratio led to form larger particles (p<0.05) but they were still below 100 nm and in acceptable range (table 1). comparing the of formulations with same molar ratio, revealed that nanoliposomes prepared by tlh method had significantly smaller particle size than those made by rev method (p<0.05). values close to zero indicate a homogeneous dispersion while those greater than 0.3 indicate high heterogeneity. pdi of all formulations in this study were approximately 0.3 which indicates acceptable uniformity and homogeneity of the formulations (p>0.05). nanoliposomal formulations 7:4 (spc : ch) rev and 7:4 (spc : ch) tlh were positively charged with zeta potential value of + 12.8 and + 10.3 mv, respectively. the tonicity of the formulations was equivalent to that of a 0.875% sodium chloride solution. particle size, polydispersity index and encapsulation efficiency of drz - nanoliposomes (mean sd, n=3) the of viscosity, surface tension, ph and ri values are presented in table 2. the showed that method of preparation and lipid molar ratio did not influence the viscosity value of nanoliposomes (p>0.05). the surface tension of the formulations in decreasing order is: biosopt > rev prepared drz nanoliposomes > tlh prepared drz nanoliposomes (p<0.05). the ph value measured for biosopt was 5.2 which is significantly lower than that of drz nanoliposomes (p<0.05). the ri values of drz nanoliposomes were 1.343 which is close to that of tears. viscosity, surface tension, ph and refractive index of formulations (mean sd, n=3) in - vitro drug release this study was carried out only with 7:4 (spc : ch) rev and 7:4 (spc : ch) tlh drz nanoliposomes which had good in - vitro properties especially regarding ee%. all of the experiments were performed without separation of entrapped and non - entrapped drz. the release profiles of drz from drz nanoliposomes and drz solution shown in figure 1 are almost the same. a burst effect of drug release observed during the first hour (about 70%) that followed by nearly complete release (over 95%) after 6 hours. release profile of drz from drz nanoliposomes and drz solution (means.d, n=3) ex - vivo drug permeability after 8 and 24 h, corneal permeability of drz for 7:4 (spc : ch) rev was 4.5 and 11%, for 7:4 (spc : ch) tlh was 6 and 14.5%, and for drz solution was 1 and 3%, respectively (figure 2). values of drz nanoliposomes were significantly higher than those of drz solution (p<0.05). significant (p<0.05) differences were observed between permeability of 7:4 (spc : ch) rev and 7:4 (spc : ch) tlh in favor of tlh prepared drz nanoliposomes. transcorneal permeation profile of drz from drz nanoliposomes and drz solution (means.d, n=3 papp, jss and lag time values are shown in table 3 . however, the permeability parameters ( papp and jss) of 7:4 (spc : ch) rev and 7:4 (spc : ch) tlh were not significantly different (p>0.05). a lag time of about 40 min was observed for drz nanoliposomes, whereas drz solution permeated through cornea without any lag time. apparent permeability coefficient, steady - state flux and lag time of formulations from ex- vivo permeation studies (mean sd, n=3 the drug leakage after 1 and 3 months storage at 4 c were found to be 1.12% and 2.86% from 7:4 ( spc : ch) rev drz nanoliposomes and 0.2% and 1.64% from 7:4 (spc : ch) tlh, respectively. the particle size of drz nanoliposomes showed no statistically significant changes over 3 months storage at 4 c (p>0.05). considering the higher permeability and the smaller particle size of 7:4 (spc : ch) tlh prepared drz nanoliposomes as compared to rev prepared ones, intraocular pressure (iop) figure 3 shows that the iop lowering activity of marketed product and drz solution within 1 h after instillation reached maximum values of 2.25 mmhg and 1.5 mmhg, respectively, but this effect quickly decreased and disappeared for both products. the maximum iop decreased value was 4.42 mmhg at the 4 hour, which was significantly higher than those of drz solution and the marketed product (p<0.05). macroscopic examination did not reveal any inflammation, redness or irritability in the rabbits eyes during the procedure. iop reduction after administration of drz nanoliposomes, drz solution and marketed product (biosopt). the fine size of nanoliposomal ophthalmic preparations tends to cause less abrasion, irritation and sensitivity. in this study, other studies revealed that the liposomes with small size and single layer such as suv, adhere more strongly to tissue and perform a better drug delivery. calculated tonicity of the formulations was nearly close to isotonic solution which is important for formulations intended for ophthalmic use. marketed drz eye drops are in a solution form, the drug is absolutely freely soluble and becomes diluted with tears and is readily discharged from the eyes. hence, viscosity enhancers such as cellulose derivatives are mostly used in marketed products to increase consistency and improve adherence in the eye. but, it should be considered that high consistency can cause problems in the movement of the eyeball, the process of optical refraction and creation of the image on the retina. the viscosity of an ophthalmic formulation should be in the range of 1550 cps. in this study, formulation 7:4 (spc : ch) tlh prepared drz nanoliposomes had a viscosity equal to 46 cps, which is in the acceptable range; while the marketed product (biosopt) had a significantly higher viscosity equal to 96 cps as measured in our lab.(p < 0.05). surface tension of drz nanoliposomes was lower than that of biosopt which is attributed to the surface activity of phospholipid amphiphilic molecules. the lower surface tension, the better spreading of the product on the cornea and the more contact between them. but, eye drops with much lower surface tension than lachrymal fluid (0.04 to 0.05 n / m) may destabilize the tear film and cause visual problems. drz has maximum stability in the range of ph 4 - 6. it should be taken into consideration that a shift from neutral ph to either acidic or basic may cause eye irritation. it is recommended that eye drops should have ri close to tears fluid (1.34 to 1.36). drz nanoliposomes in our study, had ri in the acceptable range (table 2). as cholesterol settles between phosphatidylcholine molecules, it fills empty spaces, increases rigidity of the bilayer films and prevents leakage of the drug. also, it stabilizes nanoliposomes against external pressure such as extreme shaking. in this study , the release of the drug from the drz nanoliposomes did not show a significant difference compared with the drug release from drz solution. the high - rate of nanoliposomes drug release during the first hours may be due to the presence of available free drug in the formulations and the high concentration gradient of drz between the contents in the dialysis bag and the large - volume (250 ml) receiver phase. although drz nanoliposomes and drz solution exhibited similar in - vitro drug release, drz this can be explained in terms of the conditions under which the ex - vivo study was performed. this is more similar to in - vivo conditions in relation to the receiver medium volume (10.5 ml). it is speculated that the high rate of drug permeability of nanoliposomes is a of high lipophilicity of the vehicle, which leads to increase in residence time on the surface of the cornea. the nanoliposomes can carry hydrophilic drugs (such as drz) in their cores and then attach to the surface of the corneal epithelium and gradually release the drug. other possible mechanisms such as fusion of liposome membrane with the cornea or endocytosis of liposomes may lead to the drug release in the anterior eye space after passing through the cornea. in addition, the lag time observed in ex - vivo permeation studies of drz nanoliposomes may support this hypothesis. nanoliposomes surface had a positive charge because of the cationic nature of drz at ph below 6.4. so, this would lead to their adhesion to polyanionic surface of cornea and conjunctiva and improvement in corneal retention and penetration. the greater permeability of formulation 7:4 (spc : ch) tlh as compared to 7:4 (spc : ch) rev can be attributed to their smaller particle size. it was also observed that drz nanoliposomes in contrast with drz solution and marketed product induced a remarkable decrease in iop and with respect to the duration of action, the effect of nanoliposomes was prominent. they investigated liposomal acetazolamide and concluded that acetazolamide liposomes composed of egg phosphatidylcholine: cholesterol: stearylamine (7:4:1) molar ratio prepared by a method almost similar to tlh method used in our study, revealed more prolonged iop - lowering effect compared to the solution form of the drug. in their in - vivo study on liposomal diltiazem as ocular drug delivery system for glaucoma, mokhtar ibrahim et al. , proved the system enhanced iop - reducing activity as compared to the solution form at equal concentration. hironaka et al. also investigated the uptake of carboxyfluorescein (cf) into conjuctival cells via liposomal eye drops and concluded that the amount of cf incorporated into cells increased when using liposome as a carrier; and they proposed that the increase in drug uptake was due to the affinity of phospholipid bilayer to the cell membrane that led to effective delivery of hydrophilic drug to the retina. the higher permeability coefficient and flux of drz nanoliposomes versus solution form provide a good explanation for this higher effectiveness and prolonged therapeutic effect. nanoliposomes natural structure, their positive surface charge and small vesicle size contribute to their adhesion to the corneal membrane which leads to improved clinical effect. of the two methods that were applied to prepare nanoliposomes systems, tlh method is simpler, faster, cost effective and has less toxic solvent residue than rev method. in this study, formulation 7:4 (spc : ch) tlh was selected because of its small particle size, high ee% and high corneal permeability. the characteristics of this formulation in terms of surface tension, viscosity, ph, ri and safety were all in the acceptable range required for ophthalmic preparations. the formulation was stable throughout 3-months period of storage and revealed longer and higher iop - lowering activity in comparison with drz solution and marketed eye drop. the prolonged effect may be explained in term of gradual drug release from nanoliposomes which are depot in the cornea, suspended in aqueous humor or attached to the surface of the anterior space. we recommend the addition of an antioxidant to prevent lipid oxidation and prepare a stable formulation for longer period of time. however, assay of drz in aqueous humor following drug instillation into rabbit eye should be done before clinical trials as well.

dorzolamide ophthalmic drop is one of the most common glaucoma medications but it has a short residence time in the eye. the aim of this study is to develop ocular dorzolamide hcl nanoliposomes (drz nanoliposomes) and to evaluate their potential use for the treatment of ocular hypertension. nanoliposomes were prepared using reverse - phase evaporation vesicle (rev) and thin layer hydration (tlh) method with 7:3 and 7:4 molar ratios of phosphatidylcholine: cholesterol. the physicochemical properties of the formulations were investigated. formulations with 7:4 lipid ratios were evaluated in terms of drug release, physical stability and ex - vivo permeation through the excised albino rabbit cornea. the rabbits in groups of 6 were treated with selected drz nanoliposomes or dorzolamide solution or marketed dorzolamid preparation (biosopt) and intraocular pressure (iop) was monitored. formulations with 7:4 molar ratio entrapped greater amount of drug compared to those with 7:3 lipid components ratio. drz nanoliposomes with 7:4 lipid ratio showed more transcorneal permeation than dorzolamide solution (p<0.05); and the formulation prepared by tlh method exhibited higher permeability than that prepared by rev method (p<0.05). the selected drz nanoliposomes showed greater iop lowering activity and a more prolonged effect compared to dorzolamide solution and biosopt. drz nanoliposomes prepared by tlh method with 7:4 ratios showed promising as a candidate for the treatment of ocular hypertension.

blockers, as one of secondary prevention medications, can diminish myocardial oxygen demand by reducing heart rate, blood pressure, and myocardial contractility, thereby being widely used to relieve ischemic symptoms in patients with acute coronary syndrome (acs).1 the updated american college of cardiology / american heart association (acc / aha) guidelines recommend the use of blockers for the management of stsegment elevation myocardial infarction (stemi)2 and non stsegment elevation myocardial infarction (nstemi).3 however, evidence supporting the clinical benefit of blockers is largely based on studies in patients with acute mi for stemi, and was extrapolated to patients with unstable angina pectoris (uap) and nstemi.4 in the percutaneous coronary intervention (pci) era, patients with acs, a spectrum of clinical presentations ranging from uap to nstemi and stemi, mostly constituted those undergoing pci.5 however, few studies are available to systematically describe the contemporary pattern of blocker use and determine its impact on clinical outcomes in acs patients after pci. as shachamet et al6 pointed out, many physicians remain unconvinced of either a short or longterm benefit of blocker use following pci. moreover, much less attention has been paid to specifying which subgroup of patients with acs benefits the most from blocker therapy. thus, we sought to evaluate the impact of blocker therapy on clinical outcomes in patients with acs after pci and specified subgroups in a all patients diagnosed with coronary heart disease at tongji hospital in wuhan, china, were consecutively recruited in the clinical outcomes of coronary heart diseases in tongji hospital registry from march 1, 2009. demographics, clinical profiles, and concomitant medications were collected with standardized case report forms by professional investigators in the department of cardiology, and all participants were prospectively contacted at 1, 6, and 12 months by cardiology nurses and research coordinators through patient interview, chart review, and serial telephone contacts. written informed consent was obtained from each patient at admission. between march 1, 2009, and december 30, 2014, all patients in the database were searched. for inclusion, patients were required to meet the following criteria: age older than 18 years; have an ascertained diagnosis of acs at admission, and undergoing pci. in addition, the following patients were excluded from this analysis: patients discharging unstable, patients with the absence of blocker information at discharge, or contraindication to blocker therapy such as significant bradycardia (heart rate < 50 beat per min) or hypotension (systolic blood pressure < 90 mm hg). this strategy was approved by the ethics committee of tongji medical college and conducted in accordance with the declaration of helsinki and the international conference on harmonization guidelines for good clinical practice. the study was supported by grants from the national program on key basic research project (973 program) (no . 2012cb518004), the national nature science foundation key project (no . 91439203), and the national health and family planning commission of china (no . 201202025, no . 2011bai11b04). for the purpose of calculating the proportion of blocker dose administered (daily dosage of blockers / target dose), the target dose was in line with blocker doses used in large randomized trials, defined as follows: metoprolol 200 mg / d7 carvedilol 50 mg / d,8 timolol 20 mg / d,9 bisoprolol 10 mg / d10 atenolol 100 mg / d,11 and propanolol 180 mg / d.12 in addition, patients who were diagnosed with acs must present with ischemic symptoms within 24 hours and have at least one of the following conditions: electrocardiographic changes consistent with acs, an increase in serum cardiac biomarkers (troponin or creatine kinasemb), or documentation of angina pectoris.13 successful pci was identified as a patent vessel at the treatment site with anterograde thrombolysis in myocardial infarction flow 3 and angiographic residual stenosis < 50%. in the present study, we evaluated two study end points: the primary outcome was allcause mortality, which was regarded as cardiac origin unless obvious noncardiac cause could be identified; and the secondary outcome was a composite end point of allcause death, nonfatal mi, heart failure readmission, and cardiogenic hospitalization. of these, mi referred to symptoms with new electrocardiographic changes (pathologic q waves, persistent stsegment elevation, or stsegment depression) as well as cardiac markers at least one value above the 99th percentile of the upper reference limit.14 the identification of heart failure readmission was consistent with the guidelines of the european society of cardiology.15 in addition, cardiogenic hospitalization was considered as a hospitalization for cardiovascular cause, including uap, transient ischemic attack, or revascularization procedure. we divided patients into two groups with regard to whether blocker therapy was received at discharge. categorical variables were compared using chisquare test and continuous variables were analyzed by means of wilcoxon ranksum test or student t test according to its distribution. characteristics of study participants were further compared with respect to the following: no blocker use, < 50% of target dose, and 50% of target dose. differences among groups were examined in the same way for categorical variables and 1way anova analysis or kruskal wallis rank test if deviated from normality for continuous variables. survival curves were depicted by kaplan meier method and compared with the logrank test. multivariable cox proportional hazard regression was applied to identify the independent factors associated with end points. the variables entered into the multivariate model were age, sex, hypertension, diabetes, dyslipidemia, stroke, prior infarction, recent infarction within 3 weeks, heart failure status (canadian heart class or killip heart class), arrhythmia, and medications at discharge (aspirin, clopidogrel, statins, blocker, angiotensinconverting enzyme inhibitors /angiotensin receptor blockers , nitrates). in addition, clinical factors related to treatment selection may confound the event rates, therefore, we performed propensity score matched analysis to address the issue. to estimate the propensity score, a logistic regression model developed with the variables, including age, sex, hypertension, diabetes, dyslipidemia, stroke, prior infarction, recent infarction within 3 weeks , heart failure status (canadian heart class or killip heart class), arrhythmia, and medications at discharge (aspirin, clopidogrel, statins, aceis / arb, nitrates), was used to predict the use of blockers. patients in the blocker group were 1:1 matched to patients in the no blocker group on the basis of their propensity score and the value of caliper equal to 0.2. absolute standardized differences < 10% for a given covariate indicate a relatively small imbalance. for the propensity score matched cohort, mcnemar test was used for paired categorical variables and paired t test or paired sample wilcoxon rank test for continuous variables, depending on the normality of the variables. the associations of blocker use with clinical outcomes were evaluated by use of cox regression models. spss version 20.0 (ibm corp, armonk, ny) was used for statistical analysis. the power of the study was calculated by pass version 11.0 (ncss, kaysville, ut). all patients diagnosed with coronary heart disease at tongji hospital in wuhan, china, were consecutively recruited in the clinical outcomes of coronary heart diseases in tongji hospital registry from march 1, 2009. demographics, clinical profiles, and concomitant medications were collected with standardized case report forms by professional investigators in the department of cardiology, and all participants were prospectively contacted at 1, 6, and 12 months by cardiology nurses and research coordinators through patient interview, chart review, and serial telephone contacts. written informed consent was obtained from each patient at admission. between march 1, 2009, and december 30, 2014, all patients in the database were searched. for inclusion, patients were required to meet the following criteria: age older than 18 years; have an ascertained diagnosis of acs at admission, and undergoing pci. in addition, the following patients were excluded from this analysis: patients discharging unstable, patients with the absence of blocker information at discharge, or contraindication to blocker therapy such as significant bradycardia (heart rate < 50 beat per min) or hypotension (systolic blood pressure < 90 mm hg). this strategy was approved by the ethics committee of tongji medical college and conducted in accordance with the declaration of helsinki and the international conference on harmonization guidelines for good clinical practice. the study was supported by grants from the national program on key basic research project (973 program) (no . 2012cb518004), the national nature science foundation key project (no . 91439203), and the national health and family planning commission of china (no . 201202025, no . for the purpose of calculating the proportion of blocker dose administered ( daily dosage of blockers / target dose), the target dose was in line with blocker doses used in large randomized trials, defined as follows: metoprolol 200 mg / d7 carvedilol 50 mg / d,8 timolol 20 mg / d,9 bisoprolol 10 mg / d10 atenolol 100 mg / d,11 and propanolol 180 mg / d.12 in addition, patients who were diagnosed with acs must present with ischemic symptoms within 24 hours and have at least one of the following conditions: electrocardiographic changes consistent with acs, an increase in serum cardiac biomarkers (troponin or creatine kinasemb), or documentation of angina pectoris.13 successful pci was identified as a patent vessel at the treatment site with anterograde thrombolysis in myocardial infarction flow 3 and angiographic residual stenosis < 50%. in the present study, we evaluated two study end points: the primary outcome was allcause mortality, which was regarded as cardiac origin unless obvious noncardiac cause could be identified; and the secondary outcome was a composite end point of allcause death, nonfatal mi, heart failure readmission, and cardiogenic hospitalization. of these , mi referred to symptoms with new electrocardiographic changes (pathologic q waves, persistent stsegment elevation, or stsegment depression) as well as cardiac markers at least one value above the 99th percentile of the upper reference limit.14 the identification of heart failure readmission was consistent with the guidelines of the european society of cardiology.15 in addition, cardiogenic hospitalization was considered as a hospitalization for cardiovascular cause, including uap, transient ischemic attack, or revascularization procedure. we divided patients into two groups with regard to whether blocker therapy was received at discharge. categorical variables were compared using chisquare test and continuous variables were analyzed by means of wilcoxon ranksum test or student t test according to its distribution. characteristics of study participants were further compared with respect to the following: no blocker use, < 50% of target dose, and 50% of target dose. differences among groups were examined in the same way for categorical variables and 1way anova analysis or kruskal wallis rank test if deviated from normality for continuous variables. survival curves were depicted by kaplan meier method and compared with the logrank test. multivariable cox proportional hazard regression was applied to identify the independent factors associated with end points. the variables entered into the multivariate model were age, sex, hypertension, diabetes, dyslipidemia, stroke, prior infarction, recent infarction within 3 weeks, heart failure status (canadian heart class or killip heart class), arrhythmia, and medications at discharge (aspirin, clopidogrel, statins, blocker, angiotensinconverting enzyme inhibitors /angiotensin receptor blockers , nitrates). in addition, clinical factors related to treatment selection may confound the event rates, therefore, we performed propensity score matched analysis to address the issue. to estimate the propensity score, a logistic regression model developed with the variables, including age, sex, hypertension, diabetes, dyslipidemia, stroke, prior infarction, recent infarction within 3 weeks , heart failure status (canadian heart class or killip heart class), arrhythmia, and medications at discharge (aspirin, clopidogrel, statins, aceis / arb, nitrates), was used to predict the use of blockers. patients in the blocker group were 1:1 matched to patients in the no blocker group on the basis of their propensity score and the value of caliper equal to 0.2. absolute standardized differences < 10% for a given covariate indicate a relatively small imbalance. for the propensity score matched cohort, mcnemar test was used for paired categorical variables and paired t test or paired sample wilcoxon rank test for continuous variables, depending on the normality of the variables. the associations of blocker use with clinical outcomes were evaluated by use of cox regression models. spss version 20.0 (ibm corp, armonk, ny) was used for statistical analysis. the power of the study was calculated by pass version 11.0 (ncss, kaysville, ut). between march 1, 2009, and december 30, 2014, there were 5063 patients recruited in the database, and only 3453 patients underwent the pci procedure. of these, 23 patients were discharged unstably, 183 were not diagnosed with acs at admission, 43 had a contraindication to blocker use, and 24 could not provide complete information about the administration of blockers at discharge, and were excluded from the analysis. acs indicates acute coronary syndrome; chd, coronary heart disease; pci, percutaneous coronary intervention. in the overall evaluation cohort, 2423 patients (76.2%) were discharged on blockers, while 757 patients (23.8%) were not. compared with blocker users, patients who were not administrated blockers were older (60.7910.39 versus 58.4410.47, p<0.001), had lower diastolic blood pressure (dbp) (79.0113.18 versus 80.5713.18, p=0.005), lower heart rate (74.4015.15 versus 75.1012.42, p=0.009), and were more likely to have arrhythmia (11% versus 7.1%, p=0.001). for concomitant medication use, the prescriptions of aspirin, statins, and aceis / arbs were more common in the blocker group compared with the no blocker group (99.4% versus 98.4% ; 98.6% versus 95.5% ; 81% versus 58.4%). table 1 summarizes the baseline characteristics and other medication management according to the use of blockers at discharge. the differences in the baseline characteristics in the 3 subgroups are also shown in tables 2, 3 through 4. in the propensity score matched model, there was no significant difference in the baseline characteristics between the blocker group and the no blocker group. acei indicates angiotensinconverting enzyme inhibitor; arb, angiotensin receptor blocker; dbp, diastolic blood pressure; mi, myocardial infarction; sbp, systolic blood pressure. acei indicates angiotensinconverting enzyme inhibitor; arb, angiotensin receptor blocker; dbp, diastolic blood pressure; mi, myocardial infarction; sbp, systolic blood pressure; stemi, stsegment elevation myocardial infarction. baseline characteristics in patients with nstemi variables are expressed as meansd or percentage. acei indicates angiotensinconverting enzyme inhibitor; arb, angiotensin receptor blocker; dbp, diastolic blood pressure; mi, myocardial infarction; nstemi, non acei indicates angiotensinconverting enzyme inhibitor; arb, angiotensin receptor blocker; dbp, diastolic blood pressure; mi, myocardial infarction; sbp, systolic blood pressure; uap, unstable angina pectoris. at 1 year after index admission, completed followup information was obtained in 3153 patients (99.2%). a total of 33 patients died of allcause diseases, 14 patients occurred nonfatal mi, 34 patients had heart failure, and 214 patients were readmitted for cardiogenic reasons during followup. blocker therapy was associated with a lower incidence of allcause death (unadjusted hazard ratio , 0.33 ; 95% ci, 0.170.65). after adjusting for confounders, the risk of allcause death remained consistently lower in the blocker group (adjusted hr, 0.38 ; 95% ci, 0.170.83) (table 5). in the propensity score matched cohort, the blocker group still had decreased allcause mortality (hr, 0.27 ; 95% ci, 0.080.97) (table 6). a lower rate of secondary end point was also observed in the blocker users (unadjusted hr, 0.76 ; 95% ci, 0.590.98 , although the statistical difference disappeared after adjustment ( adjusted hr, 0.87 ; 95% ci, 0.661.16). in addition, the associations of blocker use with the rate of nonfatal mi, heart failure readmission, and cardiogenic hospitalization were computed, respectively. the are illustrated in table 5, and figure 2 describes the association between the use of blockers and clinical end points. clinical outcomes and unadjusted / multivariable adjusted hrs during 1year followup the event rate at 1 year was estimated by the kaplan meier method. hf indicates heart failure; hr, hazard ratio; mi, myocardial infarction; nstemi, non stsegment elevation myocardial infarction; stemi, stsegment elevation myocardial infarction; uap, unstable angina pectoris. clinical outcomes and hrs after propensity score matching during 1year followup hf indicates heart failure; mi, myocardial infarction; nstemi, non stsegment elevation myocardial infarction; stemi, stsegment elevation myocardial infarction; uap, unstable angina pectoris. the hazard ratio (hr) and 95% ci could not be evaluated that no event occurred in the blocker group. the hazard curves for the primary and secondary end points in the overall population (a), in the patients with stsegment elevation myocardial infarction (stemi) (b), in the patients with non stsegment elevation myocardial infarction (nstemi) (c), and in the patients with unstable angina pectoris (uap) (d). the curves were described by kaplan meier methods and the p values were calculated using the logrank tests. at baseline, 728 patients (22.9%) had stemi, 576 patients (18.1%) had nstemi, and 1876 patients (59.0%) had uap. notably, a greater benefit of blocker use was found in patients with nstemi whose incidence of allcause death was significantly lower in the blocker group (0.2% versus 6.4% ; unadjusted hr, 0.04 ; 95% ci, 0.000.27), and the relationship remained even after performing multivariable cox proportional hazard regression analysis (adjusted hr, 0.00 ; 95% ci, 0.000.14). in addition, blocker use was associated with a lower risk of the secondary end point (7.8% versus 15.7% ; unadjusted hr, 0.47 ; 95% ci, 0.280.81), but no statistical difference was observed after adjustment (adjusted hr, 0.65 ; 95% ci, 0.351.21). in the patients with stemi and uap, however, there was no statistical difference between the two groups for allcause mortality (1.1% versus 1.9% ; adjusted hr, 0.40 ; 95% ci, 0.081.94 in patients with stemi and 0.7% versus 0.9% ; adjusted hr, 0.96 ; 95% ci, 0.293.10 in patients with uap) and the secondary end point (8.5% versus 16.1% ; adjusted hr, 1.13 ; 95% ci, 0.592.16 in patients with stemi and 9.0% versus 9.9% ; adjusted hr, 0.97 ; 95% ci, 0.661.41 in patients with uap ( table 5 and figure 2). the associations of blocker therapy with the clinical outcomes across the 3 subgroups were consistent in the propensity score matched cohorts (table 6). among the patients discharged on blockers, receiving < 50% of target dose was reported in 2012 patients (83.0%), while 411 patients (17%) were prescribed 50% of target dose and the administration of metoprolol accounted for the majority (85.4%). the baseline characteristics according to the treatment of blocker use are exhibited in table 1, 2, 3 through 4. for overall patients, allcause mortality was 0.7% in < 50% of target dose group, significantly lower than in the no blocker group (0.7% versus 2.1% ; adjusted hr, 0.40 ; 95% ci, 0.190.82), while the rate of allcause death was not different between 50% of target blocker dose group and no blocker group (0.7% versus 2.1% ; adjusted hr, 0.46 ; 95% ci, 0.131.59) (figure 3), and no differences were observed in the incidence of secondary end point between the three different blocker dose groups. entire cohort and subgroup analyses of clinical outcomes according to the prescribed doses of blocker therapy at discharge. the adjusted hazard ratio (hr) and 95% ci could not be evaluated that no event occurred in the 50% of target dose group. mi indicates myocardial infarction; nstemi, non stsegment elevation myocardial infarction; stemi, stsegment elevation myocardial infarction; uap, unstable angina pectoris. between march 1, 2009, and december 30, 2014, there were 5063 patients recruited in the database, and only 3453 patients underwent the pci procedure. of these, 23 patients were discharged unstably, 183 were not diagnosed with acs at admission, 43 had a contraindication to blocker use, and 24 could not provide complete information about the administration of blockers at discharge, and were excluded from the analysis. acs indicates acute coronary syndrome; chd, coronary heart disease; pci, percutaneous coronary intervention. in the overall evaluation cohort, 2423 patients (76.2%) were discharged on blockers, while 757 patients (23.8%) were not. compared with blocker users, patients who were not administrated blockers were older (60.7910.39 versus 58.4410.47, p<0.001), had lower diastolic blood pressure (dbp) (79.0113.18 versus 80.5713.18, p=0.005), lower heart rate (74.4015.15 versus 75.1012.42, p=0.009), and were more likely to have arrhythmia (11% versus 7.1%, p=0.001). for concomitant medication use, the prescriptions of aspirin, statins, and aceis / arbs were more common in the blocker group compared with the no blocker group (99.4% versus 98.4% ; 98.6% versus 95.5% ; 81% versus 58.4%). table 1 summarizes the baseline characteristics and other medication management according to the use of blockers at discharge. the differences in the baseline characteristics in the 3 subgroups are also shown in tables 2, 3 through 4. in the propensity score matched model, there was no significant difference in the baseline characteristics between the blocker group and the no blocker group. acei indicates angiotensinconverting enzyme inhibitor; arb, angiotensin receptor blocker; dbp, diastolic blood pressure; mi, myocardial infarction; sbp, systolic blood pressure. acei indicates angiotensinconverting enzyme inhibitor; arb, angiotensin receptor blocker; dbp, diastolic blood pressure; mi, myocardial infarction; sbp, systolic blood pressure; stemi, stsegment elevation myocardial infarction. acei indicates angiotensinconverting enzyme inhibitor; arb, angiotensin receptor blocker; dbp, diastolic blood pressure; mi, myocardial infarction; nstemi, non acei indicates angiotensinconverting enzyme inhibitor; arb, angiotensin receptor blocker; dbp, diastolic blood pressure; mi, myocardial infarction; sbp, systolic blood pressure; uap, unstable angina pectoris. at 1 year after index admission, completed followup information was obtained in 3153 patients (99.2%). a total of 33 patients died of allcause diseases, 14 patients occurred nonfatal mi, 34 patients had heart failure, and 214 patients were readmitted for cardiogenic reasons during followup. blocker therapy was associated with a lower incidence of allcause death (unadjusted hazard ratio , 0.33 ; 95% ci, 0.170.65). after adjusting for confounders, the risk of allcause death remained consistently lower in the blocker group (adjusted hr, 0.38 ; 95% ci, 0.170.83) (table 5). in the propensity score matched cohort, the blocker group still had decreased allcause mortality (hr, 0.27 ; 95% ci, 0.080.97) (table 6). a lower rate of secondary end point was also observed in the blocker users (unadjusted hr, 0.76 ; 95% ci, 0.590.98 , although the statistical difference disappeared after adjustment ( adjusted hr, 0.87 ; 95% ci, 0.661.16). in addition, the associations of blocker use with the rate of nonfatal mi, heart failure readmission, and cardiogenic hospitalization were computed, respectively. the are illustrated in table 5, and figure 2 describes the association between the use of blockers and clinical end points. clinical outcomes and unadjusted / multivariable adjusted hrs during 1year followup the event rate at 1 year was estimated by the kaplan meier method. hf indicates heart failure; hr, hazard ratio; mi, myocardial infarction; nstemi, non stsegment elevation myocardial infarction; stemi, stsegment elevation myocardial infarction; uap, unstable angina pectoris. clinical outcomes and hrs after propensity score matching during 1year followup hf indicates heart failure; mi, myocardial infarction; nstemi, non stsegment elevation myocardial infarction; stemi, stsegment elevation myocardial infarction; uap, unstable angina pectoris. the hazard ratio (hr) and 95% ci could not be evaluated that no event occurred in the blocker group. the hazard curves for the primary and secondary end points in the overall population (a), in the patients with stsegment elevation myocardial infarction (stemi) (b), in the patients with non stsegment elevation myocardial infarction (nstemi) (c), and in the patients with unstable angina pectoris (uap) (d). at baseline, 728 patients (22.9%) had stemi, 576 patients (18.1%) had nstemi, and 1876 patients (59.0%) had uap. notably, a greater benefit of blocker use was found in patients with nstemi whose incidence of allcause death was significantly lower in the blocker group (0.2% versus 6.4% ; unadjusted hr, 0.04 ; 95% ci, 0.000.27), and the relationship remained even after performing multivariable cox proportional hazard regression analysis (adjusted hr, 0.00 ; 95% ci, 0.000.14). in addition, blocker use was associated with a lower risk of the secondary end point (7.8% versus 15.7% ; unadjusted hr, 0.47 ; 95% ci, 0.280.81), but no statistical difference was observed after adjustment (adjusted hr, 0.65 ; 95% ci, 0.351.21). in the patients with stemi and uap, however, there was no statistical difference between the two groups for allcause mortality (1.1% versus 1.9% ; adjusted hr, 0.40 ; 95% ci, 0.081.94 in patients with stemi and 0.7% versus 0.9% ; adjusted hr, 0.96 ; 95% ci, 0.293.10 in patients with uap) and the secondary end point (8.5% versus 16.1% ; adjusted hr, 1.13 ; 95% ci, 0.592.16 in patients with stemi and 9.0% versus 9.9% ; adjusted hr, 0.97 ; 95% ci, 0.661.41 in patients with uap ( table 5 and figure 2). the associations of blocker therapy with the clinical outcomes across the 3 subgroups were consistent in the propensity score matched cohorts (table 6). among the patients discharged on blockers, receiving < 50% of target dose was reported in 2012 patients (83.0%), while 411 patients (17%) were prescribed 50% of target dose and the administration of metoprolol accounted for the majority (85.4%). the baseline characteristics according to the treatment of blocker use are exhibited in table 1, 2, 3 through 4. for overall patients, allcause mortality was 0.7% in < 50% of target dose group, significantly lower than in the no blocker group (0.7% versus 2.1% ; adjusted hr, 0.40 ; 95% ci, 0.190.82), while the rate of allcause death was not different between 50% of target blocker dose group and no blocker group (0.7% versus 2.1% ; adjusted hr, 0.46 ; 95% ci, 0.131.59) (figure 3), and no differences were observed in the incidence of secondary end point between the three different blocker dose groups. entire cohort and subgroup analyses of clinical outcomes according to the prescribed doses of blocker therapy at discharge. the adjusted hazard ratio (hr) and 95% ci could not be evaluated that no event occurred in the 50% of target dose group. stsegment elevation myocardial infarction; stemi, stsegment elevation myocardial infarction; uap, unstable angina pectoris. in this observational study, we investigated the association of blocker use with the clinical outcomes in patients with acs undergoing pci. we found that nearly 77% of eligible patients with acs undergoing pci were treated with blockers at discharge, and those not prescribed blockers were more likely to be older and have a history of arrhythmia. importantly, blocker therapy at discharge, especially a relatively low blocker dosage, were independently associated with improved survival, and the efficacy was more significant in patients with nstemi. blocker therapy also showed a trend in improved clinical outcomes in the stemi and uap patients. acs as a major cause of emergency medical care and hospitalization worldwide16 has been well improved by the introduction of pci.17 optimal secondary medication remains important after successful pci. predecessors have highlighted the importance of blocker therapy in patients with acute myocardial infarction.18, 19, 20, 21, 22, 23, 24 however, there are a few studies reporting that blocker use is not associated with improved outcome.25, 26, 27 one metaanalysis of randomized trials on the clinical outcomes of blocker use indicated no mortality benefit but reduced recurrent myocardial infarction and angina (shortterm) at the expense of increased heart failure, cardiogenic shock, and drug discontinuation.28 in this metaanalysis, data used in the reperfusion era were mainly recruited from the clopidogrel and metoprolol in myocardial infarction trial (commit)29 and the japanese blockers and calcium antagonists myocardial infarction (jcbami) trial.30 in commit, the association between metoprolol allocation and risk of clinical outcomes was only assessed in a mean period of 15 days among ami patients. on the other hand, only postmyocardial infarction patients were enrolled in the jcbami trial, which could not reflect the benefit of early blocker therapy on improvement in prognosis. yet, our study proved the benefit of early use of blockers on longterm survival among patients with acs. nevertheless, chan et al31 reported the mortality benefit of blockers in patients undergoing successful elective pci; however, they did not discuss which type of patients with acs benefited the most. in the present study , our showed that blocker use was better associated with decreased incidence of allcause death in patients with nstemi. the published can rapid risk stratification of unstable angina patients suppress adverse outcomes with early implementation of the acc / aha guidelines (crusade)32 and global registry of acute coronary events (grace)33 studies also revealed that early blocker therapy had a beneficial impact on hospital and 6month mortality in patients with nstemi. in addition, yang et al24 demonstrated that blocker therapy at discharge was associated with improved survival in stemi patients treated with primary pci and recommended longterm blocker therapy in all patients with stemi regardless of risk profile. in our analysis, the use of blockers was not statistically associated with a lower risk of allcause death in stemi patients, but the trend of improved survival was obvious. additionally, the observational data from the outcome of blocker therapy after myocardial infarction (obtain) study suggested that increased survival was not observed in patients treated with blocker doses approximating those used in previous randomized clinical trials compared with lower doses,34 which was consistent with our that relatively low blocker dose actually decrease the rate of allcause mortality. even though several investigators have studied the benefits of blocker use among patients with myocardial infarction, our study stressed the impact of blocker therapy, especially relatively low blocker dose, on reducing allcause mortality in patients after elective pci, and provided the evidence to support the idea that the benefit of oral blocker therapy might be confined to patients with nstemi.35 evidence has suggested that the benefit of blockers for patients with nstemi may be due to the multivessel disease commonly presenting in them and its sympathetic hyperactivity.36, 37, 38, 39 however, further exploration of the clinical usefulness of blocker therapy in patients with acs warrants largescale clinical trials such as a recently registered project (nct02648243). finally, we can not claim generalizability to patients with stemi / uap for it was underpowered to detect the difference. first, the nonrandomized nature of this observational study could have ed in selection bias. although randomized controlled trials are considered the highest standard for evaluating treatment efficacy, observational studies can still provide unique and valuable insights into treatment effectiveness and generalizability in practice. our findings imply that the efficacy demonstrated in randomized clinical trials can be translated into tangible clinical benefits in the real world. second, a 1year followup period may be too short for conclusive determination of the longterm efficacy of blockers in the setting of acs. third, the stemi group and the uap group were underpowered to discriminate the benefit of blocker use. first, the nonrandomized nature of this observational study could have ed in selection bias. although randomized controlled trials are considered the highest standard for evaluating treatment efficacy, observational studies can still provide unique and valuable insights into treatment effectiveness and generalizability in practice. our findings imply that the efficacy demonstrated in randomized clinical trials can be translated into tangible clinical benefits in the real world. second, a 1year followup period may be too short for conclusive determination of the longterm efficacy of blockers in the setting of acs. third, the stemi group and the uap group were underpowered to discriminate the benefit of blocker use. this large observational study has shown that the higher survival rate in patients following pci is associated with the appropriate use of blockers at discharge and this benefit is consistent in the patients with nstemi. this study was supported by grants from the national program on key basic research project (973 program) (no . 2012cb518004), the national nature science foundation key project (no . 91439203), and the national health and family planning commission of china ( no. 201202025, no.

the evidence supporting the use of blockers in patients with acute coronary syndrome after successful percutaneous coronary intervention has been inconsistent and scarce.methods and between march 1, 2009, and december 30, 2014, a total of 3180 eligible patients with acute coronary syndrome undergoing percutaneous coronary intervention were consecutively enrolled. the primary end point was allcause death and the secondary end point was a composite of allcause death, nonfatal myocardial infarction, heart failure readmission, and cardiogenic hospitalization. patients were compared according to the use of blockers at discharge. compared with the no blocker group, the risk of allcause death was significantly lower in the blocker group (hazard ratio , 0.33 ; 95% ci, 0.170.65). a consistent was obtained in multiple adjusted model and propensity score matched analysis. the use of blockers was also associated with decreased risk of composite of adverse cardiovascular events (hr, 0.47 ; 95% ci, 0.280.81), although statistical significance disappeared after multivariable adjustment and propensity score matching. furthermore, we performed post hoc analysis for the subsets of patients and the revealed that patients with non stsegment elevation myocardial infarction benefited the most from blocker therapy at discharge (hr, 0.04 ; 95% ci, 0.000.27), and the use of < 50% of target dose was significantly associated with better outcome compared with no blocker use, rather than 50% of target dose.the administration of relatively low blocker dose is associated with improved clinical outcomes among patients with acute coronary syndrome after successful percutaneous coronary intervention, especially for patients with nonstsegment elevation myocardial infarction.

there is a long history of using electron microscopes to image the motion of adatoms on a thin film, but only recent advances have made it feasible to study the chemistry of metals at the level of atomic resolution. in particular the thickness and regularity of the honeycomb network of graphene provides an exceptional support for the deposition of individual atoms and observation of their motion. however, imaging the dynamics of single atoms on graphenic materials, compared to biomacromolecules and small clusters, is technically difficult because of their subnanometric size and their fast motion. studies of the movement of mo and w atoms on graphene and their trapping by defective sites have been reported, as well as pd and te atoms which bind to the free edge states along graphene hole edges, although much of our current knowledge of such migratory behavior of single atoms relies on theoretical elucidation. recently we showed that electron beam irradiation of self - spreading polymer - encapsulated precious metal complexes can generate in situ - doped graphenic surfaces on which the dynamics of single metal atoms can be studied in real time using an aberration - corrected high resolution electron microscope. here we synthesize novel boron sulfur - doped and boron selenium - doped graphenic surfaces and study the atomic trajectories and rate of migration of single osmium atoms and individual os2 molecules. these experiments reveal the dramatic effect of two chalcogenide (group 16) dopants s and se on osmium atom migration. we synthesized two graphenic surfaces (supporting information figure s1 for a low magnification image of the b / s surface). the first was doped with heteroatoms boron and sulfur (b / s) following the procedure we described recently, while the second was doped with heteroatoms boron and selenium (b / se). this involved irradiating osms - se micelles made of encapsulated in the triblock copolymer p123 with the electron beam of an aberration - corrected high resolution hr - tem - stem with a schottky thermal field - emission source (80 kev ; 1.9 pa cm or 7.6 10 electrons nm s). in general there were regions of the surface which were single - layer (fast fourier transform in supporting information figure s1) and on which an idealized hexagonal lattice could be readily overlaid, although the surface was clearly inhomogeneous in some regions as expected from the presence of the b / s and b / se dopants. the presence of boron and chalcogen atoms as well as osmium was confirmed by a combination of energy - dispersive x - ray (edx) analysis and electron energy loss spectroscopy of energy filtered tem (eftem) (supporting information figure s2). we investigated single os atom migration by following the positions of individual os atoms on b / s (over 390 s ; 40 frames with 10 s between each frame) and b / se surfaces (over 15 s ; 31 frames with 0.5 s between each frame). the positions of each atom were extracted from a series of images from three different areas of the tem grid. successive images were aligned, and the drift was compensated by using the plugin stackreg in fiji - imagej and by using a digital micrograph(tm) script (methods section). some sections of the surface are clearly hexagonal and of graphitic nature, whereas other areas are highly distorted by the dopants. moreover, the atoms in the surface also undergo motion by absorption of energy from the beam. density functional theory (dft) calculations have predicted that the presence of an os atom adsorbed on a graphene matrix disturbs the structure of graphene when located at bridge- and edge - sites. supporting information videos (figures s3_video 1 and s4_video 2) show two examples of time - lapse tem images used to extract the coordinates of the individual atoms on both b / s and b / se surfaces. perhaps surprisingly, since the surface is doped and not homogeneous graphene, it was possible to correlate the atom trajectories with the individual positions of the atom on idealized hexagonal grids as shown in supporting information figures s5_video 3 for the b / s matrix and s6_video 4 for the b / se matrix. the experimental images highlight the existence of anchoring points on both surfaces, positions of apparent long residence times (figures 1b, c). the analysis of the various trajectories suggests that the chalcogen atoms themselves induce the differences in atomic migration and have a direct impact on the trajectory of osmium atoms on the surface. this might involve direct os chalcogen binding, but the effects could also be propagated over a longer distance on the conjugated graphenic lattice. supporting information figure s7 shows the trajectory of an individual os atom on the b / s graphenic surface (supporting information figure s7a), the superimposition of the atomic position on an ideal hexagonal monolayer graphene grid (supporting information figure s7b), the apparent trapping sites (in blue in supporting information figure s7c), and sites from which longer jumps than the average jump size are observed (acceleration sites in red in supporting information figure s7c). from these data, three hypothetical chemical structures of the doped graphenic surfaces used in this study can be proposed (supporting information figures s7d f): either the trapping sites are occupied by sulfur atoms and the accelerating sites are boron atoms (supporting information figure s7d) or the opposite (supporting information figure s7e); alternatively carbon atoms could act as acceleration sites, sulfur atoms act as trapping sites, and all the other sites where the osmium atom is seen moving to and from are occupied by boron atoms (supporting information figure s7f). these three models are hypothetical since it is technically not possible to investigate the actual atomic composition of the surface site - by - site. positions, trajectory, and experimental hopping of an os atom on the b / s- and b / se - doped graphenic surfaces. (a) example of experimental tem images and the trajectory of an individual os atom on the b / s surface recorded at different irradiation times (five frames extracted from a total of 40 pictures over a total irradiation time of 390 s). (b and c) illustrative trajectories of three individual osmium atoms on the b / s- and b / se - doped graphenic surfaces, respectively. owing to the high contrast of osmium atoms as compared to the graphenic surfaces, the extraction of the coordinates of the atom in each frame is readily achieved from a 3d surface projection for each tem picture (a). (d) cumulative apparent distances covered by an individual os atom on b / s (yellow) and b / se (blue) surfaces. (e) apparent speed of motion of an individual os atom on b / s (yellow) and b / se (blue) surfaces between each frame (t = 0 for first frame of each stack). (f) enlargement of the b / s (yellow) plot shown in (e). have reported that w atoms are mobile on a graphitic surface but are trapped by defective sites. they showed that below 250 c, when the graphene lattice was heavily damaged by the beam, almost no jumps occurred and the w atoms were pinned by larger irradiation - induced lattice defects. in the range 250500 c, jumping of w atoms was observable, and escape from a trapping center could be induced thermally or by electron irradiation, a combination of thermal and beam effects which explains the observed small oscillations. in related work the same group observed the trapping of mo atoms at defect sites in carbon nanotubes and in graphene, and recently pb and te atoms have been trapped on amorphous graphene and at edge sites of holes in graphene. our experimental data are consistent with these previous observations and also show that such an escape by os atoms from anchoring sites, at a temperature close to ambient, is dependent on the nature of the graphenic dopants. we determined the apparent rate of migration of individual os atoms on the two multidoped graphenic surfaces (average from observation of 10 single atoms). it should be noted that the real speed of motion of the atoms is at least the apparent monitored rate of migration, since we are limited by the speed of the camera and there could be more steps at intermediate times between frame captures. figure 1d shows the dependence of the cumulative path length covered by two individual os atoms (blue : b / se surface ; yellow : b / s surface) on irradiation time on the two surfaces. remarkably, the chalcogen dopant (s vs. se) dramatically influences the apparent rate of migration. the os atom travels an apparent distance of 3.5 nm in 15 s on the b / se surface, while the same distance is covered by the os atom in 390 s on the b / s surface. 26 higher speed of os atoms on the b / se surface compared to the b / s surface (233 34 pm s versus 8.9 1.9 pm s, supporting information table s1) is illustrated in figure 1e and in supporting information figure s8_video 5. on both surfaces, the apparent rate of migration is not constant but varies over time, with significant autocorrelation (figures 1e, f). this confirms the existence of anchors and suggests that the atom must receive a minimum energy from the high - energy electron beam to move from one anchoring site to another adjacent position. we found that an individual os atom required irradiation for 70 9 s on the b / s surface before hopping to another site, whereas on the b / se surface, only ca. we also investigated the dynamics of migration of two close os atoms on the b / s and b / se surfaces. we elucidated the trajectory of each atom (figure 2a) and thus the trajectory of the pair (figure 2b, c). the atomic positions were extracted from a series of time - lapse tem images, as for individual atoms. supporting information figures s9_video 6 and s10_video 7 show examples for b / se and b / s surfaces, respectively. notably, the average os os distances of 0.284 0.077 nm on the b / se surface and 0.243 0.059 nm on b / s are both close to the os os distance in metallic os (0.27048 nm as the nearest neighbor distance at 20 c) suggesting that these two os atoms form a true os2 diatomic molecule. furthermore, the two close os atoms undergo concerted migration on the multidoped graphenic surfaces. the dependence of the jumping pattern for each os atom shown in figure 2c on irradiation time is shown in figure 2e and confirms that the two atoms are indeed bonded. positions, trajectory, and experimental hopping of os2 molecules on the b / s- and b / se - doped graphenic surfaces. (a) hrtem images showing the position of the two os atoms on the b / s surface at different irradiation times (time = 0 s corresponding to the first image of the stack ; scale bar = 0.5 nm). the black circles and the black dotted line show the alignment of the surface. (b) trajectory of the hopping of the two os atoms on the b / s surface over 390 s. (c) trajectory of the hopping of the two os atoms on the b / se surface over 15 s. (d) distances covered by each os atom of a diatomic molecule on b / s (black and red) and b / se (light and deep blue) surfaces. (e) jump size against time for each atom of the molecule on the b / se surface. similar to the movement of isolated individual os atoms, the distance traveled by each atom of the os2 molecule increases linearly with irradiation time on both surfaces (figure 2d, supporting information figure s9_video 6, figure s10_video 7). the average apparent rate of migration of an os2 diatomic molecule on the b / s and b / se surfaces (7.4 2.8 pm s, and 151 45 pm s, respectively, supporting information table s1) was slightly slower (0.83 and 0.65) than for individual os atoms. again, as for single os atoms, the apparent rate of migration of os2 on the b / se - doped surface was dramatically faster (ca . 20) than on the b / s - doped surface (supporting information table s1). we note that defects within graphenic layers can themselves migrate under the influence of an electron beam. although such effects can be quantified on pure graphene, they are more difficult to map on heavily doped lattices such as those studied here. it should be borne in mind however that such movements of carbons or dopants within the support layer could also influence the movement of os atoms, as is apparent in figure 2a. it is interesting to consider the possible magnetic states of os atoms on a graphenic surface. our dft calculations on an os atom on an ideal (without defects or doping) graphene subunit, an aromatic 19-ring c54h18 system (supporting information figure s11, table s2), suggested that this model system is paramagnetic. lumo gap will go to zero, making it unlikely that paramagnetic states will be supported. experimental magnetic measurements present a challenge for future work. to further confirm that the doped graphitic surface plays a role in the observed os migration, we investigated whether the atoms follow brownian motion (bm). in bm, each jump is independent and identically distributed according to a normal distribution with variance proportional to the time between observations. we looked for evidence of this in the data, consisting of the migration of five os atoms on each kind of each surface. first we checked that the size of the jumps in the x and y directions were the same (supporting information figure s12 and table s3), pooling together all of the jumps for the five separate atoms. there appeared to be no significant difference in the dynamics of migration on the sulfur - doped surface and only a very small difference on the selenium - doped surface. the histograms shown in figures 3a, b were observed to be similar, and as such x and y jumps were pooled together for further analysis. we also assumed that the os atoms were not drifting in any particular direction and so the mean jump would be zero, which also appeared to be confirmed by the data. next, we fitted a gaussian density to the pooled jump data and plotted a normal quantile quantile plot to assess deviations from normality (figures 3c, d). analysis of the mechanism of hopping of individual os atoms on the two graphenic surfaces. (a and b) histograms of pooled jumps of individual os atoms on b / s and b / se surfaces, respectively. quantile plot of os atoms hopping on b / s and b / se surfaces, respectively. it is clear from these plots that there are more long - range jumps than would be expected for a brownian motion, but for completeness we also performed a shapiro wilk test of normality; both the selenium- and sulfur - pooled data gave p - values below 0.001, and so we can reject the hypothesis that brownian motion describes the movement of the os atoms. kinetic energy of the e - beam is the main driving force of the dynamic behavior of os atoms observed in tem. returning to the raw data, we see that no large jumps are captured for these two atoms. it is clear from supporting information table s3 that the jump sizes are larger on selenium - doped than on sulfur - doped graphene, but it is nonetheless interesting to investigate whether they come from similar distributions. supporting information figure s13 shows a comparative quantile although there is a small amount of deviation from the line in the lower tail, the plot does suggest that the distributions are indeed very similar. in , the new generations of electron microscopes with their atomic resolution capability and ultrafast cameras offer the possibility of imaging dynamic processes in real time. however, there is currently little reported data on such dynamic interactions at the level of the individual atom, owing, in particular, to the fast migration of single atoms on graphene. here , we have used a new synthetic methodology for the in situ formation of graphenic surfaces doped with boron and sulfur (b / s) or selenium (b / se) heteroatoms. the doping creates anchoring points for individual os atoms, slowing down their migration so that it is commensurate with the time scale of image capture on an aberration - corrected transmission electron microscope. we have imaged in real time the migration of individual os atoms and os2 dimers on these surfaces. the rate of movement is > 20 faster on the b / se - doped boronic graphenic surface compared to b / s. indirectly, through choice of the dopants, our methodology can provide control of atomic dynamics and might lead to a wide range of potential applications (e.g., patterning on surfaces, security labeling at the atomic level, sealing confidential documents). there is much scope for extending the fabrication to a wider range of doped - graphenic matrices, so as to further modify the surface dopants and the metal atom migration rates, and to the deposition of other metal atoms (e.g., other precious metals such as au, pt, and pd).

we deposited os atoms on s- and se - doped boronic graphenic surfaces by electron bombardment of micelles containing 16e complexes encapsulated in a triblock copolymer. the surfaces were characterized by energy - dispersive x - ray (edx) analysis and electron energy loss spectroscopy of energy filtered tem (eftem). os atoms moved ca. 26 faster on the b / se surface compared to the b / s surface (233 34 pms1versus 8.9 1.9 pms1). os atoms formed dimers with an average os os distance of 0.284 0.077 nm on the b / se surface and 0.243 0.059 nm on b / s, close to that in metallic os. the os2 molecules moved 0.83 and 0.65 more slowly than single os atoms on b / s and b / se surfaces, respectively, and again markedly faster (ca . 20) on the b / se surface (151 45 pms1 versus 7.4 2.8 pms1). os atom motion did not follow brownian motion and appears to involve anchoring sites, probably s and se atoms. the ability to control the atomic motion of metal atoms and molecules on surfaces has potential for exploitation in nanodevices of the future.

palatal rugae or transverse palatine folds are irregular mucosal elevations present in the anterior third of the palate. it is asymmetrical and made from the lateral membrane of the incisive papilla, arranged in transverse direction from palatine raphae located in midsaggital plane. they are stable throughout life following completion of growth, though there is a considerable debate in this regard. identification of a severely burnt edentulous body by rugae comparison to those on the victim's old denture is a standing proof that rugae are stable in adult life. though odontometrics serves much in post mortem dental identification, palatal rugae proves to be great supplements in post mortem identification of edentulous and severely burnt decomposed individuals. odontometrical analysis, fingerprints and dna comparisons are probably the most used techniques, allowing secure identification. however, these can not be applied in certain cases and in such situations, different, simple and less known technique can be used for personal identifications such as palatal rugoscopy study of palatal rugae. in addition, rugae pattern may be specific to racial groups, facilitating population identification which is essential in mass disasters. racial profiling using intraoral features other than the teeth may have relevance in odonto- stomatological identification in india where, credible dental anthropological data is negligible. they provide a preliminary data on rugae shape between two populations in india and its effectiveness in identifying the populations using discriminant function analysis. the present study is performed using a larger sample size to validate the findings of previous studies and to provide a stronger evidence for forensic identification using palatal rugae. thus the palatal rugae are unique in its morphology of every individual having good stability with low utilization cost providing a concrete postmortem resistant evidence for forensic purpose. therefore the present study is aimed at delineation of different types of rugae in two different populations and developing a discriminant function for the same. a total of 940 subjects were included in the present study who belong to two geographically different populations. the sample consisted of 466 south indians from tamil nadu state and 474 north indians from uttar pradesh. the subjects were enrolled by simple random sampling from ksr group of educational institutions (south india) and up college of arts and science (north india). the age group of the study subjects were 18 - 23 years, as there is a lack of consensus on rugae stability with respect to aging. informed consent was obtained from all the enrolled subjects. exclusion criteria were palatal asymmetries, cleft palate (or) a history of palatal surgery. neo colloid easy flow alginate impression of maxillary arch were made and casts were immediately poured with type iv dental stone. a sharp graphite pencil was used to delineate the rugae and patterns were recorded according to the classification given by kapali et al. the categories were straight, wavy, curved, circular and if rugae had two arms it is unification. further, differentiation of unified rugae length based categorization was not considered in our study. the delineation of all the rugae patterns was performed by the same investigator. to estimate intra - observer variability, a stepwise discriminant function analysis was also preformed between the two different populations for different types of rugae to develop a discriminant formula, di = k+di1 z1 + di2 z2.. dip zp di is discriminant function score, di is discriminant function co efficient, z is the score of the predictor variable and k is the discriminant function constant. the significance of rugae on population identification was observed for test of function using wilks' lambda statistics. neo colloid easy flow alginate impression of maxillary arch were made and casts were immediately poured with type iv dental stone. a sharp graphite pencil was used to delineate the rugae and patterns were recorded according to the classification given by kapali et al. the categories were straight, wavy, curved, circular and if rugae had two arms it is unification. further, differentiation of unified rugae length based categorization was not considered in our study. the delineation of all the rugae patterns was performed by the same investigator. to estimate intra - observer variability, a stepwise discriminant function analysis was also preformed between the two different populations for different types of rugae to develop a discriminant formula, di = k+di1 z1 + di2 z2.. dip zp di is discriminant function score, di is discriminant function co efficient, z is the score of the predictor variable and k is the discriminant function constant. the significance of rugae on population identification was observed for test of function using wilks' lambda statistics. the occurrence of different rugae shapes in the two populations in the present study is presented in table 1. circular rugae constituted less than 5% of rugae in the entire sample of 940 casts. a few non - specific rugae forms were observed. the intra - observer reliability calculated during the second examination after two months, revealed the kappa value to be 0.93. hence, the observations made at two different moments showed a negligible difference and therefore were found reliable. frequency of different rugae shapes in southern and northern indians the incidence of all the rugae patterns were more in number in north indian population compared to that of south indian population except non - specific rugae. chi- square analysis for association between rugae shape and population groups showed significant differences among all the rugae patterns at the p < 5%. chi - square analysis for assessing sex differences in the rugae shapes showed significant difference in straight, unification and circular type. chi - square analysis for assessing differences in rugae shapes between southern and northern indians chi - square analysis for assessing sex differences in the rugae shape tables 4 - 6 show the rugae shape that entered, removed and contributed to the discriminant function analysis respectively. table 7 shows that the rugae shape that contributed to the discriminant function analysis were subjected to test of function with wilks' lambda statistics and it showed overall significance among all rugae shapes. five rugae shapes curved, wavy, nonspecific, unification and circular were selected in five steps. curved rugae entered the analysis first, indicating they had the greatest ability to differentiate the population groups, followed by wavy, unification circular and non - specific rugae. straight did not contribute to the function, implying their inability to differentiate the groups. the best discriminant function was f = -4.372 + 0.901(curved) + 0.375 (wavy) + 0.354(unification) + 0.334(circular) table showing the progress of variables entered the discriminant function analysis table showing the progress of variables removed from the discriminant function analysis step - wise discriminant function analysis of the different rugae shapes wilks' lambda statistics table table 8 depicts the unstandardized and standardized coefficients, structure matrix, group centroids and sectioning point for the discriminant function. to determine the population group to which an unidentified individual belongs to, the number of each type of rugae shape is multiplied with the respective unstandardized coefficient and added to the constant. if the value obtained is greater than the sectioning point, the individual is considered as north indian; if the value obtained is less than the sectioning point, the individual is considered as south indian. discriminant function coefficients for rugae shapes that entered the analysis let us consider an unidentified individual where the number of curved rugae = 2, straight rugae = 3, wavy rugae = 3, unification = 1, circular = 0 and nonspecific = 0. multiplying the number of each rugae shape with the respective unstandardized coefficients and adding the constant, we obtain -4.372 + 0.901 + 0.375 + 0.354 + 0.334 0.289 = -1.09. since this value is less than the sectioning point -0.04, the individual is considered as south indian. the discriminant function's accuracy in population identification the expected accuracy of identifying an individual from a different population is derived from the entire sample in the original . this may be biased since the plaster casts from which the function was derived are themselves tested for population origin. to overcome this bias, jack - knifing ) where, a function is derived from all but one cast in the sample and the excluded cast tested for population origin. this procedure was performed for each of the 940 casts, i.e. a function was derived from 939 of the 940 dental casts and the 940 cast tested for population origin. classification consequently, the cross - validated accuracy of the function (87.8%) is lower than that of the original (87.9%). while cross - validation may be a theoretical construct and not a true accuracy, it gives a more realistic indication of the precision of the discriminant function. human identification is one of the most challenging subjects that man has been confronted with. sometimes it becomes necessary to apply lesser known and unusual techniques like cheiloscopy and palatoscopy. thomas and kotze studied the rugae patterns of 6 south african populations to analyse the interracial difference. their indicate that rugae were unique to each ethnic group and can be used successfully as a medium for genetic research. hence, this shows the applicability of rugae in population differentiation and it has been revealed in the present study also. hauser et al. compared the rugae patterns of swazi and greek populations and observed definite differences in the rugae pattern between the two populations. it was evident that the degree of development of rugae was dependent on the growth of the palate. according to english et al. the observation of palatal rugae is a subjective phenomenon and it poses a problem when interpreted at two different moments. hence, the intra observer variability was calculated and it was found to be k = 0.93; which shows that the difference attributed to the observation is practically inexistent. the present study showed more than 42% of wavy, curved and straight rugae forms in each population. this is consistent with finding of australian aborigines, caucasians, indians (southern and western) for wavy and curved rugae patterns. but the straight form of rugae was also found to be higher in the present study. unification, non - specific and circular was less common. however, circular forms of rugae were very few in number. thomas and kotze and nayak et al. in their study to identify two genetically similar groups, obtained a jack - knife accuracy of 61.8% and 70% respectively. the classification of jack - knife accuracy obtained from the present investigation, observing two relatively similar populations, showed a relatively higher accuracy of 87% when compared to the previous studies and hence, the type of rugae patterns used has got discriminating ability. the rugae shapes which are considered for classification in the present work are discrete variables when compared to that of rugae dimensions (continuous variables) used in previous classifications. when genetically similar populations were considered for differentiation continuous variables such as rugae measurements may have its limitations. the present analysis has revealed that incidence of all the rugae shapes were significantly higher in northern indian population when compared to southern indian population except for non - specific rugae. but the rugae shapes used as a predictor variable for southern indian population in the present study showed a variable rate of incidence when compared to the previous study. hence, we believe that even variations within the same country (uttar pradesh and tamilnadu) can show significant rugae pattern variations. the predictor variable entered the discriminant function in the present study showed a variance when compared to the study conducted by nayak et al. more number of predictor variables entered the discriminant function in our study which is attributed to the usage of larger sample size for population differentiation. the discriminant function equation obtained from the different rugae shapes in the present study was highly accurate enough to distinguish the southern and northern indian population with the classification accuracy of 87.8%. thus to identify a specific population, separate discriminant function formulae have to be developed. hence, the study of palatal rugae is one of the simple and reliable tools for population identification in forensic science.

aim: the present study is aimed at delineation of different types of rugae in two different populations and developing a discriminant function for the same.materials and methods: a total of 940 subjects were included in the present study. the sample consisted of 466 subjects from south indian population and 474 from north indian population in the age group of 18 - 23 years. neo colloid easy flow alginate impressions of maxillary arch were made and casts were immediately poured with type iv dental stone. a sharp graphite pencil was used to delineate the rugae and patterns were recorded according to the classification given by kapali et al. the association between different population and different sexes was analyzed with chi - square test and a stepwise discriminant function analysis was also performed to develop a discriminant formula.:wavy, curved and straight rugae were the most common forms in both groups. chi - square analysis for association between rugae shape and population groups showed significant differences among all the rugae patterns at the p < 5%. chi - square analysis for assessing sex differences in the rugae shapes showed significant difference in straight, unification and circular type. five rugae shapes curved, wavy, nonspecific, unification and circular were selected for discriminant function.:the discriminant function equation obtained from the different rugae shapes in the present study was highly accurate enough to distinguish the southern and northern indian population with the classification accuracy of 87.8%. thus to identify a specific population, separate discriminant function formulae have to be developed. hence, the study of palatal rugae is one of the simple and reliable tools for population identification in forensic science.

plasticity in the hippocampus has long been implicated in the etiology of mood disorders and regulation of stress. recent work has also suggested that the actions of antidepressant medications, such as selective serotonin reuptake inhibitors (ssris), on hippocampal plasticity may play an important role in alleviating symptoms of depression. serotonin, itself, is an important neurotransmitter involved in regulating the rate of hippocampal neurogenesis during adulthood, with lower levels of serotonin reducing the number of newly formed neurons in the hippocampus and elevated levels of serotonin increasing the rate of cell proliferation. more importantly, increased serotonin levels, following ssri treatment, significantly upregulate adult hippocampal neurogenesis and it has been suggested that the actions of ssri medications on hippocampal plasticity (morphology and neurogenesis) may be important for alleviating the effects of stress on affect - related behaviours. although there has been a substantial amount of work investigating the effects of ssri medications on plasticity in the hippocampus and their role in treating depressive - like behavior, very little work has taken into account the effects of these medications on hippocampal plasticity in the adult female. with women being 2 - 3 times more likely to suffer from depression, it is important to include females in research related to depression. recent work focusing on females has shown that chronic administration of imipramine, a tricyclic antidepressant, to intact female rats exhibiting depressive - like behavior increases cell proliferation in the dentate gyrus. however, others have shown that administration of fluoxetine, a popular ssri, has no effect on cell proliferation or neurogenesis in the hippocampus of adult female rats , but it does trigger spine formation in the hippocampus of the adult female rats. more work is also needed to better mimic the clinical administration method of ssris. in humans, unfortunately, the typical administration of antidepressant medications in rodent models of depression is invasive and stressful, with administration being done most often via injection, oral gavage, or minipump implant. administration method alone affects the metabolism and effects of the medication. interestingly, recent research in animal models investigating effects of environmental teratogens on development has demonstrated that administration of a solution injected in a wafer cookie may be as effective as injecting a solution into the animal and thus would reduce the stress of the animal, particularly for long - term daily treatment (greater than 4 weeks). however, it remains to be determined whether this method of drug administration is effective for ssris and is comparable to other methods of antidepressant administration that are commonly used. the aim of this project was to understand the role of fluoxetine treatment in hippocampal plasticity in the adult female rat, using two methods of ssri administration. more specifically the proposed study aims to determine the blood concentration levels of fluoxetine and its primary metabolite, norfluoxetine, in response to two administration types, cookie and minipump, at three fluoxetine doses (0, 5, and 10 mg / kg / day) and determine the effect of these forms of fluoxetine administration on neural and synaptophysin expression in hippocampus. this study will provide important information toward the goal of generating a new and reliable method of antidepressant administration that eliminates the need for surgery, lowers the stress to the animal, can be readily applied to animal models, provides accurate drug levels, and most closely models antidepressant administration in humans. this work will in a more accurate understanding of the neurobiological and physiological impact of ssris in preclinical models, ultimately improving our understanding of how to treat mood disorders in humans. thirty - six intact adult female sprague - dawley rats (250300 g ; charles river laboratories, france) were used in the present study. rats were kept under standard laboratory conditions in a 12 h:12 h light / dark schedule (lights on at 07:00 h), initially housed in pairs in clear polyurethane bins (48 cm 27 cm 20 cm) with ad libitum access to rat chow (sniff) and tap water. females were randomly divided into two conditions (cookie or minipump) with three dose options: fluoxetine (10 mg / kg / day), fluoxetine (5 mg / kg / day), and vehicle (0 mg / kg / day), for a total of 6 groups. all experiments were approved by the animal ethics board of maastricht university in accordance with dutch governmental regulations (dec 2010 - 146). all efforts were made to minimize the pain and stress levels experienced by the animals. on days 1 - 2, females in the cookie treatment groups were trained for oral ingestion of the medication treatment. for training , females were fed 1/9th of a vanilla wafer cookie (crousti fondante, delacre, belgium), filled with saline. after cookie training, females in the cookie groups were fed a cookie filled with fluoxetine (fagron, belgium : 5 mg / kg or 10 mg / kg) dissolved in vehicle (25% propylenediol in saline) or vehicle solution once per day between 8:00 and 9:00 am after cookie feeding females were monitored to ensure that cookies were eaten. females in the minipump treatment group were administered fluoxetine or vehicle via osmotic minipumps (alzet osmotic pumps, 2ml2, charles river, the netherlands) for 2 weeks. minipump implants were filled with either fluoxetine (fagron, belgium : 5 mg / kg / day or 10 mg / kg / day) dissolved in vehicle (25% propylenediol in saline) or with vehicle as previously described. minipumps were implanted subcutaneously in the dorsal region, while females were under mild isoflurane anesthesia. the weight of a full 2ml2 minipump was approximately 7.5 g. thirty minutes prior to the minipump implant, the nsaid carprofen (dose : 2.55 the first drop of blood was removed ; the second drop was placed on a test strip ( glucocard x - sensor test strips) for immediate glucose measurement with a hand - held glucose meter (glucocard tm x - meter, a. menarini diagnostics, benelux, n.v ., valkenswaard, the netherlands). to determine serum levels of fluoxetine and its active metabolite, norfluoxetine, blood collection, via the tail vein, from females treated with fluoxetine was taken twice on days 6 and 10 between 8 - 9 am, after cookie feeding, and 24 pm. blood from vehicle - treated females was taken on day 6 between 8 - 9 am only. at decapitation, blood samples were stored at 4c overnight and centrifuged at 10,000 g for 10 minutes. serum was collected and stored at 80c until analysis. to investigate whether estradiol levels affected measures of cell proliferation, 17-estradiol (e2) was measured in a subset of animals that were randomly selected (18 in total). all samples were run in duplicate using commercially available 17-oestradiol (e2) i radioimmunoassay (ria) kits from mp biomedicals (mp biomedicals, belgium). the lowest detection limit for e2 was 1.4 pg / ml. drug concentrations were determined from serum using liquid chromatography coupled with mass spectrometry (lc - chip - ms / ms) that was used as previously described. briefly, the chromatographic separation was achieved on a 1200 series lc - chip system (agilent technologies, germany) using an ultrahigh capacity chip including a 500 nl trapping column and a 150 mm 75 m analytical column, both packed with a zorbax 80sb 5 m c18 phase (agilent technologies). the mobile phase was composed of h2o / fa (100 : 0.1, v / v) (a) and acn / h2o / fa (90 : 10 : 0.1, v / v / v) (b) and used in gradient elution mode. mass spectrometric detection was performed using a 6340 ion trap equipped with a nanoelectrospray ionization source operating in positive mode (agilent technologies, waldbronn). finally, an oasis elution mcx 96-well plate (waters, uk) was used to prepare the samples for the analysis. fluoxetine and norfluoxetine levels were averaged across days to provide one morning and one afternoon value. fourteen days after treatment, females were deeply anesthetized with sodium pentobarbital, weighed, and rapidly decapitated. following extraction, the right hemisphere of the brains was stored at 4c in 4% paraformaldehyde for 24 h, then cryoprotected in 30% sucrose / phosphate - buffered saline solution for up to one week, frozen on dry ice, and kept at 80c. brain tissue was sliced in 40 m sections on a cryostat (leica). the level of cell proliferation in the granule cell layer and subgranular zone (gcl / sgz) of the hippocampus was assessed using an endogenous marker for cell proliferation, ki67. every 6th section throughout the right hippocampi was stained as previously described. sections were blocked with h2o2 and incubated overnight in rabbit anti - ki67 (1:500 ; vector laboratories) or blocked with h2o2 and ngs and incubated overnight in mouse antisynaptophysin (1:500 ; sigma aldrich). sections were then incubated for 2 h in biotinylated donkey anti - rabbit (1:500 ; jackson immunoresearch laboratories, west grove, pa) or biotinylated goat anti - mouse (1:200 vector ba-9200) secondary antibody. brain sections were further processed by using the avidin - biotin complex (abc elite kit ; 1:1000 ; vector laboratories, usa). dab (3,3-diaminobenzidine) peroxidase substrate kit (sk-4100, vector laboratories) was used as a substrate to obtain a color reaction. sections were mounted on gelatin - coated slides and dried overnight, dehydrated, stained with cresyl violet (ki67-ir only), and coverslipped with permounttm (fisher scientific, usa). the number of ki67 immunoreactive (-ir) cells in granule cell layer / subgranular zone (gcl / sgz) was counted under 40x objective using a nikon microphot sa microscope. cells were considered ki67-ir if they were intensely stained and exhibited medium round or oval nuclear bodies. ki67-ir cells were counted on half of every 6th section throughout the entire hippocampus. for representative ki67-ir cells in the gcl / sgz of the hippocampus, see figure 1. three dorsal sections of the hippocampus, located between stereotaxic coordinates bregma 2.64 mm to 4.92 mm, were analysed per animal for synaptophysin - immunoreactivity by an observer blind to conditions. photomicrographs were taken for two areas within the ca3 and gcl / sgz of the hippocampus from each of the three sections (e.g., see figure 1) for a total of 6 photomicrographs per area. immunoreactivity was examined under 40x objective using a nikon microphot sa and nikon ds - qi1mc camera with nikon nis elements f4.00 software. the software imagej64 (wayne rasband, nih, bethesda, md, usa) was used for quantification of optical densities of synaptophysin. the relative optical density was defined as the difference between optical density (grey level) measures after calibration within the area of interest and in an equivalent adjacent area (). for representative photomicrographs of synaptophysin density, the fluoxetine and norfluoxetine levels were analyzed using repeated - measures analysis of variance tests (anova) with administration type (cookie versus minipump) and dose (0, 5, or 10 mg / kg) as the between - subjects factors. repeated measure anovas were also used to assess synaptophysin density in the ca3 and gcl / sgz. anovas were conducted on the total number of ki67 cells in the gcl, the percent change in ki67-ir cells from controls, body weight, and glucose levels with administration type (cookie versus minipump) and dose (0, 5, or 10 mg / kg) as the between - subjects factors. any effects of estradiol on the number of ki67-ir and synaptophysin - ir cells were controlled for. for serum fluoxetine and norfluoxetine levels there was a significant interaction between administration type (cookie, minipump) and dose (5 mg / kg / day, 10 mg / kg / day) (fluox : f = 7.95, p = 0.012, norfluox: f = 21.16, p = 0.0002 ) with significantly higher serum levels of fluoxetine / norfluoxetine in the animals receiving 10 mg / kg / day of fluoxetine via minipump (0.000001 < p < 0.0005 ; figure 2). there was also a main effect of time (fluox : f = 57.70, p = 0.000001, norfluox: f = 7.14, p = 0.015 ) with morning levels of fluoxetine / norfluoxetine being significantly lower than afternoon levels. there was a main effect of administration method with minipump administration of fluoxetine ing in significantly higher serum levels of fluoxetine / norfluoxetine compared to cookie administration (fluox : f = 14.68, p = 0.001, norfluox: f = 43.46, p = 0.000001 ). there was also a significant main effect of dose on norfluoxetine levels (f = 22.98, p = 0.00011 ). at sacrifice, serum from trunk blood revealed significantly higher levels of fluoxetine / norfluoxetine in the animals receiving 10 mg / kg / day of fluoxetine via minipump (0.00001 < p < 0.03 ; interaction effect fluox : f = 4.52, p = 0.046, norfluox: f = 14.06, p = 0.0013, figure 2 ). there were also main effects of dose (fluox : f = 8.29, p = 0.0093, norfluox: f = 19.84, p = 0.00024 ) and a main effect of administration type (norfluox : f = 10.44, p = 0.0042 ) on drug levels in trunk blood at sacrifice. there were no other main or interaction effects (0.24 < p < 0.91). as expected, vehicle - treated animals did not have detectable serum levels of fluoxetine or norfluoxetine. minipump animals receiving the 5 mg / kg / day dose of fluoxetine had significantly more ki67-ir cells in the gcl compared to minipump animals receiving the 10 mg / kg / day dose, when looking at overall change from baseline (controls) (f = 9.64, p = 0.013; n = 5/group ). there were no other significant effects of fluoxetine dose or administration type on total number of ki67-ir cells in the gcl / sgz (0.3 < p < 0.7 ; figure 3). there was also no effect of estradiol levels on number of ki67-ir cells in the gcl / sgz (p = 0.98). synaptophysin density in the gcl / sgz was significantly greater in vehicle - treated animals, regardless of administration method (0.000001 < p < 0.03, figure 4 ; region ( ca3, gcl) by dose interaction for synaptophysin density: f = 8.48, p = 0.0012 ). synaptophysin density was also significantly greater in the gcl / sgz than in the ca3 (main effect of region : f = 40.35, p = 0.000001 ). there were no other significant main or interaction effects between groups in synaptophysin density and no effect of estradiol levels on synaptophysin density (0.15 < p < 0.99 ; table 1). as expected, animals fed with the cookie had significantly elevated blood glucose levels compared to animals with minipump implants (f = 8.0435, p = 0.008; table 2 ); however, these levels were still within the physiological range. for body weight measurements there was no significant main effect of administration type or dose on overall change in weight (0.29 < p < 0.58). findings of the present study show that two weeks of fluoxetine treatment in a significant decrease in synaptophysin expression in the dentate gyrus, but not the ca3 region, of adult female rats. in turn , we found that administration method (cookie versus minipump) and fluoxetine dose differentially affected hippocampal cell proliferation, with only females receiving the 5 mg / kg dose via a minipump having increased cell proliferation in the gcl compared to females receiving the 10 mg / kg dose via a minipump. furthermore, administration method differentially affected circulating levels of fluoxetine and its active metabolite, norfluoxetine, with the greatest levels of fluoxetine being evident at a 10 mg / kg dose administered via minipump. in the present study, fluoxetine treatment significantly decreased synaptophysin expressionin the dentate gyrus and had no effect on synaptophysin expressionin the ca3 region of the hippocampus. to our knowledge there is no previous research on the effects of fluoxetine treatment on synaptophysin expression in adult female rats. however, previous research in adult male rats has shown that 7 days of fluoxetine treatment significantly increases synaptophysin mrna levels in the granule cell layer of the hippocampus; however, they did not measure protein levels. work in hippocampal cell culture, under toxic conditions, and in male ts65dn mice (model of down syndrome) also shows that fluoxetine can rescue or improve synaptophysin expression. in addition, the decrease in synaptophysin expression in the dentate gyrus of female rats with fluoxetine administration is perhaps counterintuitive given the general idea that ssri medication enhances hippocampal neurogenesis. however, it is well documented in males only that ssri medications increase hippocampal plasticity and alleviate depressive - like behaviors, suggesting a significant role of estradiol and progesterone on the effects of ssri medications in adult females. further neurochemical and behavioural data are needed to fully understand the functional significance of ssris on hippocampal plasticity in the adult female. in the present study we did not find a significant effect of fluoxetine treatment of synaptophysin expression in the ca3 region of the hippocampus. previous work has shown that fluoxetine administration (5 mg / kg) significantly increases pyramidal spine formation in both the ca1 and ca3 regions of the hippocampus of adult female rats, with effects in the ca1 region being evident after 5 days of fluoxetine administration and effects in the ca3 region being evident after 2 weeks of fluoxetine administration. however, previous work in adult male rats shows no effect of fluoxetine treatment (10 mg / kg) on synaptophysin mrna density. discrepancies between the present study and the previous work in females may be due to methodological techniques as hajszan et al. administered fluoxetine via intraperitoneal injections and used electron microscopy to quantify spine densities, whereas, in the present study, fluoxetine was administered via a cookie or minipump and synaptophysin immunohistochemistry was measured. furthermore, hajszan et al. used ovariectomized female rats whereas female rats in the present study were cycling. estradiol alone is known to have marked effects on spine density, particularly in the ca1 region of the hippocampus , and has been shown to increase serotonin metabolite levels in the ca3 region of the hippocampus. in addition, estradiol has been shown to upregulate serotonin synthesis in the dorsal raphe in a similar manner to fluoxetine. recent work suggests that the effects of fluoxetine on spine density in adult female rats are only evident in ovariectomized females and when the endogenous actions of estradiol on hippocampal spine density are disturbed. thus, there are likely marked interactions between circulating estradiol levels and the effects of fluoxetine on spine density in the hippocampal formation. in the present study , we found effects of fluoxetine on hippocampal cell proliferation only after administration of fluoxetine via minipump. here we show that after 2 weeks of minipump administration females receiving the 5 mg / kg dose of fluoxetine had significantly more proliferating cells in the gcl compared to females receiving the 10 mg / kg via minipump. this work shows that fluoxetine levels can differentially affect cell proliferation in the adult female rat. these findings also replicate previous work showing that fluoxetine (5 mg / kg) has no effect on cell proliferation in the hippocampus of adult female rats when compared to controls. interestingly, previous work in adult male rats shows that 2 weeks of fluoxetine administration (7 mg / kg) via minipump, as in the present study, increases hippocampal cell proliferation. this work and a previous one point to marked sex differences in the effect of fluoxetine on hippocampal plasticity. sex / gender must be taken into consideration when investigating neurobiological effects of ssri medications, particularly as these medications are more often used to treat depression in women. apart from sex / gender, exposure to stress and changes in circulating corticosterone levels also play an important role in the effects of fluoxetine on hippocampal neurogenesis. for example, previous work shows the effect of fluoxetine treatment on hippocampal neurogenesis in the adult female is markedly increased in females exposed to stress. in the present study fluoxetine administration method differentially affected measures of hippocampal plasticity and also serum fluoxetine and norfluoxetine levels. here we show that serum fluoxetine and norfluoxetine levels, in a dose of 5 mg / kg, do not markedly differ between cookie and minipump administration. however, serum fluoxetine and norfluoxetine levels, after a fluoxetine dose of 10 mg / kg, were significantly elevated in animals treated with a minipump. this difference between administration methods in serum levels of fluoxetine at a higher dose is likely due to metabolism of fluoxetine by cytochrome p450 enzymes in the liver after oral (cookie) administration, thus leading to lower circulating levels of fluoxetine and norfluoxetine. in higher doses it may be advantageous to administer fluoxetine twice a day in a cookie in order to increase the circulating levels of fluoxetine and norfluoxetine. we have recently used a twice - a - day cookie administration method (total fluoxetine dose 10 mg / kg) during the postpartum period and shown significant effects on offspring development. others have also shown that voluntary fluoxetine administration in a cookie dough ball is an effective and noninvasive technique to chronically administer this, and potentially other, medication. thus, voluntary ssri medication administration is possible and may be a valuable way to further understand how these medications affect neurobiological processes. the present study investigated the effect of fluoxetine, a common ssri antidepressant medication, on hippocampal plasticity in the adult female rat. main findings show that two weeks of fluoxetine treatment in a significant decrease in synaptophysin expression in the dentate gyrus, but not the ca3 region, of the hippocampus. in turn, administration method (cookie versus minipump) and fluoxetine dose (5 mg versus 10 mg) differentially affected hippocampal cell proliferation in the gcl, with the females receiving the 5 mg / kg dose of fluoxetine via minipump having significantly more proliferating cells compared to females receiving the 10 mg / kg via minipump (when investigating overall change from baseline). furthermore, administration method differentially affected circulating levels of fluoxetine and its active metabolite, norfluoxetine, with the greatest levels of fluoxetine being evident with a 10 mg / kg dose given via minipump.

selective serotonin reuptake inhibitor medications are one of the most common treatments for mood disorders. in humans, these medications are taken orally, usually once per day. unfortunately, administration of antidepressant medications in rodent models is often through injection, oral gavage, or minipump implant, all relatively stressful procedures. the aim of the present study was to investigate how administration of the commonly used ssri, fluoxetine, via a wafer cookie, compares to fluoxetine administration using an osmotic minipump, with regards to serum drug levels and hippocampal plasticity. for this experiment, adult female sprague - dawley rats were divided over the two administration methods: cookie and osmotic minipump and three fluoxetine treatment doses: 0, 5, or 10 mg / kg / day. show that a fluoxetine dose of 5 mg / kg / day, but not 10 mg / kg / day, in comparable serum levels of fluoxetine and its active metabolite norfluoxetine between the two administration methods. furthermore, minipump administration of fluoxetine ed in higher levels of cell proliferation in the granule cell layer (gcl) at a 5 mg dose compared to a 10 mg dose. synaptophysin expression in the gcl, but not ca3, was significantly lower after fluoxetine treatment, regardless of administration method. these data suggest that the administration method and dose of fluoxetine can differentially affect hippocampal plasticity in the adult female rat.

because peripheral nerve tissue has very limited spontaneous regenerative capacity, the development of strategies to facilitate axonal regeneration is highly important for treatment of peripheral nerve injuries. although autotransplantation is considered the gold standard approach for bridging nerve gaps, autogenous grafts provide only a limited source of graft material and graft harvesting may in secondary site morbidity. allogeneic and xenogeneic nerve allografts provide an attractive alternative for patients, for whom the use of autograft is infeasible or undesirable. however, immunological rejection poses a major obstacle to clinical allogeneic and xenogeneic transplantation. unlike organ transplantation, in most cases peripheral nerve injury is not life - threatening, and therefore, peripheral nerve xenotransplantation can only be considered when its benefit is weighted against the risk associated with the therapy, such as the side effects of immunosuppressive drugs. schwann cells and fibroblasts both express mhc antigens and play an important role in eliciting immunological rejection after allogeneic nerve transplantation. however, immunologic responses following nerve xenotransplantation are relatively poorly understood. in this study, we investigate the immunological response and graft survival in rats after nerve xenotransplantation from mice. we show that mouse nerve xenografts were vigorously rejected in rat recipients, and that the rejection was associated with severe intragraft infiltration by mononuclear cells despite only a transient and moderate increase in th1 cytokines detected in the recipients. female sd rats (weighed between 150 and 200 g) and adult female balb / c mice were used as the recipients and donors, respectively. all animals were purchased from the second affiliated hospital of harbin medical university (haerbin, china). all surgical procedures and postoperative care of the animals all surgical procedures were performed under anesthesia with ketamine (50 mg / kg ; i.m .). to prepare donor nerve grafts, the mouse sciatic nerve was exposed through a dorsal gluteal muscle splitting incision, and a segment (1 cm) of sciatic nerve was harvested and used immediately. the skin over the recipient right hindlimb was incised, and the muscle was bluntly dissected to expose the superficial peroneal nerve, and a 1 cm gap was created. the mouse sciatic nerve graft was interposed to the transected nerve and immediately repaired with 10 - 0 nylon epineurial sutures. for autograft recipients, the right superficial peroneal nerve was exposed in an identical fashion, transected with microscissors, and tension - free repair of the nerve gap (1 cm) was then performed with 10 - 0 nylon epineurial sutures. sera were collected from recipient mice at various times, and the levels of il-2, il-4, ifn-, and tnf- were determined using elisa kits (westang biotechnology, shanghai, china) according to the manufacturer's instructions. animals were sacrificed at various time points after transplantation, and the nerve grafts were harvested and cut in half horizontally. one segment was immersed in a 10% formaldehyde solution, dehydrated in ethanol, and embedded in paraffin. the paraffin nerve sections were stained with hematoxylin and eosin (h&e) and by immunohistochemistry for s-100. briefly, sections for s-100 staining were first incubated with polyclonal rabbit anti - s-100 antibodies (westang biotechnology), washed, and followed by staining with biotinylated goat antirabbit antibodies (westang biotechnology). another segment of the nerve graft was immersed in a 3% cold gluteraldehyde solution, postfixed with osmium tetroxide, dehydrated in ethanol, and embedded in araldite 502. ultrathin sections were obtained with an ultramicrotome (ultracut e, reichert - jung, vienna, austria), stained with lead citrate and uranyl acetate, and observed under a jeol transmission electron microscope (jem-1220 ; jeol ltd ., tokyo, japan). differences between group means were tested using student's t - test by microsoft excel software. recipient rats were sacrificed 2, 4, and 8 weeks after transplantation and the nerve grafts were examined macroscopically and histologically (n = 5 per group at each time point). all autografts showed no distension or adhesion to the ambient tissues throughout the 8-week observation period. however, xenografts removed at week 2 markedly dilated and adhered to the surrounding tissues. while some xenografts showed moderately reduced distension at week 4, graft adhesion to the ambient tissues was progressively overwhelming in all animals during the 8-week observation period. histological examination at weeks 2 and 4 revealed very mild mononuclear cell infiltration with otherwise normal peripheral nerve structure in autografts (figures 1(a)-1(b) and 2(a)-2(b) ). however, most autografts showed signs of wallerian degeneration at week 8, with moderate axonal degeneration and nerve fiber fractions (figures 1(c) and 2(c) ). consistent with the light microscopic analysis, electron microscopy confirmed the preservation of normal fascicular structure in autografts at weeks 2 and 4 (figures 3(a)-3(b) ), but schwann cell cytoplasmic degeneration, chromatin condensation, and nuclear collapse were identified in the autografts at week 8 (figure 3(c) ). severe mononuclear cell infiltration, distension and necrosis were detected in all xenografts as early as 2 weeks after transplantation (figures 1(d)1(f) ). the number of axons in the grafts markedly reduced by week 2 and became almost undetectable at week 8 (figures 2(d)-2(e) ). electron microscopy revealed inflammatory infiltrates in nerve fibers, schwann cell nuclear pycnosis and mitochondrial damage, and progressive destruction and loss of myelinated fibers in all xenografts (figures 3(d)2(f) ). sera were collected from xenograft recipients prior to 2, 4, and 8 weeks after transplantation, and the levels of il-2, il-4, ifn-, and tnf- were determined by elisa (table 1). while the serum levels of il-2, ifn-, and tnf- at week 2 were statistically higher in the xenograft recipients than in the control rats, these increases were relatively less marked and transient. the serum levels of all these cytokines in the xenograft recipients returned to normal by week 4 after transplantation. xenotransplantation provides a possible solution to the severe shortage of allogeneic donors that limits clinical transplantation , but the virulent xenoimmune responses pose a strong barrier to clinical xenotransplantation. transplantation of discordant xenografts in hyperacute rejection due to the presence of natural antibodies (nabs) in the recipient sera. the major nabs that mediate hyperacute rejection are those which recognize the 1, 3gal antigens. however, anti-1, 3gal abs are unlikely to mediate the rejection of nerve xenografts, as neural cells do not express 1, 3gal. thus, cellular immune responses, which play a critical role in nerve allograft rejection, are likely to be more important than humoral immune responses in nerve xenograft rejection. indeed, nerve xenograft rejection has been relatively poorly studied compared to other cellular or organ xenografts. here , we observed that mouse nerve xenografts can be vigorously rejected in rats, and the rejection is associated with a severe mononuclear cell infiltration in the grafts. schwann cells are critical for nerve regeneration, but also the major cells trigger the rejection of nerve allografts because of their mhc expression. it has been reported that cold preservation can decrease the number of schwann cells and reduce the immunogenicity of nerve allografts. although recipient schwann cells may migrate into the graft and support axon regeneration, donor schwann cells in the graft are required for the regeneration of a long nerve graft. given the possibility that the recipient schwann cells may be less efficient in migrating into nerve xenografts than into allografts, cold preservation could have an even greater limitation for preventing nerve xenograft rejection. although histological analysis confirmed severe mononuclear cell infiltration and complete nerve xenograft rejection, the recipient rats showed only a transient and moderate increase in th1 cytokine production. further studies are needed to determine whether peripheral nerve xenografts are less efficient than other types of xenografts in eliciting the systemic t cell immune responses. it is possible that other effector cells, such as th17 cells, nk cells, and macrophages, may play an important role in the rejection of mouse nerve xenografts in rats. previous studies have shown that nk cells and macrophages contribute significantly to xenograft rejection due to the genetic incompatibility in mhc class i and cd47 , which are needed for inhibiting nk cell and macrophage activation, respectively. further studies are clearly needed to identify the major effector cells in the rejection of peripheral nerve xenografts.

xenotransplantation offers a potentially unlimited source for tissues and organs for transplantation, but the strong xenoimmune responses pose a major obstacle to its application in the clinic. in this study, we investigate the rejection of mouse peripheral nerve xenografts in rats. severe intragraft mononuclear cell infiltration, graft distension, and necrosis were detected in the recipients as early as 2 weeks after mouse nerve xenotransplantation. the number of axons in xenografts reduced progressively and became almost undetectable at week 8. however, mouse nerve xenotransplantation only led to a transient and moderate increase in the production of th1 cytokines, including il-2, ifn-, and tnf-. the data implicate that cellular immune responses play a critical role in nerve xenograft rejection but that further identification of the major effector cells mediating the rejection is required for developing effective means to prevent peripheral nerve xenograft rejection.

bread wheat (triticum aestivum l.) is one of the most important crop species providing the staple food for 40% of the world's population. as with other crops, the yields of wheat are annually significantly reduced due to attack of a large variety of pests and diseases. supplementation of conventional wheat breeding for pathogen resistance with marker - assisted breeding and direct gene transfer by molecular methods promises to enhance the efficiency of plant breeding. prerequisites of this approach are the saturation of genetic maps in the region of interest and isolation of the resistance gene. positional or map - based cloning is an experimental approach to isolate unknown genes based on their position in the genome. this approach involves construction of a high - density genetic map covering the target locus and a physical map spanning the region of interest. physical maps are produced by ordering dna clones from large - insert (usually bac) libraries into contigs on the basis of clone fingerprint pattern. whereas some wheat genes can be mined from smaller genomes of related or model species, many agronomically important genes, including those for pathogen resistance, yield, or grain quality factors, however, only a few bread wheat genes, including those conferring pathogen resistances, grain protein content, vernalization requirement, and domestication traits , have been successfully cloned. t. aestivum is an allohexaploid species (2n = 6x = 42, aabbdd genome), which originated from a spontaneous hybridization of three diploid wheat ancestors, donors of the a, b, and d genomes, respectively. they contributed to the enormous size of the bread wheat genome (1c17 gbp), which is composed of ~1% of genes interspersed by huge amounts of repetitive elements. a variety of genomic resources has been developed to enable positional cloning in bread wheat including a number of bac libraries. while fingerprinting so many clones is technically feasible using the snapshot - based hicf procedure and recent indicate that existing computational techniques may allow for reliable assignment of fingerprinted bac clones to particular homoeologous chromosomes, handling and screening libraries composed of more than million clones remain expensive and tedious tasks. these obstacles can be overcome by working with a library derived from a smaller part of the wheat genome. one option is to use diploid genome - donor species or their relatives as surrogates, taking advantage of smaller genome size and absence of polyploidy. several bac libraries of diploid wheat progenitors have been constructed including those of t. monococcum and ae. tauschii physical map (see, http://www.wheat.ucdavis.edu) were employed in cloning agronomically important genes. however, clone numbers in these libraries are still too large to be screened easily. moreover, wheat genomes have undergone revolutionary changes following the polyploidization events including losses of dna. this partial diploidization and other genomic changes suggest that physical maps and genomic sequences of wheat diploid ancestors, although useful resources for wheat genomics, can not fully substitute for the genomic sequence of hexaploid wheat itself. recently, we proposed an alternative approach utilizing flow cytometry to dissect the hexaploid wheat genome into small fractions chromosomes or chromosome arms, which represent only a few percent of the hexaploid wheat genome. the chromosome - based strategy was made possible by developing procedures for purification of particular wheat chromosomes by flow sorting and a protocol for preparation of intact dna from sorted chromosomes suitable for cloning. this allowed us to create the first ever bac library derived from a single chromosome of a higher eukaryote. our ability to purify single chromosome arms from hexaploid wheat enabled us to construct bac libraries from 13 of the 21 wheat chromosomes thus far (see , http://olomouc.ueb.cas.cz/dna-libraries/cereals), as well as from the short arm of rye 1r chromosome, which is a component of a number of wheat varieties. here, we report on the construction of bac libraries from both arms of wheat chromosome 7d, which harbors numerous genes underlying agronomically important traits, mainly resistance genes. the long arm of 7d is known to carry several greenbug (schizaphis graminum) resistance genes as well as qtls influencing russian wheat aphid (rwa, diuraphis noxia) resistance. the short arm of 7d comprises several major genes and qtls for rwa resistance , the recently dissected lr34 locus underlying resistance to leaf rust, yellow rust, stem rust and powdery mildew, genes stb4 and stb5 underlying resistance to septoria tritici blotch as well as several yield - related qtls. in order to demonstrate how the chromosome - arm - specific bac libraries can facilitate positional gene cloning in a supersized plant genome , we report on a hybridization - based screening of the 7dl library with markers for the greenbug resistance gene gb3, and a pcr - based screening of the 7ds library with markers for russian wheat aphid resistance gene dnci2401. chinese spring carrying both arms of chromosome 7d as telosomes (2n = 40 + 2t7ds + 2t7dl) was used to sort the arms. four thousand two hundred seeds subdivided into 184 batches of 2025 were germinated in the dark at 25c on moistened filter paper for 5560 h to reach root length of 2 - 3 cm. cell - cycle synchronization, accumulation of metaphases in root tips and preparation of chromosome suspensions were performed as described. briefly, chromosome suspensions were prepared by mechanical homogenization of 2025 root - tip meristems enriched for metaphase cells in 1 ml ice - cold isolation buffer (ib,). chromosomes in suspension were stained with 2 g / ml dapi (4,6-diamidino-2-phenylindole) and analyzed using a facsvantage se flow cytometer (becton dickinson, san jose, usa). both arms were sorted separately from the same sample in batches of 200,000 into 320 l of 1.5xib. the purity in sorted fractions was checked regularly by fish with probes for telomeric and gaa repeats as described. preparation of high molecular weight dna (hmw dna) and library construction were performed as described with some modifications. briefly, each batch of flow - sorted arms was spun down and the pelleted chromosomes were mixed with low - melting point agarose to form 20-l miniplugs, which were incubated in lysis buffer containing proteinase k to purify the chromosomal dna. the isolated hmw dna was partially digested with hindiii (new england biolabs, beverly, mass ., usa) and subjected to two rounds of size selection. at the first round, the partially digested dna was size - separated in 1% seakem gold agarose gel (lonza, rockland, ill ., usa) in 0.25x tbe under the following conditions of pulsed - field gel electrophoresis (pfge): voltage 6 v / cm, switch time 150 s, run time 17 hours. size fraction of 100210 kb was excised from the gel and split into two parts. fraction b comprised fragments of 100150 kb whereas fraction m represented a fraction of 150210 kb. both fractions were subjected to a second round of size selection in 0.9% seakem gold agarose gel in 0.25x tbe under the following conditions: voltage 6 v / cm, switch time 3 s, run time 17 h. a gel zone corresponding to 100150 kb was excised from the lane containing the b fraction and was subdivided into two slices comprising a fraction of 100120 kb (b1) and 120150 kb (b2), respectively, whereas only one gel slice (~150200 kb) was excised from the m lane. the dna of particular fractions was electroeluted from the gel and ligated into hindiii - digested dephosphorylated pindigobac-5 vector (epicentre, madison, wisc ., usa) in 1: 4 molar ratio (dna : vector). the recombinant vector was used to transform escherichia coli electromax dh10b competent cells (invitrogen, carlsbad, calif . the library was ordered by qbot ( genetix, new milton, uk) into 384-well plates filled with 75 l freezing medium consisting of 2yt, 6.6% glycerol and 12.5 g / ml chloramphenicol. the clones were stored at 80c. a total of 121 bac clones from the 7dl (63 from the b1, 32 from the b2, and 27 from the m fraction) and 184 clones from the 7ds library (56 from the b1, 76 from the b2 and 52 from the m fraction) were analyzed to estimate average insert size and percentage of empty bac clones. the bac dna was isolated after overnight incubation of particular bac clones in 1.5 ml 2yt supplemented with 12.5 g / ml chloramphenicol using a standard alkaline lysis method. ing dna fragments were separated in a 1% agarose gel in 0.25x tbe buffer by pfge. the size of the fragments was estimated by comparing with two size markers: lambda ladder pfg marker and midrange marker i (new england biolabs). high - density colony filters were prepared from the 7dl - specific bac library by spotting bac clones in duplicate in a 4 4 gridding pattern onto hybond n+ nylon membranes (ge healthcare, piscataway, nj, usa) using the genetac g3 robot (genomic solutions, ann arbor, mich . an est - derived sts marker ( sts - aug-08 - 28) was mapped 0.08 cm proximal to the greenbug resistance gene gb3 (azhaguvel et al . the pcr product was separated by agarose gel electrophoresis and a fragment of desired size was eluted from the gel using qiaquick gel extraction kit ( qiagen, germantown, md . the fragment was then p - datp radio - labeled by neblot kit ( new england biolabs) using manufacturer's protocol. the labeled probe was purified in a sephadex g50 column (ge healthcare) and denatured at 100c for 10 min. for prehybridization, overnight incubation of colony filters in hybridization solution (2x sspe, 0.5%sds, 5x denhardt 's reagent, 40 g / ml herring sperm dna) was done in rotary glass tubes at 65c. the labeled probe was mixed with 5 ml of hybridization solution and colony filters were incubated at 65c overnight. to remove the unbound probe, we washed the filters twice in washing solution containing 2x sspe and 0.5% sds and rinsed with 1x ssc. the washed filters were exposed to x - ray film for one to three days based on the signal intensity to identify positive clones. to complete the assembly of the contig spanning the gb3 region, we conducted three rounds of 7dl bac library screening. the sts - aug-08 - 28 marker was used for the first round of the screening whereas probes derived from protruding ends of bac no. 25, respectively, were used for the second and third round of screening (figure 3). the positive bac clones ing from each bac library screening were grown overnight in lb media supplemented with 12.5 g / ml chloramphenicol. bac dna was extracted by standard alkaline lysis procedure and subjected to hindiii digestion to create a fingerprint for each positive clone. five micrograms dna of all positive clones in each screen were extracted with qiagen plasmid kit (qiagen) and used for direct cycle sequencing of bac ends using t7 (5taa tac gac tca cta tag gg 3) and m13r (5 cag gaa aca gct atg acc 3) primers. moreover, three bac clones (bac 22, bac 25 and bac 72) were sequenced completely by sanger technology at the genome center, washington university, st. the alignment of bac end sequences with the fully sequenced bac clones was done with seqman module in the software dnastar (lasergene corp ., the insert size of the positive bac clones was determined by aligning bac end sequences with the reference sequences of the completely sequenced bac clones . bac end sequences were used to develop sequence tagged site ( sts) or cleaved amplified polymorphic sequence (caps) markers, which were used for further screening of the library to identify and/or confirm overlapping bacs to span the region of interest. to enable pcr screening of the 7ds library , we generated three types of bac pools: plate pools, superpools and three - dimensional (3d) pools. first, 128 plate pools were prepared by pooling clones from each of 384-well plates comprising the library. rectangular dishes (nunc, rochester, ny, usa) containing agarose (1.6%) 2yt medium with 12.5 g / ml chloramphenicol were inoculated with all 384 clones of single plates using a genetac g3 robot. after 16-h incubation at 37c, the clones were suspended in 510 ml te buffer (10 mm tris, 1 mm edta, ph 8.0), bacteria were pelleted by centrifugation and subsequently resuspended in 600 l get buffer (50 mm glucose, 25 mm tris, 10 mm edta, ph 8.0). dna was isolated using standard alkaline lysis protocol supplemented with rnase treatment and precipitation of contaminating compounds by ammonium acetate. the dna of each aliquot was dissolved in 20 l sterile deionized water; the aliquots were combined and stored at 20c. for pcr screening these stocks were diluted to 10 ng/l dna. to prepare the 3d pools we created 48 pools (8 plate, 16 row and 24 column pools) for each stack, thus totally 768 pools represented the entire library. to prepare the pools, we spotted bacterial clones in duplicate on plates with agarose medium as described above. bacteria grown for 48 h were suspended in 5 ml te buffer and boiled for 30 min to release dna. remnants of bacteria were pelleted at 3000 g for 60 min and 1.4 ml of the supernatants were deposited at 20c as the particular pools. for pcr superpools were created for each of the 8-plate stacks by combining all clones from the respective 8 plates. spotting, growth of bacteria and dna isolation were done as for the 3d pools. the 7ds - specific library was screened with microsatellite markers xcfd68, xgwm473 and xbarc214 closely linked to the dn gene. primer sequences were retrieved from the graingenes database (http://wheat.pw.usda.gov/gg2/index.shtml). the pcr reaction mix (10 l volume) consisted of 2 l template dna, 1 buffer for dynazyme dna polymerase (finnzymes, espoo, finland), 0.01% cresol red, 1.5% saccharose, 0.2 mm dntps, 1 m primers and 0.4 u dynazyme ii dna polymerase (finnzymes). the pcr reaction was performed under the following conditions: denaturation for 5 min at 95c followed by 35 cycles of denaturation at 95c for 30 s, annealing at the appropriate temperature for 30 s, extension at 72c for 30 s, and a final extension at 72c for 10 min. the presence of pcr products was detected by electrophoresis in 1.2% agarose gel run in 0.5x tbe buffer. the precise size of individual pcr products was determined using abi 3730xl sequencer (applied biosystems, foster city, calif . the fragment sizes were estimated relative to the genescan-500 liz size standard ( applied biosystems) by genemarker v1.75 (softgenetics, llc, usa). all clones of the 7ds - specific library were fingerprinted using the snapshot - based high - information - content fingerprinting technology. the fingerprints were automatically edited with the computer program package fpminer (www.bioinforsoft.com) and genoprofiler and assembled using fpc v9.3 at an initial cutoff of 1 10, followed by various steps of dqing and end merging. the short and the long arms of the 7d chromosome were sorted simultaneously from the 7d double - ditelosomic (ddt) 7d line. the flow karyotype obtained from this line consisted of two peaks representing 7dl and 7ds telocentric chromosomes, respectively, three composite peaks comprising groups of various wheat chromosomes and the rightmost peak corresponding to the largest wheat chromosome 3b (figure 1). the leftmost peak, representing a chromosome with the smallest relative dna content, actually comprised telosome 7dl and not 7ds, as the arm designated 7ds based on homology with the wheat chromosome arms 7as and 7bs is physically longer and hence has greater relative dna content. the purity of sorted telosomes as estimated by fish varied between 8492% (average 88.8%) for the 7dl and between 7986% (average 84.1%) for the 7ds telosome. the 7d chromosome is metacentric and the 7dl and 7ds arms are of similar size (2.04% of the wheat genome alias 346 mbp for 7dl and 2.25% of the wheat genome alias 381 mbp for 7ds,). the 7ds constituted 1.3% of the sorted 7dl whereas 7dl represented 1.1% of the sorted 7ds fraction. the remaining contamination was composed of a mixture of chromosomes without a prevalence of a particular type. in total, 31 agarose miniplugs comprising 5,900,000 7dl telosomes corresponding to ~4.18 g dna were prepared. in parallel, 6,000,000 7ds telosomes (~4.67 g) were embedded in 30 agarose plugs. bac libraries were created from both telosomes using the hindiii cloning site. the library derived from the long arm of the chromosome 7d called taacsp7dlha comprised 50,304 clones ordered in 131 384-well plates. the average insert size of the whole library reached 116 kb and, excluding 0.5% empty clones and considering the 11% contamination by other chromosomes and the size of 346 mbp, the library provided 14.9x coverage of 7dl. the library derived from the short arm of the chromosome 7d named taacsp7dsha consisted of 49,152 clones ordered in 128 plates. having 113 kb mean insert size and 1.4% empty clones, the library constituted 12.1 arm equivalents if the 16% contamination with other chromosomes and 7ds molecular size of 381 mbp were considered. the representation and mean insert size of the particular fractions are shown in table 1. the overall distribution of insert sizes, which were estimated for 121 clones from the 7dl library and 184 clones from the 7ds library, ranged from 10 to 200 kb and differed slightly between the libraries as shown in figure 2. whereas most clones (39%) were found in the fraction of 125149 kb in the 7dl library, the most numerous fraction (42% of clones) of the 7ds library is in the size range of 100124 kb. this was due to greater representation of b2 compared with b1 fraction in the 7dl library. on the other hand, the 7ds library contained a greater percentage of large clones more than 150 kb (9% versus 7% for 7dl) although the average insert size of the m fraction was significantly greater in the 7dl library (147 kb for 7dl - m versus 130 kb for 7ds - m). this seeming discrepancy was due to a greater proportion of the m fraction in the 7ds library (11% for 7dl - m versus 18% for 7ds - m). the portion of bac clones smaller than 50 kb, which are usually noninformative in hicf analysis applied in construction of physical maps, was negligible in both libraries (below 2%). on the other hand, presence of short clones less than 75 kb in all fractions of both libraries suggested that the ligation ratio between the wheat dna and the vector was set up correctly preventing ligation of more than a single dna fragment into one vector due to excess wheat dna. an improper ratio would have led to the creation of chimeric clones, which significantly compromise the quality of libraries. absence of chimeric clones in the 7d libraries was further supported by the fact that no inserts exceeding sizes excised from the gel at the size selections were found for the b1, b2 and m fractions, respectively. the mean insert size was estimated to be 116 kb for the 7dl and 113 kb for the 7ds library; both were significantly larger than for early wheat chromosome - specific bac libraries, which reached 82 kb in the 1bs - specific library, 85 kb in a composite library constructed from chromosomes 1d, 4d and 6d and 103 kb in the 3b - specific library. the increase in insert size was achieved by including a second size - selection step in the bac library construction procedure. available genomic bac libraries created from hexaploid wheat, which can be used as a standard for evaluating the quality of the 7d - specific libraries, varied significantly in their average insert size, which ranged from 75 kb for a library constructed from wheat cv. however, in a comparison of the coverage of the libraries, even the most representative of the wheat genomic libraries constructed by allouis et al. providing 9.3x wheat genome coverage does not reach the coverage of our chromosome - specific libraries (14.9 and 12.1x for the 7dl and 7ds libraries, resp .). est - derived marker sts - aug-08 - 28 (azhaguvel et al . in preparation) tightly linked with the gb3 gene was used as a hybridization probe to screen the 7dl - specific bac library (figure 3, screen i) providing 14 positive clones. marker sts - bac 22-r developed from the protruding end of bac 22 clone was used as a probe for a second round of library screening and detected 22 positive bac clones including two selected in the previous screen (figure 3, screen ii). 25-t7 derived from the protruding end of bac no. 25 (figure 3, screen iii). this marker identified 23 positive bac clones (including five bac clones detected in the screen ii). three bac clones (bac 22, bac 25 and bac 72) were fully sequenced and assembled. the sizes of bac 22, bac 25 and bac 72 were 200.56 kb, 110.95 kb and 117.09 kb, respectively. markers that were continuously derived during the contig assembly from available bac sequences cosegregated with the resistance gene. only a marker derived from the end of the bac 72 showed recombination with the gene and flanked the gb3 region from the opposite side. thus three rounds of screening the 7dl library were sufficient to build a contig spanning the region between markers flanking the gb3 gene. positional cloning of gb3 and a detailed annotation of this contiguous sequence will be described elsewhere (azhaguvel et al . the number of positive clones selected by hybridization with single - copy probes and confirmed by contig assembly and sequencing outputs indicates library coverage greater than estimated based on insert size analysis . in fact, the number of positive clones confirmed by fingerprinting was 20 or more in all screens ( 21.7 at average) but in the screen i, bac - end sequences were available for 14 of 20 clones, so only these clones were included in the final contig assembly. as all these screens were conducted in one region of the 7dl arm, we do not conclude that this number (21.7) shows evidence of overall underestimation of coverage based on insert size analysis but rather indicated overrepresentation of this region in the library. differences in local coverage were observed also in the 7ds library for particular markers (table 2 and figure 4). the above mentioned numbers of positive bac clones were obtained after screening only three high - density filters that comprised the whole 7dl library. for comparison, bac libraries of diploid wheat relatives t. urartu, ae. speltoides and ae. tauschii representing 3.7, 5.4 and 4.1 equivalents of the respective genomes occupied 9, 13 and 10 high - density filters, respectively. consequently, one filter comprising 18,432 clones of a diploid library represents only 0.41x genome coverage. an even greater workload would be required in hybridization screening of the polyploid wheat libraries. the bac library of tetraploid t. turgidum with 5.8x coverage occupies 28 high - density filters; thus one filter carries just 0.2x genome equivalents. glenlea was spotted on 24 filters with a greater density of 27,360 clones per filter. despite of that, one filter represented only 0.13x genome equivalents. on the other hand, the 7dl library with coverage of 14.9x was placed on only three filters and thus the 18,432 clones spotted on one filter cover the 7dl arm 5.46x. such coverage would be sufficient for positional cloning providing 99.5% probability of recovering any sequence present on the arm and poses a strong argument for the chromosome - based genomics in wheat. microsatellite markers xcfd68, xbarc214 and xgwm473 were found to be tightly linked to the dn gene (fazelnajafabadi and lapitan, unpublished). these markers were applied to screen the 7ds library using the set of bac pools. as the first step , pcr was run on the superpools representing stacks of 8 plates, followed by screening on the plate pools and finally 3d pools. although some of the superpools did not provide a pcr product, screening was performed on all plate pools to verify the reliability of the information obtained from superpools. the from superpools and plate pools, respectively, were in full accordance for all markers tested. this implies the superpools can be used to preselect stacks of plates to be subjected to further screening and thus reduce the number of pcr reactions needed for library screening. however, considering the small number of 3d pools and the high coverage of the library due to which 37.575% of the superpools (depending on the locus) were found to be positive, this step is not essential. similarly, pcr was run on all plate pools and 3d pools, respectively, to compare the . in 6 of 47 cases (12.8%) the information from the plate pools helped in identifying the positive plates in the 3d pool set. these were cases when the pcr product obtained from 3d pools, which were prepared by a simplified procedure, was too weak for an unambiguous identification of the positive plate. thus the set of plate pools prepared by a more advanced dna - isolation procedure proved to be useful in resolving the position of the positive clones. however, as demonstrated below, its role can be substituted by availability of data from the bac contig assembly. among the positive bac clones selected by xbarc214 and xgwm473, several bac clones were found that provided products of an unexpected size. by comparing these products with products obtained by pcr on dna of flow - sorted 7ds arms we verified that these products did not relate to the 7ds arm. these bac clones remained as singletons in the contig assembly, which implies that they come from contamination of the sorted fraction by other chromosomes. such clones were not considered positives. however, screening the 7ds library with the xgwm473 marker provided an even more complex spectrum of pcr products. besides clones bearing a double - band of 220 and 226 bp, respectively, which has been characteristic of the dnci2401-linked marker, and a few clones of unexpected size, we also found numerous clones that generated a pcr product of 200 bp. this product was also visible as a weaker band when amplifying dna of flow - sorted 7ds. this presumption was supported by finding a bac contig ctg135 comprising clones with the 200 bp pcr product (figure 4(d) ). sequencing the locus both from positive clones of ctg285 and ctg135 confirmed that the two loci differed. comparing sequences of the 226 and the 200 bp band revealed a 26 bp deletion and 92% homology in the remaining sequence. this locus is probably not polymorphic in available mapping populations as there is no evidence about it in databases as graingenes or gramene. the of the screening the 7ds library with xcfd68, xbarc214 and xgwm473 are shown in figure 4 and table 2. most of the positive clones were identified unambiguously based on pcr screening of plate and 3d pools. the xcfd68 marker selected nine positive clones in the screening and two additional bac clones were revealed after integrating data from the bac contig assembly as row or column information for these clones was missing (figure 4(a) ). similarly, for xbarc214 15 positive bac clones were identified by library screening whereas bac addresses of another two clones could be completed only after considering data from the bac contig assembly (figure 4(b) ). in case of the xgwm473 marker, nine and 16 positive clones, respectively, were identified in ctg285 and 135, respectively (figures 4(c) and 4(d) ). all bac addresses were complete; however, three of ten bac clones providing the 220 and 226 bp products were absent from the contig assembly, thus their position in ctg285 could not be verified. they might have been missing in the library replica used for fingerprinting or excluded by the genoprofiler software due to a low quality of fingerprint or cross - contamination. our imply that the proposed system of bac pools is sufficiently powerful to reveal positions of positive bac clones. having contig assembly data in hand makes the deconvolution of bac addresses easier and enables further reducing the number of clones to be screened. the local coverage differed among the tested loci and ranged between 10 and 17x, averaging 13.5x. this is in agreement with the coverage estimate based on insert size (12.1x). various pooling strategies have been proposed for pcr - based screening of wheat libraries for cloning of genes. employed the analogous pooling strategy as applied in this study involving superpools (combining clones from ten consecutive plates) and 3d (plate, row, and column) pools created for stacks of ten plates. the glenlea wheat library comprising 3.1 genome equivalents was represented by 171 superpools and 8,550 3d pools. in their case , assaying superpools played a substantial role in reducing the overall number of pcr reactions. in this two - step screening, a single positive bac clone could be reached in 221 (171 superpools + 50 3d pools for the selected superpool) pcr reactions. however, for obtaining several clones, which is preferable in positional cloning, significantly more pools would have to be assayed. a different pooling strategy was proposed by febrer et al. who screened a part of the wheat genomic library constructed by allouis et al. for a wheat dwarfing gene rht. the screen of 715,776 clones from the library (~5 genome equivalents) was based on pooling of dna from bac clones into 675 superpools arrayed in a three - dimensional configuration. a second round of pcr was used to detect a specific bac clone within the candidate plate that corresponded to the gene of interest. so assaying at least three 384-well plates was needed to identify a single copy of the target gene. for identifying all three homoeologues of the rht gene, 17 candidate plates were screened providing 13 rht - containing clones. for comparison, assaying one 384-well plate with 3d pools of the 7ds library (representing half of the library) supplemented with deconvolution of the positive clones was sufficient to identify four to 12 copies of a particular microsatellite locus. once a bac contig assembly has been completed, screening can be further simplified by preparing bac pools from mtp clones only. in case of the wheat 3b - chromosome - specific bac library consisting of 67,968 clones, thus the library was represented by 60 three - dimensional pools, which were sufficient for screening the whole library. in the case of a library from the short arm of chromosome 3d containing 36,864 clones, the mtp comprised 3,823 bac clones occuring in 50 three - dimensional pools. screening this reduced number of pools can identify a bac contig containing the marker; however, as the mtp may not comprise the complete chromosomal sequence and its coverage is ~1x, the risk of losing some information is relatively high and thus screening the 3d mtp pools of the 3b library was combined with screening plate pools prepared from the whole library. microsatellites represent a frequently used marker system as they can be easily extracted from genomic (e.g., bac - end or shotgun) sequences, are abundant and highly polymorphic. they are a typical marker of choice in efforts to place genes of interest on a genetic map. however, microsatellites proved to be of limited use in large - scale screening of a soybean genomic bac library to anchor the physical contig map because of their multilocus character. screening by microsatellites in a hexaploid wheat library would be even more complicated due to the presence of homoeologous genomes. this view is supported by findings of nilmalgoda et al. who screened a hexaploid wheat library of 3.1x coverage both with gene - derived and microsatellite markers. whereas gene sequences identified 2.7 positive bac clones on average, the average number of clones hit by microsatellites was twofold (5.5 clones). our small - scale screening detected two loci relating to one of the microsatellite markers (xgwm473) even on one chromosome arm. similarly, multiple loci were found for another microsatellite marker (xgwm44) when screening the 7ds library and analyzing the selected clones using the available contig assembly (imkov, unpublished). this indicates that of screening bac libraries with microsatellite markers must be interpreted with caution even in the case of chromosome - arm specific libraries. the screening of the library with markers for the dnci2401 gene is a first step towards positional cloning of this gene. the work is in progress to derive new markers from colinear regions of related genomes (rice, brachypodium) that were identified based on sequencing bac clones from contigs containing the markers. two bac libraries specific for both the long and the short arms of the bread wheat chromosome 7d were constructed with parameters challenging available wheat genomic libraries. the libraries represent the first subgenomic bac resources available for wheat homoeologous chromosome group 7 and will facilitate advancement in many areas of wheat genomics, including physical map construction and map - based cloning of genes. due to the small number of clones, high genome coverage and absence of clones from homoeologous genomes, these libraries make screening with markers for genes of interest highly efficient and cost - effective. for example, one high - density filter prepared from the 7dl library provided coverage of 5.4x, which ensures 99.5% probability of finding any sequence located on the 7dl chromosome arm. a simple pooling strategy was elaborated based on which 6x equivalents of the 7ds arm could be screened by 384 pcr reactions. after completing physical map assembly of the 7ds arm, the number of pools to be screened can be further reduced. all these features and the of screening these libraries with markers for aphid resistance genes indicate that chromosome - arm - specific bac libraries are a powerful resource for cost - effective positional gene cloning in bread wheat.

positional cloning in bread wheat is a tedious task due to its huge genome size and hexaploid character. bac libraries represent an essential tool for positional cloning. however, wheat bac libraries comprise more than million clones, which makes their screening very laborious. here, we present a targeted approach based on chromosome - specific bac libraries. such libraries were constructed from flow - sorted arms of wheat chromosome 7d. a library from the short arm (7ds) consisting of 49,152 clones with 113 kb insert size represented 12.1 arm equivalents whereas a library from the long arm (7dl) comprised 50,304 clones of 116 kb providing 14.9x arm coverage. the 7ds library was pcr screened with markers linked to russian wheat aphid resistance gene dnci2401, the 7dl library was screened by hybridization with a probe linked to greenbug resistance gene gb3. the small number of clones combined with high coverage made the screening highly efficient and cost effective.

it generally is accepted that wear and loosening depend on proper alignment of the components and the limb, however, review of the methods of previous research of limb alignment in tka has revealed two limitations that cause us to question the relationship between wear and loosening. these limitations include the use of a short film of the knee instead of a whole leg view to assess limb alignment, and the study of a knee component with size, shape, and fixation features that no longer are deemed desirable. the use of the standard short knee film as a substitute for the whole leg view for assessing component and limb alignment is commonplace in everyday practice but controversial. limb alignment in the coronal plane is quantified by the hip - knee - ankle axis. neutral alignment is said to exist when the line connecting the center of the femoral head and center of the ankle passes through the center of the knee, and varus or valgus alignment is said to exist when this line passes medially or laterally, respectively, to the center of the knee. one study concluded short knee images can not substitute for whole leg views when accurate assessment of the hip - knee - ankle axis is essential, whereas other studies suggested the anatomic axis of the knee on short knee film appears to be a valid alternative to the hip - knee - ankle axis of the limb on full leg radiographs. for the short knee film to accurately substitute for the whole leg view, the variability of the angle formed by the anatomic and mechanical axes in the tibia and femur and the variability of the bow of the tibia and femur should be small. studies have qualitatively reported variability in the bow of the tibia and femur; however, these studies did not describe a method for quantifying the bow. a previous study of normal limbs found that only 2% of subjects have a neutral hip - knee - ankle axis, which might be explained by variability in the angle formed by the anatomic and mechanical axes and the bow in the tibia and femur and a lack of correlation between the bow of the tibia and femur in a given limb. accordingly, we hypothesized the angle formed by the anatomic axis of the proximal fourth of the tibia and the mechanical axis of the tibia and the angle formed by the anatomic axis of the distal fourth of the femur have wide variability; the bow of the tibia and femur has wide variability; there are no differences in the angle and bow between females and males; and there is no correlation between the bow of the tibia and femur in a given limb. we considered all patients who underwent tkas from june 19 to december 1, 2007, for inclusion. the inclusion criterion was the presence of primary arthritis of the knee with or without previous open or arthroscopic meniscectomy or ligament reconstruction. we excluded patients with a treated leg with evidence of a hip disorder (ie, developmental dysplasia, perthes, or slipped epiphysis), an osteotomy, a healed fracture, internal fixation, arthroplasty of the hip, knee, or ankle, or a malaligned computer tomogram of the leg. four patients were excluded because of developmental dysplasia of the hip (n = 1), tha (n = 1), internal fixation of a femoral neck fracture (n = 1), and a malaligned computer tomogram of the leg (n = 1). therefore, the study consisted of 138 patients (90 women, 48 men) with an average age of 68 10 years. all patients received an unconstrained tka (vanguard ; biomet, inc, warsaw, in). , we acquired a postoperative, anteroposterior scanogram with a field of view from the hip to the ankle with use of ct (ge lightspeed 16 ; ge healthcare, www.gehealthcare.com). because simultaneous flexion of the knee and rotation of the leg causes large changes in projected angles , we limited the projection error of the mechanical axis to approximately 1 by iteratively rotating the limb and repeating the scanogram until the two augment holes in the posterior condyles were at least partially visible on either side of the flange of the femoral component. one of us (kk) made measurements under a magnified view using screen measurement software (screen caliper, compass, and protractor ; iconico inc, www.iconico.com ; adobe photoshop, adobe inc, www.adobe.com) with a reported accuracy of 0.5. because the short knee film often is used intraoperatively and postoperatively to assess component position, especially when access to full limb radiographs is limited , and because the short knee film typically shows only the proximal fourth of the tibia and the distal fourth of the femur , we defined the anatomic axes of the tibia and femur based on axes centered in the proximal fourth of the tibia and the distal fourth of the femur. the anatomic axis of the tibia was a line joining the midpoints of the tibia at the joint line and at the junction of the proximal one - fourth and distal three - fourths of the tibia (fig . 1). the mechanical axis of the tibia was a line joining the midpoint of the tibia at the joint line and the center of the talus. the angle formed by the mechanical axis and the anatomic axis of the tibia was measured. we defined the bow of the tibia in the coronal plane as the offset in centimeters of the anatomic axis of the tibia and the center of the talus (fig . 1). a positive value (+) indicated a valgus tibia with the apex of the bow pointing medial and with the anatomic axis passing medial to the center of the talus. a negative value indicated a varus tibia with the apex of the bow pointing lateral and with the anatomic axis passing lateral to the center of the talus. the anatomic axis of the femur was a line joining the midpoints of the femur at the joint line and at the junction of the distal one - fourth and proximal three - fourths of the femur (fig the mechanical axis of the femur was a line joining the midpoint of the femur at the joint line and the center of the femoral head . the angle formed by the anatomic axis and the mechanical axis of the femur was measured . we defined the bow of the femur in the coronal plane as the offset in centimeters of the anatomic axis of the femur from the center of the femoral head . the larger the offset, the larger the bow with a positive value ( +) indicating the anatomic axis passed lateral to the center of the femoral head and a negative value indicating the anatomic axis passed medial to the center of the femoral head.fig. 1a bthe anatomic axis of the tibia (longitudinal white line) was a line joining the midpoints of the tibia at the joint line and at the junction of the proximal one - fourth and distal three - fourths of the tibia (transverse black line). the mechanical axis of the tibia was a line joining the midpoint of the tibia at the joint line and the center of the talus (longitudinal black line). the bow of the tibia was quantified by the offset (d) measured from a line (transverse white line) drawn perpendicular from the anatomic axis of the tibia to the center of the talus. a positive value (+) indicated a valgus tibia with the apex of the bow pointing medial and with the anatomic axis passing medial to the center of the talus. (a) the offset of the tibia with the greatest valgus bow was 3.5 cm. a negative value indicated a varus tibia with the apex of the bow pointing lateral and with the anatomic axis passing lateral to the center of the talus. (b) the offset of the tibia with the greatest varus bow was 2.2 cm. the use of an extramedullary tibia guide that references the ankle would place the tibial cut in six additional degrees of varus in the valgus tibia (left) and four additional degrees of valgus in the varus tibia (right) requiring lateral and medial soft tissue release to balance the knee, respectively.fig. 2a bthe anatomic axis of the femur (longitudinal white line) was a line joining the midpoints of the tibia at the joint line and at the junction of the distal one - fourth and proximal three - fourths of the femur (transverse black line). the mechanical axis of the femur was a line joining the midpoint of the femur at the joint line and the center of the femoral head (longitudinal black line). the bow of the femur was quantified by the offset (d) measured from a line (transverse white line) drawn perpendicular from the anatomic axis of the femur to the center of the femoral head. the larger the offset, the larger the bow with a positive value (+) indicating the anatomic axis passed lateral to the center of the femoral head and a negative value indicating the anatomic axis passed medial to the center of the femoral head. (a) the offset of the femur with the greatest lateral bow was 6.4 cm. (b) the offset of the femur with the least lateral bow was 0.4 cm. because of the variability in the lateral bow of the femur, the mechanical axis of the femur does not form a 5 to 6 angle with the anatomic axis of the distal fourth of the femur in either leg. the anatomic axis of the tibia (longitudinal white line) was a line joining the midpoints of the tibia at the joint line and at the junction of the proximal one - fourth and distal three - fourths of the tibia (transverse black line). the mechanical axis of the tibia was a line joining the midpoint of the tibia at the joint line and the center of the talus (longitudinal black line). the bow of the tibia was quantified by the offset (d) measured from a line (transverse white line) drawn perpendicular from the anatomic axis of the tibia to the center of the talus. a positive value (+) indicated a valgus tibia with the apex of the bow pointing medial and with the anatomic axis passing medial to the center of the talus. (a) the offset of the tibia with the greatest valgus bow was 3.5 cm. a negative value indicated a varus tibia with the apex of the bow pointing lateral and with the anatomic axis passing lateral to the center of the talus. (b) the offset of the tibia with the greatest varus bow was 2.2 cm. the use of an extramedullary tibia guide that references the ankle would place the tibial cut in six additional degrees of varus in the valgus tibia (left) and four additional degrees of valgus in the varus tibia (right) requiring lateral and medial soft tissue release to balance the knee, respectively. the anatomic axis of the femur (longitudinal white line) was a line joining the midpoints of the tibia at the joint line and at the junction of the distal one - fourth and proximal three - fourths of the femur (transverse black line). the mechanical axis of the femur was a line joining the midpoint of the femur at the joint line and the center of the femoral head (longitudinal black line). the bow of the femur was quantified by the offset (d) measured from a line (transverse white line) drawn perpendicular from the anatomic axis of the femur to the center of the femoral head. the larger the offset, the larger the bow with a positive value (+) indicating the anatomic axis passed lateral to the center of the femoral head and a negative value indicating the anatomic axis passed medial to the center of the femoral head. (a) the offset of the femur with the greatest lateral bow was 6.4 cm. (b) the offset of the femur with the least lateral bow was 0.4 cm. because of the variability in the lateral bow of the femur, the mechanical axis of the femur does not form a 5 to 6 angle with the anatomic axis of the distal fourth of the femur in either leg. we used the arithmetic mean, standard deviation, 95% confidence interval, frequency distribution, and quantile box plot to describe the variability of the angle formed by the anatomic and mechanical axes of the tibia and femur and the bow of the tibia and femur. the small ticks indicate the 10% and 90% quantiles, the intermediate ticks indicate the 2.5% and 97.5% quantiles, and the large ticks indicate the 0% and 100% quantiles. an unpaired t - test was used to determine whether the angle formed by the anatomic and mechanical axes of the tibia and femur and the bow of the tibia and femur were different between females and males. a correlation coefficient was computed to assess the correlation between the bow of the tibia and the bow of the femur. each analysis was performed with software (version 7.0.2, jmp for macintosh ; spss, chicago, il ; www.jmp.com). the angle formed by the anatomic axis and the mechanical axis of the tibia and the bow of the tibia varied widely (fig . 3). the angle formed by the anatomic axis and the mechanical axis of the femur and the bow of the femur also varied widely (fig . the angle formed by the anatomic and mechanical axes of the tibia varied 11 from 4 to 6 and averaged 1.1 1.4 ( valgus tibia). the angle was less than 1 or greater than 1 in 61%, less than 2 or greater than 2 in 34%, and less than 3 or greater than 3 in 13% of the subjects. the angle formed by the anatomic and mechanical axes of the femur varied 10 from 1 to 8, averaged 3 1.6, and the 95% confidence interval (2.83.4) did not include 5 or 6.fig. 3a bfrequency distributions of the (a) angle formed by the anatomic and mechanical axes of the tibia and (b) bow of the tibia as quantified by the offset of the anatomic axis from the center of the talus are shown. descriptive statistics include the quantile plot and the number of patients in each column.fig. 4a bfrequency distributions of the (a) angle formed by the anatomic and mechanical axes of the femur and (b) bow of the femur as quantified by the offset of the anatomic axis of the femur from the center of the femoral head are shown. frequency distributions of the (a) angle formed by the anatomic and mechanical axes of the tibia and (b) bow of the tibia as quantified by the offset of the anatomic axis from the center of the talus are shown. frequency distributions of the (a) angle formed by the anatomic and mechanical axes of the femur and (b) bow of the femur as quantified by the offset of the anatomic axis of the femur from the center of the femoral head are shown. the bow of the tibia, defined by the offset of the anatomic axis from the center of the talus, varied 5.7 cm from 2.2 cm medial to 3.5 cm lateral to the center of the talus and averaged 0.3 1.1 cm. the bow of the femur, defined by the offset of the anatomic axis from the center of the femoral head, varied 7.2 cm from 6.8 cm lateral to 0.4 cm medial to the center of the femoral head and averaged 2.5 1.3 cm. we observed no difference in the angle formed by the anatomic and mechanical axes of the tibia (p = 0.8784) and femur (p = 0.7706) and the bow of the tibia (p = 0.8578) and femur (p = 0.8101) between females and males (table 1).table 1comparison of the angle formed by the anatomic and mechanical axes and the bowdependent variablefemale (n = 90)male (n = 48)p valueangle of the anatomic and mechanical axes in the tibia1.2 1.50.9 1.40.8784, nsangle of the anatomic and mechanical axes in the femur3.0 1.53.2 1.70.7706, nsbow of the tibia (ie, offset of the anatomic axis from the center of the talus)0.4 1.1 cm0.2 1.1 cm0.8578, nsbow of the femur (ie, offset of the anatomic axis from the center of the femoral head)2.5 1.2 cm2.7 1.4 cm0.8101, nsns = nonsignificant. comparison of the angle formed by the anatomic and mechanical axes and the bow we found no correlation between the bow of the tibia and femur in a given limb (r = 0.0185, p = 0.8286), which means the degree and direction of the bow in the tibia is not related to the degree of bow of the femur in a given limb. for the short film of the knee to accurately substitute for the whole leg view, the variability of the angle formed by the anatomic and mechanical axes in the tibia and femur and the variability of the bow of the tibia and femur should be small. we hypothesized the angle formed by the anatomic axis of the proximal fourth of the tibia and the mechanical axis of the tibia and the angle formed by the anatomic axis of the distal fourth of the femur has wide variability; the bow of the tibia and femur has wide variability; there are no differences in the angles and bows between females and males; and there is no correlation between the bow of the tibia and femur in a given limb. we examine several limitations that might have affected the observed variability of the angle formed by the anatomic and mechanical axes and the bow of the tibia and femur. first, while we used nonweightbearing, rotationally controlled ct scanograms obtained in knees with osteoarthritis after tka instead of weightbearing scanograms of normal limbs, we believe this unlikely affected the variability because the weightbearing status, presence or absence of components, and removal of osteophytes back to the normal edge of the joint at the time of surgery do not affect the shape of the tibia and femur. second, the variability from our study of a western population is likely to be different from the variability of an occidental population, which has a high prevalence of tibias with a varus bow and a high prevalence of femurs with a large lateral bow. the wide variability of the angle formed by the anatomic and mechanical axes of the tibia and femur in our study agrees with some previous studies , but does not agree with the historic, pioneering principles in tka. in terms of the tibia, the principle of aligning the varus / valgus osteotomy of the proximal tibia in tka was based on an idealized depiction of the tibia in which the shaft of the tibia was straight and the mechanical and anatomic axes of the tibia were assumed to be the same. we found only 11% (16 of 138) of the tibias were straight with 34% of the subjects having an anatomic axis that diverged greater than 2 varus or valgus from the mechanical axis of the tibia. in terms of the femur , the principle of aligning the varus / valgus osteotomy of the distal femur in tka was based on the assumption that the angle formed by the anatomic and mechanical axes of the femur is consistently between 5 and 6. in contrast to these previous studies, we found the angle averaged 3 with only 6% of the femurs having an angle of 5 to 6. the difference might be explained by our use of the distal one - fourth of the femoral shaft to define the anatomic axis, which is a shorter linear length for defining the anatomic axis of the femur than used in other studies. the clinical consequences of this variability are the use of a short knee film to check the varus / valgus osteotomy intraoperatively and component alignment and to predict the hip - knee - ankle axis postoperatively is unreliable because of the inconsistent relationship between the anatomic and mechanical axes of the tibia and femur (fig b(a) the short view of the knee suggests the tibial component is malaligned in varus. (b) however, the long leg view shows the limb has a neutral hip - knee - ankle axis. the use a short knee radiograph to assess component and limb alignment is not recommended. (a) the short view of the knee suggests the tibial component is malaligned in varus. (b) however, the long leg view shows the limb has a neutral hip - knee - ankle axis. the use a short knee radiograph to assess component and limb alignment is not recommended. the offset of the anatomic axis of the tibia and femur from the center of the talus and femoral head, respectively, is a new method for quantifying the degree of bow of these two bones. in the tibia, the bow typically starts at the junction of the proximal one - fourth and distal three - fourths of the tibia (fig . 1). the extramedullary tibial guide strives to reproduce the mechanical axis of the tibia by referencing the ankle and making a classic tibial osteotomy that centers the ankle on the knee. making a classic tibial osteotomy with an extramedullary guide only maintains the normal plane between the knee and ankle when there is no bow in the tibia (ie, anatomic and mechanical axes of the tibia are the same), which occurred in only 11% of the patients in our study. the use of an intramedullary tibial guide in a less than ideal cut in a bowed tibia because aligning the intramedullary tibial guide parallel to the mechanical axis of the tibia is impossible. in the femur, the factors determining the level of the bow are more complex than in the tibia. the bow in the femur is affected by variations in the bow of the shaft of the femur, the neck - shaft angle, and the length of the femoral neck (fig . much has been written about how the degree of bow affects the starting point for insertion of the intramedullary rod and the accuracy of alignment of the varus / valgus osteotomy of the distal femur . the wide variability of the bow of the femur in our patients further underscores the need to determine, for each patient, the best angle for making the distal femur varus / valgus osteotomy . determining the best angle for each patient is a challenge because guidelines for placing the starting point for insertion of the intramedullary rod in the distal femur and selecting the angle, although available, lack agreement . we identified no difference in the variability of the angle formed by the anatomic and mechanical axes and the bow in the tibia and femur between females and males . the lack of a gender difference between the angle formed by the anatomic and mechanical axes in the femur has been confirmed in the chinese population . a previous study of normal limbs found that only 2% of subjects have a neutral hip - knee - ankle axis, which can be explained by the wide variability of the bow in the tibia and femur and the lack of correlation between the bow of the tibia and femur in a given leg observed in the current study . a leg with a valgus hip - knee - ankle axis typically has a tibia with a medial bow combined with a femur with a small lateral bow ( ie, offset). a leg with a varus hip - knee - ankle axis typically has a tibia with a lateral bow combined with a femur with a large lateral bow (ie, offset) (fig . the independent pairing of any shaped tibia with any shaped femur does not agree with the classic assumption that the bow of the tibia and femur compensate for each other to form a neutral hip - knee - ankle axis of the limb .fig . 6a f(a, d) the anatomic axis (longitudinal white lines) of the knee measured on the radiographic view of these two knees are similar; however, (b, e) the hip - knee - ankle axis of these two limbs are dissimilar. (c, f) the reason the anatomic axis of the knee does not predict the hip - knee - ankle axis of the limb is because of the wide variability in the bow of the tibia of the femur and because of the lack of correlation between the bow of a tibia and femur in a given limb. (a, d) the anatomic axis (longitudinal white lines) of the knee measured on the radiographic view of these two knees are similar; however, (b, e) the hip - knee - ankle axis of these two limbs are dissimilar. (c, f) the reason the anatomic axis of the knee does not predict the hip - knee - ankle axis of the limb is because of the wide variability in the bow of the tibia of the femur and because of the lack of correlation between the bow of a tibia and femur in a given limb. the variability in the angle formed by the anatomic and mechanical axes and bow in the tibia and femur has changed our method for choosing the correct angle for making the proximal tibia and distal femoral cut. because few normal limbs have a neutral hip - knee - ankle axis (ie, 2%), and because changing the hip - knee - axis from normal to neutral changes the three kinematic axes of the knee from normal and requires collateral ligament and retinacular releases that can lead to instability, we align the components kinematically. three interrelated axes, none of which share any relationship to the center of the femoral head or the center of the ankle, describe the kinematics of the knee. the primary axis is a tibial - femoral axis in the femur about which the tibia flexes and extends and is nonorthogonal to the three anatomic planes (ie, sagittal, coronal, and axial). one secondary axis is the patellofemoral axis in the femur about which the patella flexes and extends that is aligned parallel to the primary axis. the other secondary axis is a tibial - femoral axis in the tibia about which the tibia internally and externally rotates on the femur that is perpendicular to the tibial - femoral axis and the patellofemoral flexion axis in the femur. therefore, the foundation for restoring normal kinematics in tka is aligning the axis of the femoral component coincident with the axis in the femur about which the tibia flexes and extends. virtual, preoperative planning is used to align the axis of the femoral component coincident with the tibial - femoral axis in the femur by shape - matching the articular surface of the femoral component to the articular surface of the normal femur. a three - dimensional model of the normal femur is reconstructed from mr images of the patient s knee, which are each restored by filling in areas of focal wear. the surgical technique uses custom - made tibial and femoral cutting guides machined to the topography of the patient s knee to position the femoral and tibial components in all six degrees of freedom. this technique, which surface - matches the components to a knee that virtually has been restored to normal, adheres to the measured resection principle of tka and restores the hip - knee - ankle axis to the natural prearthritic alignment of the limb the subject had before arthritis and deformity developed. conventional instrumentation and the current iterations of surgical navigation systems do not account for the wide intrasubject and intersubject variations in the angle formed by the anatomic and mechanical axes and the bow of the tibia and femur that are important in the selection of the ideal surgical planes in tka. use of the short knee film to judge placement of intramedullary and extramedullary rods, component alignment, and predict the hip - knee - ankle axis of the limb can not be justified.

in general practice, short films of the knee are used to assess component position and define the entry point for intramedullary femoral alignment in tkas; however, whether it is justified to use the short film commonly used in research settings and everyday practice as a substitute for the whole leg view is controversial and needs clarification. in 138 long leg ct scanograms we measured the angle formed by the anatomic axis of the proximal fourth of the tibia and the mechanical axis of the tibia, the angle formed by the anatomic axis of the distal fourth of the femur and the mechanical axis of the femur, the bow of the tibia (as reflected by the offset of the anatomic axis from the center of the talus), and the bow of the femur (as reflected by the offset of the anatomic axis from the center of the femoral head). because the angle formed by these axes and the bow of the tibia and femur have wide variability in females and males, a short film of the knee should not be used in place of the whole leg view when accurate assessment of component position and limb alignment is essential. a previous study of normal limbs found that only 2% of subjects have a neutral hip - knee - ankle axis, which can be explained by the wide variability of the bow in the tibia and femur and the lack of correlation between the bow of the tibia and femur in a given limb as shown in the current study.

the aging of a population leads to a higher incidence and prevalence of various health and social problems related to age, as well as the increased rates of dependency, comorbidity, and economic and social costs associated to those entities. it is necessary to identify individuals at risk in order to delay the onset of health problems and the economic problems related to aging. frailty appears as a clinical syndrome associated with greater vulnerability of the individual to adverse health outcomes, a higher level of disability, and increased mortality. sarcopenia is considered the factor that initiates the pathophysiological cycle of frailty, generating structural, and metabolic changes through insulin resistance, together with the tendency to inflammatory conditions and an increased cardiovascular risk. the lack of a definition of frailty common to different authors makes it difficult to calculate the real prevalence of the syndrome, which is said to be around 10.7% of the elderly, with 41.6% in the prefrailty situation. fatigue, resistance, ambulation, illnesses, and loss of weight (frail) scale may be used to detect frailty. it establishes three frailty levels according to the responses obtained to five questions on comorbidity, fatigue, resistance, aerobic capacity, and unintended weight loss. patients with 0 points are considered nonfrail, with 1 or 2 points for prefrail, and frail are those with 3 or more points. frail people have less capacity for daily living activities than nonfrail patients, but this relation is not well defined since it is not known if these entities are time - related. the possibility of using frailty as a screening method to detect people at risk of being unable to cope with their daily life activities might be useful to establish interventions to prevent or delay this loss. the objective of this study was to describe the functional capacities of the elderly in each frailty state according to frail scale. a time - related relationship between these entities may indicate when to start rehabilitation measures by detecting the frailty level. a descriptive observational study was accomplished, taking as target population 70-year - olds or older patients assigned to a single primary care center. the chosen primary care center was located in an urban area of the eastern part of the city of madrid, of a low - medium socioeconomic status. patients who did not sign the informed consent, were institutionalized and/or had language barriers or cognitive impairment and could not understand the information provided and required were excluded. based on an estimated prevalence of frailty of 10.7% in previous studies with similar tools, in a similar population, the calculated sample size was 145 subjects from a reference population of 3806 patients, (5% precision, confidence interval ( ci) - 95% ). these individuals were selected using consecutive not randomized sampling during the months of october and november 2014 until the sample was complete. subjects selected were verbally informed about the study features and were asked to complete the informed consent in writing by a letter. this enabled the investigators to consult their medical records and proceed with a personal interview. the following variables were recorded: sex, age (grouped into 5-year groups), frailty level according to frail scale, comorbidities, and pluripathology following the instructions for development of clinical guidelines of the spanish primary care societies (illnesses are classified in eight groups, depending on the organ or system affected), instrumental daily life activities capacity through lawton - brody scale, and the social risk following the gijn social risk scale adapted to the spanish population. age was also measured as a continuous quantitative variable, calculating the median and standard deviation. bivariate analysis was performed with chi - square test for measuring the association between variables, taking frailty as the dependent variable, and using the pearson's regression ratio to quantify the association power if any. a logistic regression was performed in order to reject those variables that worked as confounding factors. spss 22.0 (spss 22.0, copyright by ibm, usa) program was used for the statistical analysis. ethical issues were considered, respecting personal privacy, obtaining patients consent for the recruitment and the use of the data only for the purpose of this study. this investigation was approved by the investigation committee of the east health assistance area of madrid. classified as frail was 17.81% (ci 1224%) of the population and 39.72% (ci 3248%) as prefrail. the median age of frail individuals was 84 years with a standard deviation of 1-year. the median age of prefrail individuals was 81, with a standard deviation of 1-year. prevalence of frailty by age group of the population, 45.20% were male and 54.8% were female. the distribution of age groups and variable frequencies by sex is shown in table 1. percentage distribution and 95% ci for sociodemographic characteristics of the study population by gender total disability for instrumental daily living activities comprised 3.42% (ci 0.5 - 6.4%) of the population, severe 8.22% (ci 3.9 - 12.7%), moderate 15.07% (ci 9.8 - 20.9%), and mild 27.4% (ci 20.3 - 34.6%). there was no limitation in those activities for 45.20% (37.1 - 53.3%) of the population. a marked increase in the level of dependence occurred when the nonfrail and prefrail groups were compared as shown in figure 2. distribution of dissability for instrumental activities of daily live by different levels of frailty of the total sample, 0.68% (ci 0.2 - 2.02%) presented a high social risk, 23.29% (ci 16.4 - 30.1%) moderate, and for 76.03% (ci 69.1 - 82.9%) there was no risk at all. the bivariate analysis between frailty and the different variables showed p values which allowed the establishment of a possible association between the frailty and the level of dependence in instrumental activities of daily living, the presence of comorbidity such as cardiovascular, musculoskeletal and digestive diseases, and age. the only association found was between frailty and disability, age dependency and diseases of the digestive system. p - values for association between frailty (dependent variable) and the rest of the study variables in a bivariate analysis p - values for association between frailty (dependent variable) and variables previously associated in the bivariate analysis within a multivariate model the prevalence of frailty similar to the previous studies with similar scales and population (5.827.3%) was found although the criteria for the diagnosis of this entity are not the same in all of the literature. further research in this field must be done to establish common criteria so that the real impact of this syndrome can be described. although we have used a different methodology in sampling, the use of the same scales makes the comparison with other studies plausible. the are applied to the population studied, but further research will help us to compare the differences with the general population. it is important to attend primary care to detect frailty properly as certain studies have demonstrated differences in the way family physicians and geriatricians detect this entity. in a descriptive analysis, frailty has been more frequent in relative terms in men than in women although they have presented greater relative frequency of prefrailty. our findings are in agreement with those studies that indicate that frailty is more common in women and that they persist in this condition longer than men. this may be due to the attendance of frail women at a different health - care center from the rest of population, delegating some activities to other family members. the ideal time for screening frailty can be set between 75 and 81 although the sample size limits the interpretation of , and other studies have shown that there may be younger frail individuals. the transition from a normal situation to prefrailty status entails deterioration in instrumental activities of daily living. this relationship has been described in other articles, but the temporal association between the onset of prefrailty and the start of the loss of functional capacities has not been recounted in the consulted literature. indicate that frailty can be used as a method that allows targeted interventions to retard disability. mijnarends et al. reflected on the relationship between frailty and sarcopenia, recognizing the similarities between the frail scale and fried's frailty phenotype, and encouraged the study of the relationship of these entities with a disability. cardiovascular diseases showed no significant relationship with frailty, but this may change with further research, in accordance with previous literature and remove the limitations of this investigation. detection of frailty syndrome, especially its intermediate stage or prefrailty status, identifies individuals with greater risk of disease and negative prognosis. this condition can assist in making clinical decisions based on the timing of benefits of the different diagnostic and therapeutic procedures. establishment of the relationship between frailty and functional dependency as well as the identification of those individuals more likely to require the use of social resources later are vital so that social and health policies can be determined. it is necessary to establish the temporal relationship of the events associated with the onset of frailty, such as the time of evolution from the prefrail status to frailty, or the prognosis of frail and prefrail patients. studying the relationship between frailty and different pathology groups can strengthen the understanding of the pathophysiology of the syndrome in order to find the possible ways of prevention and treatment. it is possible that intervention in the gastrointestinal or cardiovascular field can delay the development of the syndrome, relying in each case on their pathophysiology. patients included in the study did not cover the entire population spectrum because of the sampling. using charts and data collection in a medical environment can produce biases that have to be borne in mind when the are interpreted. detecting frailty in the early stage called prefrailty may be useful to identify patients at risk of losing their functional capacities. this relationship is important not only to maintain the best health level in the population, but also for the organizational implications that can be developed from it. the identification of prefrail individuals may be useful in the implementation of health and social programs that prevent the development of frailty and disability, and its economic and social costs. further research might be done to describe the effects of different interventions on prefrail patients.

introduction: in an aging population, new strategies are required to identify individuals at risk of adverse health outcomes. frailty syndrome is related to negative health events. this increased risk may be used to identify individuals in which interventions can delay the onset of physical and functional complications. the aim of the study was to determine the relationship between the onset of frailty and the beginning of functional disability.materials and methods: this was a cross - sectional observational study with consecutive sampling to analyze 146 patients aged seventy and older who come to the primary care center. the level of frailty was registered according to fatigue, resistance, ambulation, illnesses, and loss of weight scale. disability for instrumental activities of daily live dependency, comorbidity, and social risk factors was registered too.:the prevalence of frailty and prefrailty was 17.8% and 39%, respectively, and were associated with age, level of disability, and the presence of gastrointestinal disease. prefrail patients had initial levels of dependency, while those who were not frail were mostly independent.:frailty syndrome is easily detectable. the intermediate stage known as prefrailty is related to the start of the functional disability. the syndrome screening identifies individuals at risk in whom we can potentially intervene to delay the onset of the syndrome and delay functional disability. control of comorbidity in frail patients must be studied. screening age could be set in patients between 75 and 81 years old.

a voice is the most important and effective medium employed not only in daily communication but also in logical discussions. only humans are able to use words as means of verbal communication, although almost all animals have voices. vocal sounds are generated by the relevant operations of the vocal organs such as a lung, trachea, vocal cords, vocal tract, tongue, and muscles. the airflow from the lung causes a vocal cord vibration to generate a source sound, and then the glottal wave is led to the vocal tract, which works as a sound filter as to form the spectrum envelope of a particular voice. the voice is at the same time transmitted to the auditory system so that the vocal system is controlled for the stable vocalization. different vocal sounds are generated by the complex movements of vocal organs under the feedback control mechanisms using an auditory system. as infants grow they acquire these control methods pertaining to the vocal organs for appropriate vocalization. these get developed in infancy by repetition of trials and errors concerning the hearing and vocalizing of vocal sounds. any disability or injury to any part of the vocal organs or to the auditory system may in an impediment in vocalization. people who have congenitally hearing impairments have difficulties in learning vocalization, since they are not able to listen to their own voice. a speech therapist helps themtotrain their speech by teaching the vocal organs to learn vocalization and clear speech. we are developing a talking robot by reproducing a human vocal system mechanically and based on the physical model of the vocal organs in the human. the fundamental frequency and the spectrum envelope determine the principal characteristics of a voice. fundamental frequency is a characteristic of the voice source that is generated by the vibration of vocal cords. the resonance effects that get articulated by the motion of vocal tract and nasal cavity cause the spectrum envelope. for the autonomous acquisition of vocalization skills by the robot, an adaptive learning using an auditory feedback control the robot consists of motor - controlled vocal organs such as vocal cords, a vocal tract, and a nasal cavity to generate a natural voice imitating a human vocalization. by introducing auditory feedback learning with an adaptive control algorithm of pitch and phoneme, the robot is able to autonomously acquire the control skill of the mechanical system to vocalize stable vocal sounds imitating human speech. in the first part of the paper, the construction of vocal cords and vocal tract for the realization of the robot is briefly presented, and then the analysis of the autonomous learning of how the robot acquires the vocalization skill by using the neural network will be described. then, a robotic training system for the hearing - impaired people is introduced, together with the evaluation of the interactive speech training conducted in an experiment. the talking robot mainly consists of an air pump, artificial vocal cords, a resonance tube, a nasal cavity, and a microphone connected to a sound analyzer, which, respectively, correspond to a lung, vocal cords, a vocal tract, a nasal cavity, and an audition of a human, as shown in figure 1. an air from the pump is led to the vocal cords via an airflow control valve, which works for the control of the voice volume. the resonance tube as a vocal tract is attached to the vocal cords for the modification of resonance characteristics. the nasal cavity is connected to the resonance tube with a sliding valve between them. it realizes the pitch extraction and the analysis of resonance characteristics of generated sounds in real time, which are necessary for the auditory feedback control. the system controller manages the whole system by listening to the vocalized sounds and calculating motor control commands, based on the auditory feedback control mechanism employing a neural network learning. the relation between the voice characteristics and motor control parameters is stored in the system controller, which is referred to in the generation of speech and singing performance. vocal cords with two vibrating cords molded with silicone rubber with the softness of human mucous membrane were constructed in this study. two - layered construction (a hard silicone is inside with the soft coating outside) gave the better resonance characteristics, and is employed in the robot. the vibratory actions of the two cords are excited by the airflow led by the tube, and generate a source sound to be resonated in the vocal tract. the tension of cords can be manipulated by applying tensile force to them. by pulling the cords, the relationship between the tensile force and the fundamental frequency of a vocal sound generated by the robot is acquired by the auditory feedback learning before the singing and talking performance, and pitches during the utterance are kept in stable by the adaptive feedback control. the human vocal tract is a non - uniform tube about 170 mm long in man. its cross - sectional area varies from 0 to 20 cm under the control for vocalization. a nasal cavity with a total volume of 60 cm is coupled to the vocal tract. in the mechanical system, a resonance tube as a vocal tract it works as a resonator of a source sound generated by the vocal cords. it is made of a silicone rubber with the length of 180 mm and the diameter of 36 mm, which is equal to 10.2 cm by the cross - sectional area as shown in figure 1. the silicone rubber is molded with the softness of human skin, which contributes to the quality of the resonance characteristics. in addition, a nasal cavity made of a plaster is attached to the resonance tube to vocalize nasal sounds like /m/ and /n/. a sliding valve as a role of the soft palate is settled at the connection of the resonance tube and the nasal cavity for the selection of nasal and normal sounds. for the generation of nasal sounds /n/ and /m/, the motor - controlled sliding valve is open to lead the air into the nasal cavity. by actuating displacement forces with stainless bars from the outside of the vocal tract, the cross - sectional area of the tube is manipulated so that the resonance characteristics are changed according to the transformations of the inner areas of the resonator. compact servo motors are placed at 8 positions xj (j = 18) from the lip side of the tube to the intake side, and the displacement forces pj(xj) are applied according to the control commands from the motor - phoneme controller. an adaptive learning algorithm for the achievement of a talking and singing performance is introduced in this section. first in the learning phase, the system acquires two maps in which the relations between the motor positions and the features of generated voices are established and stored. one is a motor - pitch map, which associates motor positions with fundamental frequencies. it is acquired by comparing the pitches of vocalized sounds with the desired pitches, which cover the frequency range of speech. the other is a motor - phoneme map, which associates motor positions with phonetic features of vowel and consonant sounds. second in the performance phase, the robot speaks and sings by referring to the obtained maps, while pitches and phonemes of generated voices are adaptively maintained by hearing its own output voices. the neural network (nn) works to associate the sound characteristics with the control parameters of the nine motors settled in the vocal tract and the nasal cavity. in the learning process , the network learns the motor control commands by inputting 10th - order linear predictive coding (lpc) cepstrum coefficients derived from vocal sound waves as teaching signals. the network acquires the relations between the sound parameters and the motor control commands of the vocal tract. after the learning, the neural network is connected in series into the vocal tract model. by inputting the sound parameters of desired sounds to the nn, the corresponding form of the vocal tract is obtained. in this study, the self - organizing neural network (sonn) was employed for the adaptive learning of vocalization. figure 2 shows the structure of the sonn consisting of two processes, which are an information memory process and an information recall process. after the sonn learning, the motor control parameters are adaptively recalled by the stimuli of sounds to be generated. the information memory process is achieved by the self - organizing map (som) learning, in which sound parameters are arranged onto a two - dimensional feature map to be related to one another. weight vector vj at node jin the feature map is fully connected to the input nodes xi, where 10th - order lpc cepstrum coefficients are given. a competitive learning is used, in which the winner c as the output unit with a weight vector closest to the current input vector x(t) is chosen at time t in learning. by using the winner c, the weight vectors vj - s are updated according to the rule shown below; vj(t+1)=vj(t)+hcj(t),hcj(t)={(t)exp(rcrj222(t))(inc),0(i nc). here, rc rj is the distance between units c and j in the output array, and nc is the neighborhood of the node c. (t) is a learning coefficient which gradually reduces as the learning proceeds. then, in the information recall process, each node in the feature map is associated with motor control parameters for the control commands of nine motors employed for the vocal tract deformation, by using the three - layered perceptron. in this study, a conventional back - propagation algorithm was employed for the learning. with the integration of the information memory and recall processes, the sonn works to adaptively associate sound parameters with motor control parameters. in the current system, 25 25 arrayed map v = is used as the som. for testing the mapping ability, after the self - organizing learning, five japanese vowels vocalized by six different people were mapped onto the feature map. same vowel sounds given by different people were mapped close with each other, and five vowels were roughly categorized according to the differences of phonetic characteristics. we found that, in some vowel area, two sounds given by two different speakers fell in a same unit in the feature map. it means that the two different sounds could not be separated, although they have close tonal features with each other. redundant sound parameters which were not used for the japanese speech were buried in the map, since the 150 inputted sounds were generated randomly by the robot. furthermore, two different sounds given by two different speakers were occasionally fallen in the same unit. after the sonn learning, five japanese vowel sounds given by 6 different speakers with normal audition were applied to the supervised learning as the reinforcement signal to be associated with the suitable motor control parameters for the japanese vocalization. figure 3 shows the of the reinforcement learning with five japanese vowels given by five speakers no. 1 to 5. the distribution of same vowel sounds concentrated with one another, and the patterns of different vowels were placed apart. after the learning of the relationship between the sound parameters and the motor control parameters, we inputted human voices from microphone to confirm whether the robot could speak autonomously by mimicking human vocalization. with the comparison of spectra between human vowel vocalization and robot speech, we confirmed that the first and second formants f1 and f2, which present the principal characteristics of the vowels, were formed properly as to approximate the human vowels, and the sounds were well distinguishable by listeners. the transition between two different vowels in the continuous speech was well acquired by the sonn learning, which means that all the cellsoninthe som are associated with motor control parameters properly to vocalize particular sounds. voices of hearing - impaired people then were given to the robot so as to confirm that the articulatory motion would be reproduced by the robot. figure 4 shows the mapping of six different voices given by hearing - impaired speakers no. a, no. f. the same colors indicate the vocal sounds generated by the same vowels. in figure 5, vocal tract shapes estimated by the robot from voices of hearing - impaired person no. a are presented, together with the comparison of the vocal tract shapes estimated by the able - bodied speaker no. 1 voices. from the observation of the robot's reproduced motions of the vocal tract, the articulations of auditory - impaired people were apparently small, and complex shapes of vocal tract were not sufficiently articulated. furthermore, in the map shown in figure 4, /u/ sound given by the hearing - impaired speaker no. a is located inside the /e/ area of able - bodied speakers, and his /o/ vowel is located close to the /u/ area of able - bodied speakers. these articulatory characteristics also appear in the vocal tract shapes shown in figure 5. in the figures, the vowel /u/ shape of speaker no. a shown in (b-2) is almost the same with the /o/ shape of speaker no. 1 presented in (c-1). likewise, the /o/ shape shown in (c-2) appears close to the shape of (b-1). thus, these proved that the topological relations of resonance characteristics of voices were well preserved in the map, and the articulatory motion by the robot was successfully obtained to reproduce the speech articulation by listening arbitrary vocal sounds. in the speech training, the robot interactively shows the articulatory motion of vocal organs as a target to a trainee so thats / he repeats his / her vocalization and the observation of the robot motion. the trainee is also able to refer to the som to find the distance to the target voice. the training of speech articulation by an auditory - impaired subject is shown in figure 7. an experiment of speech training was conducted by six hearing - impaired subjects: no. a f (four males and two females), who study in a high school and a junior high school. in figure 8, the training of three subjects no. f are shown by presenting the trajectories of voices appeared in the som during the training experiments. figure 8(a) shows a of successful training with less trials conducted by the subject no. a. by observing the articulatory motion instructed by the robot, this subject recognized the difference in his articulation and effectively learned the correct motion. e, however, he had achieved the vocalization by repeating several trials and errors, especially for the vowels /i/ and /e/ as presented by the arrows from i1 to i5 and e1 to e5, respectively. in the case of the training conducted by the subject no. the clarity of his voices was quite low, and the original voices were mapped far from the area of clear voices. he could not understand the shape of the robot's vocal tract, nor realize the correspondence between the robot's motion and the motion of his inner mouth. this subject tried to articulate his vocal tract following the articulatory motion indicated by the robot, however, his voice moved to the different direction in the som as shown by arrows in figure 8(c). he failed the acquisition of vocalization skill and could not achieve the training. in the questionnaire after the training, he pointed out the difficulties of moving a particular part of the inner mouth so as to mimic the articulatory motion of the robot. by the experimental training, five subjects could mimic the vocalization following the directions given by the robotic voice simulator, and acquired the better vocal sounds. in the questionnaire after the experiment, two subjects commented that the correspondence between robot's vocal tract and human actual vocal tract should be instructed, so that they could easily understand which part inside the mouth should be intensively articulated for the clear vocalization. a robotic voice simulator and its articulatory reproduction of voice of hearing - impaired people were introduced in this paper. by introducing the adaptive learning and controlling of the mechanical model with the auditory feedback, the voice robot was able to acquire the vocalization skill as a human baby does in speech training. the robot was applied to introduce a training system for auditory - impaired people to interactively train the speech articulation for learning proper vocalization. the robotic voice simulator reproduces the articulatory motion just by listening to actual voices given by auditory - impaired people, and they could learn and know how to move their vocal organs for the clear vocalization, by observing the motions instructed by the talking robot. the use of som for visually presenting the distance between target voice and trainee's voice is also introduced. we confirmed that the training using the talking robot and the som would help hearing - impaired people learn the articulatory motion in the mouth and the skill of clear vocalization properly. in the next system , the correspondence between robot's vocal tract and human actual vocal tract should be established so that a subject could understand which part inside the mouth should be intensively articulated in the training. by analyzing the vocal articulation of auditory - impaired people during the training with the robot, we will investigate the factor of unclarity of their voices originated by the articulatory motions.

a talking and singing robot which adaptively learns the vocalization skill by means of an auditory feedback learning algorithm is being developed. the robot consists of motor - controlled vocal organs such as vocal cords, a vocal tract and a nasal cavity to generate a natural voice imitating a human vocalization. in this study, the robot is applied to the training system of speech articulation for the hearing - impaired, because the robot is able to reproduce their vocalization and to teach them how it is to be improved to generate clear speech. the paper briefly introduces the mechanical construction of the robot and how it autonomously acquires the vocalization skill in the auditory feedback learning by listening to human speech. then the training system is described, together with the evaluation of the speech training by auditory impaired people.

diabetes mellitus (dm) is a chronic metabolic condition that affects 8.3% of the world population and causes significant morbidity and mortality. the number of people suffering from diabetes is expected to increase beyond 592 million people over the next 25 years. endothelial damage in diabetes leads to damage of multiple organs and an increased risk of myocardial infarction, stroke, and peripheral vascular disease, along with other chronic complications such as kidney disease or retinopathy. diabetes also increases the risk of cognitive dysfunction and both vascular dementia and alzheimer's disease. this association is more prominent in elderly diabetics, although mild cognitive impairment may be present also in relatively younger diabetics. the impact of diabetes in cognitive function may become more apparent as the life expectancy has significantly increased over the past years. a recent meta - analysis determined that type 2 diabetics had worse performance in neuropsychological tests when compared to normal subjects. as for type 1 diabetes, which is less common and there is, however, evidence of an overall decrease in pediatric cognitive performance for diabetic children except in the memory and language domains. a more recent study showed a nonstatistically significant reduction of intellectual function for type 1 diabetics when compared to normal children. although recent data has found that intensive glucose control could be associated with increased mortality among diabetics, the impact on cognitive function is less understood. we conducted a meta - analysis to determine if intensive glucose control can actually prevent or delay the onset of cognitive decline both in type 1 and in type 2 diabetics. as we move to achieve patient centered care, having information for patients regarding the balance between quantity and quality of life will be useful. pubmed (medline) database was searched for randomized controlled trials published from january 1, 1980, to june 1, 2014, using mesh terms and keywords. search terms used included type 1 diabetes mellitus, type 2 diabetes mellitus, drug therapy, and cognitive function. the full search including mesh terms was (( ( diabetes mellitus, type 1/drug therapy or diabetes mellitus, type 2/drug therapy) or diabetes mellitus, type 1/therapy ) or diabetes mellitus, type 2/therapy ) and (cognitive and ( physiology or physiology or physiology or function) ) and (( clinical trial or randomized controlled trial) and (1980/01/01 : 2014/12/31) ). we also reviewed the reference list of the identified articles looking for additional studies that might be included in this meta - analysis. we included randomized controlled trials (rct), which analyzed patients with either type 1 or type 2 diabetes, had at least one group of patients receiving intensive glucose control and another receiving conventional antidiabetic treatment, and provided information regarding assessment of cognitive function after a follow - up period using a standardized method. the exclusion criteria we used were as follows: studies which included patients already diagnosed with cognitive dysfunction or established dementia, studies that used only the minimental score examination (mmse) as an assessment of cognitive function, and studies that utilized a cognitive testing method which was not comparable to those used in any of the other articles included. we defined interventions as intensive if they tailored care to reach a glycated hemoglobin (hba1c) goal of less than 7% or a fasting glucose level of less than 130 mg / dl. conventional treatment was defined simply as the continuation of the regular treatment the patient was already receiving. four of them intended to achieve levels of hba1c below 6%, while another one targeted hba1c levels below 7%. two more studies did not report a goal level of glycated hemoglobin, one of them targeted preprandial glucose levels below 130 mg / dl instead, and the last one adjusted goals of glycaemia and hba1c individually with each patient. the methods used to achieve these goals ranged from multifactorial behavioral interventions to adjusted doses of oral antidiabetics to 3 or more insulin injections per day or continuous insulin infusion with an external pump. the main outcome was cognitive dysfunction classified into the following domains based on standard domain definitions: information processing speed, executive function, attention / concentration, verbal memory, and motor function. information processing speed was assessed through the digit - symbol substitution subtest (dsst) of the wechsler assessment of intelligence scale (wais), in which the participant is initially shown a key containing symbol - digit pairs and must later copy the corresponding symbol under a series of numbers with empty boxes below. as measures of executive functioning, participants were assessed using the trail making test part b (tmt - b) and the similarities subtest of the wais. the tmt - b measures the time a subject needs to draw lines connecting 25 encircled letters and numbers distributed over a sheet of paper in alternating order. for the similarities subtest, subjects are asked in what way two words are alike (i.e., poem and statue). memory function was evaluated using the rey auditory verbal learning test (ravlt), a verbal learning task where the participant is given a list of 15 unrelated words repeated over 5 trials. a delayed - recall trial is administered 30 minutes after the initial learning phase and the number of freely recalled words is recorded. reaction time to auditory and visual stimuli was measured through computerized tasks where participants had to press a key immediately after presentation of visual (light) or auditory (tone) stimuli. the finger tapping test was administered as a measure of motor function. in this test , participants place their hand on a board with a lever and tap their index finger on the lever as quickly as possible, using their dominant and nondominant hands, within a 10-second time interval. scores are calculated by averaging the number of taps over five consecutive trials within a 5-point range with each hand. the stroop test is a measure of selective attention, cognitive flexibility, and cognitive inhibition. read a list of color names printed in black ink. in the second part they must name the color of a list of x's or color patches, depending on the version used. in the third part of the task the subject must name the color of a color word written in nonmatching ink color (e.g., the word green printed in red). a stroop interference effect occurs when color - naming speed is significantly reduced as the subject must inhibit an automatic reading response to produce a more effortful color - naming response. we reported relevant baseline characteristics for each study as mean and percentage as reported. to aggregate unweighted for all studies we report the median and interquartile range for continuous variables and for hba1c we report the mean values before and after the intervention per randomized group. to assess for heterogeneity across studies we used the cochran q chi - square (significance level < 0.10) and the i - squared statistic (> 50%). for the mathematical pooling we stratified the analysis by type of diabetes and calculated the standardized mean difference (smd) with the corresponding 95% confidence interval and p value. the smd represents the difference between the mean and standard deviation of the cognitive test in the intensive control group minus that of the conventional group for each study. the smd was weighted by the sample size of each individual study per randomized group. our search strategy yielded 82 articles, from which we excluded 73 abstracts because they were not rct or did not meet inclusion criteria. from the remaining 9 studies from the original search, we removed 3 more articles after exclusion criteria were applied. one additional study was retrieved from the references of the articles reviewed and was included for analysis as it did not meet exclusion criteria. a total of 7 articles were finally included in the meta - analysis, of which 4 analyzed type 1 diabetics and 3 studied type 2 diabetics (figure 1). the combined sample size was 6056 patients (3011 under intensive glucose control and 3045 under conventional treatment). the median age was 27 years for type 1 diabetics and 62.4 years for type 2 diabetics. median baseline levels of hba1c were 9.24% for the intensive treatment group and 9.07% for the conventional treatment patients, while hba1c levels after treatment follow - up were 7.43% for the intensive group and 8.17% for the conventional treatment patients. the most commonly reported tests where the dsst, trail making, finger tap, and ravlt. the univariate show that on each test there is a difference between the intensive treatment group and the control group. diabetes the dsst smd had a positive direction (0.71), while the trail making test, stroop test, and ravlt had a negative direction. however, a negative direction on the smd for trail making test also favors intensive glucose control due to the nature of the test. these are summarized in figure 2, where for tmt have been mirrored to a positive sense for a better presentation. our meta - analysis demonstrates that tight glucose control is not superior to conventional care at preventing cognitive decline among type 1 diabetics and has a positive impact only on the information processing speed and executive functions among type 2 diabetics. the lack of effect seen for type 1 diabetics could be related to the nonsignificant differences described to date in cognitive function between diabetics and healthy control groups. among young diabetic patients who are free of multiple comorbidities, the effect of hyperglycemia may not be severe enough to cause a significant cognitive impairment, and thus the glucose lowering regime used to treat diabetes becomes unimportant regarding prevention of cognitive function loss. an alternative explanation is that the small cognitive decline reported among type 1 diabetics is related to the effect of repeated hypoglycemic events which may cause white matter damage but would not be reduced by tight glucose control. in contrast, the effect of tight glucose control varied across cognitive domains among type 2 diabetics. the intensive control group performed significantly better on the dsst and tmt but did worse than the conventional treatment group on the stroop test and the ravlt. from these we can conclude that tight glucose control favors the domains of information processing speed and executive function, but at the cost of negatively affecting attention and memory functions. the presence of comorbidities at the age of onset of diabetes, which is much later than that for type 1 diabetics, may help explain these . also, it has been described that insulin is one of the molecules that regulate tau protein phosphorylation in neurons, and thus insulin resistance may disrupt this process, causing tau to bind to microtubules, giving rise to the pathogenesis of alzheimer's disease and dementia. educational level is also an important confounding factor in this population, as it has been observed that cognitive performance correlates directly with the amount of years of completed study. however, there are no enough data to test the impact of these confounders in the current meta - analysis. in regard to the higher risk of mortality previously reported for tight glucose control regimes, only two of the studies included reported a mortality outcome. thus, evaluating the relationship between cognitive decline, mortality, and tight glucose control was not possible. to date, age, the increased risk of hypoglycemia, and the presence of important comorbidities are factors that favor the increased incidence of deaths in type 2 population, while in type 1 diabetics, though there was an increased risk of hypoglycemic events, the great majority were nonfatal. first, while there is significant evidence on the relationship of diabetes and cognitive decline, very few trials have addressed the impact of different glucose control regimes on cognitive function. more so, many studies evaluating this question could not be included because they either used noncomparable tests or reported cognitive decline using only the mmse. also, the large variation in sample size among type 2 diabetes studies caused one of the studies to carry more significant weight than the others. in , we observed that there is no benefit from intensive glucose lowering regarding cognitive function for the young type 1 diabetics, while the older type 2 diabetics benefit from this therapy in the domains of information processing speed and executive function but find their attention and memory hindered. these findings provide insight into the pathophysiology of different types of cognitive impairment and possible therapeutic avenues in the future. some studies have shown an increased risk of cardiovascular mortality and hypoglycemia when using intensive glucose control regimes. thus, each case should be evaluated individually to assess the benefits of a tight glycemic control against the observed risks. since these complications are more common in older diabetic patients, intensive control of the glucose levels might be safer and more recommendable in type 1 diabetics, most of which are children or young adolescents, regarding noncognitive benefits.

diabetes mellitus is associated with cognitive decline and impaired performance in cognitive function tests among type 1 and type 2 diabetics. even though the use of tight glucose control has been limited by a reported higher mortality, few reports have assessed the impact of treatment intensity on cognitive function. we conducted a meta - analysis to evaluate if an intensive glucose control in diabetes improves cognitive function, in comparison to standard therapy. we included 7 studies that included type 1 or type 2 diabetics and used standardized tests to evaluate various cognitive function domains. standardized mean differences (smds) were calculated for each domain. we found that type 1 diabetics get no cognitive benefit from a tight glucose control, whereas type 2 diabetics get some benefit on processing speed and executive domains but had worse performances in the memory and attention domains, along with a higher incidence of mortality when using intensive glucose control regimes.

mercury is a common contaminant in the environment due to both natural and anthropogenic sources. it is of environmental concern because elevated concentrations can be toxic to living organisms. mercury has no known metabolic function in animals and is not easily eliminated by organisms, including humans. areas around mined and unmined deposits of hg contain some of the highest concentrations of hg worldwide. abandoned and inactive hg mines are of environmental concern because of the high concentration of hg present in discarded wastes at these sites. there are presently no hg mines operating in the united states, primarily because of low demand for hg and environmental concerns. cinnabar (hexagonal, hgs) is the dominant hg - bearing ore mineral in hg mines worldwide, although minor ore minerals such as elemental hg (hg), metacinnabar, (isometric hgs, metastable relative to cinnabar), calomel (hg2cl2), kleinite (hg2n(cl, so4)n(h2o) ), montroydite (hgo), and terlinguaite (hghgclo) ) are found in some deposits. extraction of hg during mining is generally carried out in a retort or a rotary furnace where hg ore is heated to 600700 c, which converts cinnabar to elemental hg, the final hg product that is sold commercially. retorting of hg - bearing ore is known to be an incomplete process, and as a , calcine found at most hg mines contains unconverted cinnabar, elemental hg, metacinnabar, and other ionic hg byproduct compounds formed during ore retorting. one consequence of hg mining is that considerable calcine is generated during ore processing and this calcine is typically discarded on the surface at mined sites. even after many years of inactivity, hg mining areas continue to be characterized by highly elevated hg concentrations as a of the incomplete hg extraction process. microscopic fine - grained cinnabar and several byproduct hg compounds found in calcine can adversely affect surrounding environments due to sediment and water runoff containing high concentrations of hg. minor byproduct hg compounds such as chlorides, oxychlorides, and sulfates, which are water - soluble are generally released downstream in mine runoff, potentially contaminating nearby ecosystems. unprocessed ore and tailings containing cinnabar also remain at some mines, and can be a significant source of hg to surrounding environments. new analytical methods for isotopic analysis have been developed to help characterize environmental cycling of hg. measurements of the isotopic composition of hg hold promise as a basis for tracing sources of hg contamination in the environment and evaluation of isotopic fractionation during geochemical processes. before hg isotopes can be evaluated as a tracer of hg in mined areas, the variability in hg isotopic composition of the hg sources involved must be measured. therefore, the objective of this study was to measure the isotopic compositions of hg in cinnabar from two areas of past hg mining to evaluate the variability of hg isotopic composition within hg deposits; the minor hg ore minerals metacinnabar, calomel, kleinite, montroydite, and terlinguaite to evaluate potential isotopic fractionation during formation of these hg minerals; calcine to evaluate potential fractionation of hg ing from retorting of ore during mining; and, water leachates of calcine to evaluate the variability in isotopic composition of the hg in simulated runoff from hg mines. two mining districts of significant past hg production, mcdermitt, nevada, and terlingua, texas were selected for study. mines of the terlingua district are found in southwest texas, in and around the town of terlingua (29 19.170, 103 37.000). total production from this region was > 5 000 000 kg of hg (150 000 flasks of 34.5 kg each). consequently, of the hg mines in the u.s., the terlingua district produced the third largest amount of hg and only mines in the california coast ranges (120 000 000 kg of hg) and mcdermitt, nevada (10 000 000 kg of hg) produced more. the terlingua district includes over 30 separate mines, but our work focused on the mariscal, study butte, mariposa, and terlingua (also known as the chisos) mines. as is true for most hg mines worldwide, the hg ore in the terlingua district is dominantly cinnabar, but minor hg minerals, including metacinnabar, elemental hg, calomel, montroydite, terlinguaite and kleinite were identified at some mines. the district has been estimated to contain over 2 000 000 m of calcine. this calcine contains hg concentrations as high as 19 000 g / g, some of which are water - soluble. leaching, runoff, and downstream transport of hg from calcine potentially deliver significant hg to surroundings streams, which eventually flow into the rio grande. mercury was mined at mcdermitt (also known as cordero, 41 54.970, 117 48.600) from 1935 to 1992, when the mine closed. mcdermitt was the largest hg mine in nevada, and production of hg was about 10 000 000 kg. ore was dominantly cinnabar, but minor elemental hg and oxychlorides (e.g., hg2clo and hg4cl2o) were found locally. numerous piles of calcine are scattered throughout the mcdermitt mine site, but have been estimated as > 1 000 000 m. similar to the terlingua mines, high hg concentrations (up to 1400 g / g) in calcine at mcdermitt potentially lead to hg leaching and transport of hg downstream from the mined areas. all of the calcine and most of the cinnabar samples from the mcdermitt mine and terlingua district analyzed in this study were collected during previous studies. all calcine was collected as grab samples about 2550 cm below the surface to avoid the highly oxidized near - surface environment, and generally at this depth, the calcines were dark gray in color as opposed to more oxidized red - brown at the surface. all mcdermitt mine cinnabar samples were physically separated from bulk ore by crushing and hand picking. samples of metacinnabar, calomel, cinnabar, terlinguaite, kleinite, and montroydite from the terlingua district were obtained from the colorado school of mines (csm, golden, colorado) geology museum. these minerals were physically separated from the host rock and other minerals. previously separated and powdered calomel, montroydite, and a mixed terlinguaite - kleinite sample from the terlingua district were obtained from a u.s. geological survey (usgs, denver, colorado) the terlinguaitekleinite sample from the usgs was fine - grained and these minerals could not be separated, thus, this sample was analyzed as a mixed mineral. the concentration of total hg (thg) was determined in pulverized ore and calcine samples using a 3:1 hcl: hno3 (aqua - regia) leach (1.0 g sample leached in 10 ml aqua - regia) and cold - vapor atomic fluorescence spectrometry following epa method 1631. quality control for thg was established using method blanks, blank spikes, matrix spikes, standard reference materials (srms), and sample duplicates. for the srm s, nist 2704 and pacs-2 analyzed in this study, recoveries ranged from 96 to 107% of certified values for thg. method blanks were below the thg limit of determination, which was 0.005 g / g. pulverized calcine and hg mineral samples were leached in aqua - regia (3:1 hcl : hno3) overnight in borosilicate glass test tubes (1.0 g sample leached in 10 ml aqua - regia) at room temperature and diluted immediately prior to analysis with 3% (v / v) hcl to a final hg concentration of 1 g / l (all minerals and calcines) or 2 g / l (calcine leachates). a blank digestion was performed, diluted to 50 ml with 3% (v / v) hcl and confirmed to contain no hg. hg was introduced into a multiple collector inductively coupled plasma mass spectrometer (mc - icp - ms) (nu plasma hr, nu instruments, wrexham, north wales, uk) as a cold vapor, which was generated from hg in solution using stannous chloride in a commercially available cold vapor (cv) generation system (hgx-200, cetac, omaha, ne). argon carrier gas (sample gas) was introduced into the cv generator at a flow rate of 5060 ml / min and stannous chloride reductant and sample solution, each at a rate of 1.5 ml / min were mixed with the ar gas. a makeup ar gas flow of 0.91.0 l / min was added prior to introduction into the plasma. signal (3% ( v / v) hcl blank ) was measured on each isotope peak for 60 s and subtracted from the peak integrated data. signal data were measured via time - resolved analysis software for approximately eight minutes and data were integrated for approximately five minutes. the sensitivity of the mc - icp - ms for hg was 7001000 mv (g / l) and data were collected using solution thg concentrations of 12 g / l. each sample was bracketed by nist 3133 hg standard at the same thg as the sample and sample data were corrected for mass bias using standard sample bracketing correction. the standard sample bracket method was optimized to ensure that the precision was comparable to previously published studies that used either standard - sample bracketing or tl correction for mass bias. sensitivity for hg was comparable to or greater than that of other published methods. studies were performed to determine how closely standard and sample concentrations and matrix should to be matched to obtain optimal precision. common matrix constituents did not cause mass bias in the measurements when no matrix matching between standards and samples was performed. no mass bias was introduced when sample and standard concentrations were varied over a 25-fold range, but method precision decreases as sample concentration is decreased. also, when concentrations greater than 2 g / l were used washout times increased. isotopic ratioshg / hg, hg / hg, hg / hg, and hg / hg were measured and hg isotopic compositions are reported in standard delta notation as hg in per mil , referenced against nist 3133 where x = 199, 200, 201, or 202. values of hg are calculated as follows: all hg values and 2 standard deviation (sd) reproducibility of replicate isotopic measurements are reported in supporting information (si) table s-1, but only hg data are discussed below. all data were assessed for mass independent fractionation (mif) by calculating hg, hg, and hg using a method previously described. the long - term reproducibility of the method was determined to be 0.09 (2sd) for hg and 0.05 (2sd) for hg by measurement of the isotopic composition of 2 g / l thg in secondary standard um - almadn, but was 0.24 (2sd) for hg and 0.10 (2sd) for hg at a thg concentration of 1 g / l (si table s-1 contains all and values for this standard). laboratory water leaching studies were conducted on samples of calcine using an operationally defined procedure modified from epa method 1312. eight mine - waste calcine samples (seven from terlingua and one from mcdermitt) of 3050 g were leached with 150 ml of deionized water acidified with ultrapure h2so4/hno3 to a ph of 5.0. leachate fluid of ph 5 is recommended to simulate rainwater ph in the western u.s. the samples were leached on a shaker table using 250 rpm for 18 h. the leachate was isolated and filtered using 0.45 m nitro - cellulose disposable filters. concentrated ultrapure hcl was added to the filtrate to yield 3% (v / v) hcl content. immediately prior to analysis, leachates were diluted with 3% (v / v) hcl to yield a hg concentration in the range of 12 g / l. to establish the variability of hg isotopic composition of cinnabar within a single hg deposit, hg isotopic compositions were determined in several cinnabar crystals separated from a single sample of ore from the mcdermitt mine and several additional cinnabar samples collected throughout the mcdermitt mine site. the total range in the isotopic composition of hg measured for a single sample of mcdermitt cinnabar was 0.42 to 0.69, which is indistinguishable within analytical precision (0.24, si tables s-1 and s-2). a smaller range in isotopic hg compositions was found for the multiple samples of cinnabar, 0.57 to 0.70, also indistinguishable within analytical precision. previous studies report hg compositions for various hg - bearing mineralized rocks and ore deposits in california and nevada as 3.88 to 2.10, a variation of 5.98. other studies reported a smaller variation in hg values for cinnabar samples collected from numerous hg mines and deposits worldwide, 1.73 to 1.33, a variation of 3.06. it is unclear if the hg compositions measured in hg - bearing mineralized rocks show a wide variation due to isotopic hg sources from varying rock types or isotopic fractionation during ore formation. although the above studies reported a wide variation in hg compositions for various hg deposits worldwide, our hg isotopic analysis of numerous samples of cinnabar collected throughout the mcdermitt mine indicates that cinnabar at the mcdermitt mine has an isotopically narrow range of hg composition of 0.60 0.20. the range in hg was 1.60 to 1.72, indicating a statistically insignificant variation of 0.12, for various cinnabar samples from the terlingua district (figure 1). the hg of other hg - bearing ore minerals from the terlingua district (montroydrite, kleinite, terlinguaite, metacinnabar, and calomel) ranged from 2.70 to 1.39, a variation of 4.09 (figure 1), a much wider variation than found for the terlingua district cinnabar samples. in addition, mass - independent fractionations in hg ranging from 0.09 to 0.36 were observed in these hg minerals (si table s-1, figure 2). the differences in hg isotopic composition among the various hg minerals from the terlingua district are statistically significant (figure 1). isotopic compositions of hg for various sources of hg from the mcdermitt mine and the terlingua district. data shown for cinnabar, metacinnabar, terlinguaite, and kleinite are isotopic measurements made in duplicate or triplicate on one sample of each mineral and symbols represent the average. for calomel and montroydite, duplicate or triplicate isotopic measurements were made on two separate samples of each mineral from different sources (colorado school of mines museum and u.s . hg vs hg of minerals, mine waste calcine, and leachates of mine waste calcine . the slope of the line is 1.66 for the minerals and 1.11 for the calcines . the slope of the line for the leachates is 1.35 although the mif is not statistically significant for any of the leachates based on the method reproducibility and 2 of the mean . the hg - bearing ore minerals of the terlingua district are primary, hypogene in origin, formed by hydrothermal mineralizing fluids. deposition of ore minerals from a hydrothermal fluid is complex, potentially ing from several geochemical processes including changes in ph, temperature, fluid boiling and mixing, redox reactions, and reactions with surrounding wallrocks. formation conditions are unknown for the individual hg ore minerals in the terlingua deposit, and thus, it is not possible to explain their wide varying isotopic compositions. however, possible factors controlling mass - dependent isotopic variability in these minerals include varying formation temperatures, paragenesis, or variable redox states or bond strengths among the different minerals. mass independent isotopic variability is generally caused by the nuclear field shift effect or the magnetic isotope effect. previous studies have shown mif due to photochemical reduction of hg and methyl - hg to hg. when hg vs hg is plotted for each of these photochemical reduction processes a slope of 1.36 is obtained for methyl - hg and 1.00 for hg reduction. the same plot of the terlingua hg minerals has a slope of 1.66 indicating that the source of mif for these minerals is not photochemical reduction (figure 2). the hg values of two samples of calomel vary significantly between the two samples analyzed (0.75 and 2.05). it is formed under a variety of temperatures, and is also known to contain microscopic amounts of elemental hg. the hg values of calomel suggest isotopic fractionation as a of variable formational processes, formation temperatures, or redox conditions. the difference in hg values also could indicate the presence of elemental hg in different proportions to calomel in the samples. this hg would be expected to have a different isotopic composition than calomel, causing variations in the measured bulk hg isotopic composition of the calomel samples. the hg data obtained from the analysis of calcine in the terlingua district show isotopic fractionation of the cinnabar due to the retorting process. the hg values for terlingua calcine vary from 1.34 (study butte mine) to 1.52 (mariscal mine), a variation of 2.86 (figure 1). thus, the hg values for samples of terlingua calcine are isotopically heavier than the original cinnabar, recording hg isotopic fractionation. mif in hg range from 0.17 to 0.23 in the terlingua calcines (figure 2), which is small or statistically insignificant. during the retorting process, hg in cinnabar the retorting process is often incomplete, and not all of the cinnabar is converted to hg. thus, some of the original cinnabar survives the retorting process and is present as microscopic grains in calcine, often as fine - grain cinnabar encapsulated in quartz and calcite gangue, but there is also metacinnabar present in calcine. concentrations of thg up to 19 000 g / g in samples of calcine from the terlingua district probably are due to the presence of residual cinnabar (see si table s-3 for hg concentrations in calcines and calcine leachates). therefore, calcines with hg values (i.e., study butte mine calcine sample 03sb3, 1.34) that are isotopically similar to terlingua cinnabar (1.66 0.12), may contain significant residual cinnabar that has not been altered by the retort process. conversely, terlingua calcines that are enriched in the heavy isotopes of hg (relative to cinnabar) require that the volatilized hg was enriched in the light isotopes. however, no correlation between thg in calcines and isotopic composition was found (si figure s-1). the lack of correlation between thg in calcines and hg values is partly due to a cinnabar nugget effect. for example, a calcine containing cinnabar may have the isotopic composition of cinnabar, but samples with more residual cinnabar will have a higher thg, but also have the isotopic composition of cinnabar, thus, hg will not generally correlate with higher thg in many calcine samples. calcine contains byproduct hg compounds formed when cinnabar is converted to elemental hg during the retorting process and these newly formed hg compounds are enriched in the heavier isotopes of hg. consequently, previous research has shown that there are several natural and anthropogenic processes that will fractionate hg isotopes and in the formation of an isotopically light hg(gas) including biotic, photochemical, and chemical reduction of hg to hg, volatilization of hg(gas) from aqueous solution and hg(liquid), and retorting of hg ore. the for calcines in this study are consistent with previous studies that have shown that the hg remaining after incomplete reduction and volatilization will be enriched in the heavier isotopes of hg. in addition, theoretical calculations indicate that heavier isotopes of hg will be preferentially partitioned into the oxidized species (hg) relative to the reduced species (hg). if a kinetic process during retorting causes rayleigh - type fractionation, then a range in hg of 0.289.25 represents fractionation between the ore and calcine for retort efficiencies between 50 and 99%, using published fractionation factors for reduction and volatilization of hg over a temperature range of 2237 c. a much smaller range of fractionation is observed in this study for retorting, which is expected since the retorting process is carried out at much higher temperatures. while the observed fractionation of hg in the calcine relative to cinnabar is consistent with reduction and volatilization as the relevant processes, there could be other processes that fractionate hg in the calcine. for instance, formation of other hg minerals such as metacinnabar during retorting also could cause fractionation. similar to the for the terlingua calcine samples, the hg data obtained for calcine samples from the mcdermitt mine also reflect fractionation processes due to ore retorting. the hg values vary from 0.64 to 0.22 for three of the five mcdermitt calcine samples (figure 1); these samples are isotopically similar to the mcdermitt cinnabar (0.60 0.20). mif in hg range from 0.05 to 0.17 and are not statistically significant (figure 2). these three hg compositions indicate the presence of significant residual cinnabar that has not been altered isotopically by the retort process in these mcdermitt calcine samples. another mcdermitt calcine sample was isotopically heavier (hg = 0.28) than mcdermitt cinnabar, and similar to the for the terlingua calcine samples, this is likely a of hg isotopic fractionation during retorting (as discussed above). conversely, the fifth mcdermitt calcine sample has a significantly lighter hg value of 1.49. previous studies have reported the presence of elemental hg in calcine from the mcdermitt mine due to inefficient removal of elemental hg during retorting. we interpret the lighter hg in this calcine sample to be due to the presence of elemental hg. leaching experiments were designed to examine the isotopic composition of hg minerals that are soluble in weakly acidic water (ph 5, which is similar to rainwater ph in this region of the us) and likely to be mobilized during weathering of the calcine piles. the hg values for leachates of terlingua calcine vary from 0.17 to 2.09, and in general, the leachates have hg values that are isotopically heavier than the associated bulk calcine (figure 3). in only one sample (03mar3, mariposa mine) was the leachate hg (0.20) isotopically lighter than the bulk calcine sample (0.46). the of the calcine leachate studies suggest the possibility that minor, soluble, byproduct hg compounds, formed during retorting and present in the calcine, were leached during these experiments. this study was designed to determine the isotopic composition of rainwater leachate of the calcine, not that of any particular soluble compounds present in the calcine, thus, our data only indicate that these leachates are isotopically heavy in comparison to the hg of the associated calcine and cinnabar from an individual mine. for example, for sample 03sb1 (study butte mine) the leachate hg was 0.71, whereas the calcine hg was 0.46. thus, the hg measured for the leachate was 1.17 heavier than that of the calcine sample that was leached. variability in isotopic composition of total hg in mine - waste calcine (open box) and ph 5 water - leachable hg (filled box) of those calcines from mines of the terlingua district and mcdermitt mine. mar = mariposa mine, msm = mariscal mine, sb = study butte mine, and mcd = mcdermitt mine. cinnabar from the terlingua district and mcdermitt mine (gray box) are shown for reference. because byproduct hg compounds are a minor component of the calcines, these compounds are often difficult to identify. however, previous studies using edge extended x - ray absorption fine structure analysis have identified several hg oxides, chlorides, oxychlorides, and sulfates in various calcine samples, and many of these hg compounds are water - soluble. in this study, calcine samples were leached with weak acidic water and compounds such as sulfates, chlorides, and oxychlorides were dissolved, whereas cinnabar has limited solubility at this ph. in summary, the of our leachate studies indicate that runoff from the hg mines studied is most likely to carry a hg isotopic composition that is similar to or heavier than that of calcine present at the site due to the presence of the minor, byproduct hg compounds formed during or after retorting of the calcine. because of the high solubility of these compounds at the ph of the leachate used, they dominate the hg composition in the water runoff. recent studies reported that the hg isotopic compositions of river sediment collected downstream from the idrija hg mine, slovenia, were similar to that of cinnabar ore at the mine upstream. in the idrija study, downstream river sediments were found to contain large amounts of cinnabar, and thus, isotopic hg tracing was used successfully to link downstream hg contamination to cinnabar from the idrija mine. we have not measured the isotopic hg compositions of stream sediments proximal to the mines studied, however, we focused on examining anthropogenic hg isotopic fractionation in calcine at these sites. calcines are voluminous and primary cinnabar ore minor at the mines studied (e.g., > 2 000 000 m in the terlingua district), and sediment transport away from such mines, especially in dry, desert environments also is generally minor. therefore, due to the large volume of calcine at these hg mines, the hg isotopic composition of water runoff is most likely to be dominated by the hg isotopic composition of calcine, not the primary cinnabar ore. studies using hg isotopic tracing in hg mined areas should include the measurement of the hg isotopic composition of calcine in areas where calcine predominates volumetrically over primary cinnabar ore. the measurement of the hg isotopic compositions of cinnabar and leachates of the calcine are also necessary to adequately trace hg sources from areas mined for hg. in addition, tracing of mining - related hg may be complicated by microbial, chemical, and photochemical transformations in water and sediment that potentially fractionate hg isotopes. such fractionation processes have not been well characterized in ecosystems downstream from areas mined for hg.

the isotopic composition of mercury (hg) was determined in cinnabar ore, mine - waste calcine (retorted ore), and leachates obtained from water leaching experiments of calcine from two large hg mining districts in the u.s. this study is the first to report significant mass - dependent hg isotopic fractionation between cinnabar ore and ant calcine. data indicate that 202hg values relative to nist 3133 of calcine (up to 1.52) in the terlingua district, texas, are as much as 3.24 heavier than cinnabar (1.72) prior to retorting. in addition, 202hg values obtained from leachates of terlingua district calcines are isotopically similar to, or as much as 1.17 heavier than associated calcines, most likely due to leaching of soluble, byproduct hg compounds formed during ore retorting that are a minor component in the calcines. as a of the large fractionation found between cinnabar and calcine, and because calcine is the dominant source of hg contamination from the mines studied, 202hg values of calcine may be more environmentally important in these mined areas than the primary cinnabar ore. measurement of the hg isotopic composition of calcine is necessary when using hg isotopes for tracing hg sources from areas mined for hg, especially mine water runoff.

the et2ds is a population - based cohort study designed to investigate potentially modifiable risk factors for cognitive decrements in type 2 diabetes. the study commenced in 2006/2007 as a cross - sectional survey of 547 men and 519 women aged 6075 with type 2 diabetes. participants were recruited at random, in 5-year age bands, from the lothian diabetes register, which is a computerized database containing clinical details on over 20,000 patients with known type 2 diabetes living in lothian, scotland. the lothian research ethics committee approved the study, and fully informed written consent was obtained from each participant. details of the study recruitment and examination procedures have previously been described in detail. study participants have been shown to be representative of the target population of older men and women with type 2 diabetes living in the general population. specially trained research assistants administered a detailed battery of cognitive tests in a standard order during a single session in a quiet and well - lit room following tests for adequate visual acuity and capillary blood glucose levels (> 4.0 mmol / l). cognitive ability was assessed using tests of immediate and delayed nonverbal memory and verbal declarative memory (faces and family pictures subtest and logical memory i ( lm) from the wechsler memory scale - iii ); nonverbal reasoning, working memory, information processing speed (matrix reasoning , letter - number sequencing , and digit symbol test from the wechsler adult intelligence scale 3rd edition); executive function (borkowski verbal fluency test) and mental flexibility (trail making test - part b). vocabulary (crystallized intelligence) was measured using the combined version of the junior and senior form a synonyms of the mill hill vocabulary scale . as on vocabulary - based tests vary little with aging, they can be used to estimate peak prior cognitive ability. late - life cognition adjusted for vocabulary correlates highly with actual cognitive change. the mini - mental state examination (mmse) is often used as a screening for dementia and was included as a general mental assessment for possible cognitive pathology. a self - administered questionnaire was used to collect data on age, sex, educational attainment, diabetes history and treatment modality, smoking history, alcohol consumption, medication use, and history of cardiovascular disease, including the world health organization (who) chest pain and edinburgh claudication questionnaires. clinical measurements included plasma a1c and fasting total and hdl serum cholesterol, height, weight, waist and hip circumferences, a resting 12-lead electrocardiogram, and brachial blood pressures as previously described. additional information on macrovascular disease was obtained from the information and services division of nhs scotland on all medical and surgical discharges from scottish hospitals since 1981 (smr01 scheme), and any k09 or k010 codes indicating cardiovascular or cerebrovascular disease were extracted. retinal photography was performed approximately 23 weeks after each subject's initial visit to the research clinic for cognitive and physiological testing. of the 1,046 participants who underwent both cognitive and retinal examinations, two were excluded who did not have gradable photographs for dr severity in either eye, leaving 1,044 who provided data for this analysis. after pupillary dilatation, standard seven - field nonstereoscopic color photographs were taken of both eyes at 35 using a high - resolution digital retinal camera. all photographs were graded by two trained optometrists, working independently and according to the scale described by the early treatment diabetic retinopathy study (etdrs) research group. inter- and intraobserver variations and the validity of this grading system have previously been evaluated. for each eye, the maximum grade in any of the seven photographic fields was determined for each of the characteristic lesions of dr and was used in defining the final retinopathy levels, varying from level 10 (no retinopathy) to level 81 (advanced proliferative retinopathy). for the purpose of this study, a score of 81 was added for panretinal photocoagulation scars only if the laser treatment was for dr. the retinopathy level for a participant was assigned on the basis of the severity scores of the worse eye. if the photographs from an eye could not be graded, the scores for the other eye were used. discrepancies in the final level score at subject level between the graders were resolved in the first instance by discussion between them. the average weekly alcohol intake was defined as standard drinks per week. diabetes treatment was classified into three groups: 1 ) diet control only, 2 ) oral antidiabetes agents without insulin, and 3 ) insulin injection with or without oral agents. waist - to - hip ratio (whr) was calculated as the waist circumference divided by the hip circumference in centimeters. bmi was defined as weight in kilograms divided by the square of height in meters. mmol / l or use of medication prescribed by a doctor to lower blood lipids level. hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 85 mmhg or use of medication prescribed by a doctor to lower blood pressure. coronary heart disease (myocardial infarction and/or angina) was defined if two of the first three of the following criteria were met or if both the first and last criteria were met: 1 ) subject recall of a doctor's diagnosis of myocardial infarction or angina, 2 ) positive who chest pain questionnaire, 3 ) electrocardiogram evidence of ischemia, and 4 ) prior hospital discharge (icd) code for ischemic heart disease. cerebrovascular disease (stroke and/or transient ischemic attack) was defined if two of three of the following criteria were met: 1 ) subject recall of a doctor's diagnosis of stroke or tia, 2 ) prior hospital discharge code consistent with stroke or tia, and 3 ) confirmation by clinical notes review. intermittent claudication was based on a positive response to the edinburgh claudication questionnaire. any macrovascular disease was defined as a history of myocardial infarction, angina, stroke, tia, or peripheral arterial disease. dr was defined as an etdrs level of 20, i.e., the presence of microaneurysms alone or with any of the following lesions: hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading, venous loops and/or reduplication, fibrous proliferations, preretinal hemorrhage, vitreous hemorrhage, and new vessels. this was further divided into mild nonproliferative dr (npdr) (levels 2035), moderate - to - severe npdr (levels 4353), and proliferative dr (levels 6181). due to small numbers in the proliferative retinopathy group, severity of dr was collapsed into three categories (none, mild, and moderate severe); the two most severe categories were combined into moderate a measure of general cognitive ability, representing the variance common to all the cognitive tests except mhvs, was generated. this was done by subjecting the seven cognitive tests (faces, lm, mr, lns, dst, vft, and tmtb) to a principal - components analysis. scree slope analysis and the eigenvalues - greater - than-1 rule both indicated a single component (which accounted for 44% of the total variance, on which all tests had high loadings), thus validating the use of a general factor. scores on this first unrotated principal component were saved as standardized scores (mean sd 0 1). all continuous variables were normally distributed except for the tmtb for which a natural logarithmic transformation of scores was used, as well as depression scores (normalized through a n + 1 logarithmic transformation), duration of diabetes, and alcohol intake (square root transformations). severe ) were compared on all demographic and medical variables using tests of linearity included in anova for continuous data and analyses for categorical data. ancova was used for comparing cognitive test scores in subjects according to severity of dr and in assessing the effects of retinopathy severity on the imputed cognitive change from estimated peak prior cognitive function (mhvs scores) at the same time as adjusting for possible confounders. adjustment variables were introduced into the models in three cumulative steps. in the first step finally, education level, vascular risk factors (alcohol intake, smoking status, whr, systolic blood pressure, and total cholesterol), the presence of macrovascular disease, and depression symptoms were additionally adjusted. these variables were selected as possible confounders if they showed a significant association with dr on univariate analysis or if, in previous literature, a variable was reported to be associated with both dr and cognitive test performance. we did not include duration of diabetes or a1c in the main multivariate model because it is likely that prolonged exposure to hyperglycemia underlies the development of dr and their inclusion in the model could in overadjustment. all tests were two tailed, and a two - sided p value 0.05 was taken to indicate statistical significance. all analyses were performed using spss, version 14.0, for windows. specially trained research assistants administered a detailed battery of cognitive tests in a standard order during a single session in a quiet and well - lit room following tests for adequate visual acuity and capillary blood glucose levels (> 4.0 mmol / l). cognitive ability was assessed using tests of immediate and delayed nonverbal memory and verbal declarative memory (faces and family pictures subtest and logical memory i ( lm) from the wechsler memory scale - iii ); nonverbal reasoning, working memory, information processing speed (matrix reasoning , letter - number sequencing , and digit symbol test from the wechsler adult intelligence scale 3rd edition); executive function (borkowski verbal fluency test) and mental flexibility (trail making test - part b). vocabulary (crystallized intelligence) was measured using the combined version of the junior and senior form a synonyms of the mill hill vocabulary scale . as on vocabulary - based tests vary little with aging, they can be used to estimate peak prior cognitive ability. late - life cognition adjusted for vocabulary correlates highly with actual cognitive change. the mini - mental state examination (mmse) is often used as a screening for dementia and was included as a general mental assessment for possible cognitive pathology. a self - administered questionnaire was used to collect data on age, sex, educational attainment, diabetes history and treatment modality, smoking history, alcohol consumption, medication use, and history of cardiovascular disease, including the world health organization (who) chest pain and edinburgh claudication questionnaires. clinical measurements included plasma a1c and fasting total and hdl serum cholesterol, height, weight, waist and hip circumferences, a resting 12-lead electrocardiogram, and brachial blood pressures as previously described. additional information on macrovascular disease was obtained from the information and services division of nhs scotland on all medical and surgical discharges from scottish hospitals since 1981 (smr01 scheme), and any k09 or k010 codes indicating cardiovascular or cerebrovascular disease were extracted. retinal photography was performed approximately 23 weeks after each subject's initial visit to the research clinic for cognitive and physiological testing. of the 1,046 participants who underwent both cognitive and retinal examinations, two were excluded who did not have gradable photographs for dr severity in either eye, leaving 1,044 who provided data for this analysis. after pupillary dilatation, standard seven - field nonstereoscopic color photographs were taken of both eyes at 35 using a high - resolution digital retinal camera. all photographs were graded by two trained optometrists, working independently and according to the scale described by the early treatment diabetic retinopathy study (etdrs) research group. inter- and intraobserver variations and the validity of this grading system have previously been evaluated. for each eye, the maximum grade in any of the seven photographic fields was determined for each of the characteristic lesions of dr and was used in defining the final retinopathy levels, varying from level 10 (no retinopathy) to level 81 (advanced proliferative retinopathy). for the purpose of this study, a score of 81 was added for panretinal photocoagulation scars only if the laser treatment was for dr. the retinopathy level for a participant was assigned on the basis of the severity scores of the worse eye. if the photographs from an eye could not be graded, the scores for the other eye were used. discrepancies in the final level score at subject level between the graders were resolved in the first instance by discussion between them. the average weekly alcohol intake was defined as standard drinks per week. diabetes treatment was classified into three groups: 1 ) diet control only, 2 ) oral antidiabetes agents without insulin, and 3 ) insulin injection with or without oral agents. waist - to - hip ratio (whr) was calculated as the waist circumference divided by the hip circumference in centimeters. bmi was defined as weight in kilograms divided by the square of height in meters. mmol / l or use of medication prescribed by a doctor to lower blood lipids level. hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 85 mmhg or use of medication prescribed by a doctor to lower blood pressure. coronary heart disease (myocardial infarction and/or angina) was defined if two of the first three of the following criteria were met or if both the first and last criteria were met: 1 ) subject recall of a doctor's diagnosis of myocardial infarction or angina, 2 ) positive who chest pain questionnaire, 3 ) electrocardiogram evidence of ischemia, and 4 ) prior hospital discharge (icd) code for ischemic heart disease. cerebrovascular disease (stroke and/or transient ischemic attack) was defined if two of three of the following criteria were met: 1 ) subject recall of a doctor's diagnosis of stroke or tia, 2 ) prior hospital discharge code consistent with stroke or tia, and 3 ) confirmation by clinical notes review. intermittent claudication was based on a positive response to the edinburgh claudication questionnaire. any macrovascular disease was defined as a history of myocardial infarction, angina, stroke, tia, or peripheral arterial disease. dr was defined as an etdrs level of 20, i.e., the presence of microaneurysms alone or with any of the following lesions: hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading, venous loops and/or reduplication, fibrous proliferations, preretinal hemorrhage, vitreous hemorrhage, and new vessels. this was further divided into mild nonproliferative dr (npdr) (levels 2035), moderate - to - severe npdr (levels 4353), and proliferative dr (levels 6181). due to small numbers in the proliferative retinopathy group, severity of dr was collapsed into three categories (none, mild, and moderate severe); the two most severe categories were combined into moderate severe dr and used in all subsequent analyses. a measure of general cognitive ability, representing the variance common to all the cognitive tests except mhvs, was generated. this was done by subjecting the seven cognitive tests (faces, lm, mr, lns, dst, vft, and tmtb) to a principal - components analysis. scree slope analysis and the eigenvalues - greater - than-1 rule both indicated a single component (which accounted for 44% of the total variance, on which all tests had high loadings), thus validating the use of a general factor. scores on this first unrotated principal component were saved as standardized scores (mean sd 0 1). all continuous variables were normally distributed except for the tmtb for which a natural logarithmic transformation of scores was used, as well as depression scores (normalized through a n + 1 logarithmic transformation), duration of diabetes, and alcohol intake (square root transformations). severe ) were compared on all demographic and medical variables using tests of linearity included in anova for continuous data and analyses for categorical data. ancova was used for comparing cognitive test scores in subjects according to severity of dr and in assessing the effects of retinopathy severity on the imputed cognitive change from estimated peak prior cognitive function (mhvs scores) at the same time as adjusting for possible confounders. adjustment variables were introduced into the models in three cumulative steps. in the first step finally, education level, vascular risk factors (alcohol intake, smoking status, whr, systolic blood pressure, and total cholesterol), the presence of macrovascular disease, and depression symptoms were additionally adjusted. these variables were selected as possible confounders if they showed a significant association with dr on univariate analysis or if, in previous literature, a variable was reported to be associated with both dr and cognitive test performance. we did not include duration of diabetes or a1c in the main multivariate model because it is likely that prolonged exposure to hyperglycemia underlies the development of dr and their inclusion in the model could in overadjustment. all tests were two tailed, and a two - sided p value 0.05 was taken to indicate statistical significance. all analyses were performed using spss, version 14.0, for windows. characteristics of the total et2ds population (n = 1,066) and comparisons with nonparticipants from the target population have previously been presented. these subjects did not differ significantly from subjects without photographs either available or suitable for grading (n = 22) with respect to diabetes duration or treatment, mean a1c, cardiovascular risk factors, or prevalence of macrovascular disease (data not shown). however, they did perform significantly better on tests of nonverbal memory (mean sd faces scores 66.0 7.8 vs. 58.3 7.0 ; p < 0.001), verbal declarative memory (mean lm scores 25.4 8.2 vs. 20.0 5.9 ; p = 0.003), and general cognitive ability (mean g scores 0.00 1.00 vs. 0.53 0.77 ; p = 0.014). of the 1,044 type 2 diabetic participants, 339 (32.5%) subjects of these, 292 (86.1%) had mild npdr, 32 (9.4%) had moderate - to - severe npdr, and 15 (4.4%) had proliferative retinopathy (n = 19 and n = 8 for men in the latter two categories, respectively). subjects with dr were more likely to be receiving insulin treatment and had higher mean a1c, diabetes duration, and whr. characteristics of study participants according to severity of dr data are means sd, % (n), or median (interquartile range) unless otherwise indicated. age- and sex - adjusted general cognitive ability (g) was significantly lower in the group with moderate - to - severe dr (mean 0.44) compared with the group without dr (0.05 ; p = 0.003), with an intermediate score for the mild dr group (0.04) (p for trend = 0.003). no significant associations were found between dr and estimated premobid cognitive ability (mhvs score). the association between dr and current general cognitive ability (g) remained statistically significant after adjustment for mhvs, but in this model there was a significant interaction between sex and retinopathy for g (p = 0.030). the total study population was stratified by sex, and the effects of retinopathy on g and individual cognitive tests were examined separately for men and women (table 2). following age adjustment, significant associations were only found in men between dr and g, vft and tmtb, and in both sexes with dst. when adjusted for mhvs to estimate lifetime change in cognitive ability, the retinopathy - dst association lost statistical significance in women but remained significant in men. when further adjusted for a wide range of potentially confounding variables, including macrovascular disease, associations in men persisted between dr and g (p for trend < 0.001 ; = 0.020), vft (p for trend = 0.001 ; = 0.020), tmtb (p for trend = 0.009 ; = 0.012), and dst (p for trend = 0.001 ; = 0.032) tests (assessing executive function, mental flexibility, and processing speed, respectively). no statistically significant differences across any of the retinopathy categories were found for the lm, mr, or lns tests for either sex. there was not any significant interaction of retinopathy and any of the variables other than sex. estimated mean (2 se) of general factor (g) scores of men and women according to severity of dr. multivariable - adjusted mean (se) of cognitive scores by severity of dr in men and women further adjusted for education, alcohol intake, smoking status, whr, systolic blood pressure, total cholesterol, major macrovascular disease, and depression symptoms. * effect size of dr as indexed by 2 (the proportion of variance) in the fully adjusted model. analyses were repeated after excluding individuals with prevalent stroke or history of tia and those with mmse scores < 24. none of the associations reported were essentially altered by these exclusions (data not shown). in a final analysis adjustment for both attenuated the association between dr and tmtb to borderline significance (p = 0.059) in men but did not change other . in this representative population of older people with type 2 diabetes, increasing severity of dr was associated with poorer general cognitive ability. when analyzed separately by sex, a significant dose - response relationship was found only in men and for individual tests of verbal fluency, information processing speed, and mental flexibility (but not memory and nonverbal reasoning). these associations persisted after adjustment for estimated premorbid cognitive ability (vocabulary scores), suggesting that in men, dr was not only associated with cognitive ability in later life but also with increased estimated lifetime cognitive decline. to estimate cognitive decline , it was not possible to adjust for the same cognitive test applied at an earlier age, but all the nonvocabulary tests were significantly correlated and vocabulary scores were strongly loaded on general cognitive ability (coefficient 0.57 ; p < 0.001). after additional control for education, vascular risk factors, macrovascular disease, and mood, associations between dr and estimated cognitive decline remained significant, suggesting that they were not simply due to confounding by other vascular mechanisms. to our knowledge , no previous studies have specifically examined the relationship between dr and cognitive functioning in a large group of older people with type 2 diabetes from the general population using comprehensive and detailed assessment of retinal signs. two previous studies did not show an association between dr and cognitive function in older diabetic patients, but these were based on either a direct ophthalmoscopic examination of the retina or a single - field retinal photograph. these methods image only a small portion of the retina and with considerably less rigor compared with multiple - field photographs and as a , the prevalence of dr signs may have been underestimated. our findings are consistent with those from studies of predominantly nondiabetic older subjects from the general population. in the blue mountains eye study , subjects with hypertension aged 49 years and older who had evidence of retinopathy signs were significantly more likely to score poorly (< 23) on the mmse, and in the cardiovascular health study there was a significant association between retinopathy and worse performance on information - processing speed in subjects aged > 69 years. the atherosclerosis risk in communities study (aric) also reported that retinopathy was independently associated with poorer cognitive function in middle - aged people free of stroke, both in those with and in those without diabetes and hypertension. longitudinal findings from the 14-year follow - up of the same cohort showed that people with retinopathy had a greater 10-year decline in verbal fluency and information - processing speed (but not in delayed verbal memory) compared with those without retinal microvascular abnormalities. due to the considerable homology between retinal and cerebral microvasculature, retinal vascular changes are likely to provide an indirect marker of concomitant changes in the brain microvasculature. it is possible that accelerated cognitive aging associated with type 2 diabetes may arise, at least in part, from the cumulative impact of a disruption of blood - brain barrier and/or the ischemic injuries leading to diverse changes in brain parenchyma. as our findings persisted after subjects with recognized clinical stroke were excluded, perivascular brain damage and/or ischemia from asymptomatic cerebral small vessel disease (svd) could explain the cognitive associations. for example, microinfarcts and mri signs of cerebral svd (e.g., lacunar infarcts and white matter lesions) have been shown to contribute importantly to cognitive impairment and vascular dementia. furthermore, increased blood - brain barrier permeability has been demonstrated in people with type 2 diabetes, and alteration in the blood - brain barrier secondary to microvascular endothelial dysfunction may be an important pathophysiological mechanism in the initiation or worsening of cerebral svd. cerebral svd predominantly affects the subcortical associative areas of deep gray matter (the basal ganglia and thalamus) and white matter structures, with disruption of integrity of frontal subcortical circuits. our in type 2 diabetes are consistent with a cognitive profile that is associated with cerebral svd. a striking finding of the present study was a significant interaction of dr with sex such that the negative associations of dr with several cognitive measures were statistically significant only in men. , no previous study has examined whether sex modifies the relation of retinopathy with cognition. the sex - specific effect of dr on cognitive functioning could be influenced by lower prevalence of dr (29.7 vs. 35.1% ; p = 0.058), coronary heart disease (24.4 vs. 37.8% ; p < 0.001); and stroke and/or tia (5.5 vs. 11.6% ; p < 0.001) in women compared with men, but adjusting for macrovascular disease did not alter the sex - specific effects. it is also possible that other unknown risk factors that were not examined in this analysis (e.g., subclinical atherosclerosis, estrogen use, or physical activity) could play a mediating role. although several hypotheses have been proposed in an attempt to explain sex difference observed in some studies, including male - female differences in brain reserve and differential susceptibility to normal or pathological age - associated changes, there is no clear empirical evidence on the effect of sex on cognition in the general population or in people with diabetes. despite the representativeness of our study participants to the target population in terms of demographic and clinical characteristics, indeed, it has been hypothesized that diabetes may be a more potent risk factor for cognitive dysfunction in women than in men due to a higher risk of diabetes - related macrovascular disease and early loss of possible protective effects of estrogen (due to earlier menopause) on cognitive functioning. it is possible that women with more severe diabetes (those more likely to have retinopathy and cognitive decline) may have died of macrovascular disease before they could be recruited into the study, thereby leading to conservative estimates of associations between retinopathy and cognitive decline. however, the degree of variation of g scores in men and women in the study population was similar and, as shown in table 1, people with any retinopathy were only slightly more likely to be male. it therefore seems unlikely that survival bias could be the full explanation for the sex difference. given that we had no specific a priori reason for assuming that our association of dr with cognition would be different between males and females, and given that other studies have not suggested the existence of such a sex difference, interpretation of the interaction between sex and dr must be treated with caution until it has been replicated in other large studies. one of the main strengths of this study was the use of high - quality retinal photographs and detailed grading of dr using full seven - field photography and the low amount of missing retinal grading data. other strengths included the application of a detailed cognitive test battery covering the major cognitive domains, although use of a single test to assess each particular cognitive domain could mean that only limited aspects of what may be considered complex mental functions were actually determined. the study population had a verified clinical diagnosis of type 2 diabetes and has been shown to be representative of the target population of elderly men and women with the full range of severity of type 2 diabetes living in the general population. the use of a general cognitive factor, g, helped avoid potential problems caused by multiple testing and residual confounding by peak prior mental ability was minimized by controlling for performance on a test of vocabulary rather than relying solely on level of education as in many previous studies. however, the presence of microvascular complications other than retinopathy in the no dr group (e.g., neuropathy and nephropathy), leading to shared microvascular risk burden between the retinopathy and retinopathy - free subjects, may have tended to reduce differences toward the null. the effect size for retinopathy in these older men with type 2 diabetes was small after multivariate adjustment. such a small effect size for risk factors associated with cognitive decline is not uncommon, including the effects of other potentially important factors (e.g., apolipoprotein e genotype and smoking). it is possible that even very modest effects of a risk factor may lead to larger cognitive deficits and possibly dementia with further aging. furthermore, reduction of such a risk factor could have a significant impact on cognition in the diabetic population as a whole by producing a positive shift in the overall population distribution of cognitive ability. however, while our findings support the hypothesis that reduction of microvascular disease might reduce the rate of cognitive decline, the clinical significance of our findings remains to be established. the major limitation of the study is that analyses were cross - sectional, with measures of retinal and cognitive function made almost simultaneously, limiting our ability to determine whether dr preceded or occurred in parallel with cognitive decline. such a follow - up project involving the present study population for the actual cognitive change is underway. in , dr was independently associated with estimated lifetime cognitive decline in older men with type 2 diabetes, supporting the hypothesis that cerebral microvascular disease may contribute to the accelerated age - related cognitive decline observed in diabetic populations. if the above findings are substantiated, diabetes - associated cognitive dysfunction may be amenable to therapeutic and preventive strategies that are targeted specifically at protecting the cerebral microvasculature and reducing the risk of developing even mild microvascular disease in an aging diabetic population.

objectivecerebral microvascular disease associated with type 2 diabetes may exacerbate the effects of aging on cognitive function. a considerable homology exists between the retinal and cerebral microcirculations; a hypothesized association between diabetic retinopathy (dr) and cognitive decline was examined in older people with type 2 diabetes.research design and methodsin the population - based edinburgh type 2 diabetes study, 1,046 men and women aged 6075 years with type 2 diabetes underwent standard seven - field binocular digital retinal photography and a battery of seven cognitive function tests. a general cognitive ability score (g) was generated by principal components analysis. the mill - hill vocabulary scale was used to estimate premorbid cognitive ability. dr was graded using a modification of the early treatment of diabetic retinopathy scale.after age and sex adjustment, a significant relationship was observed with increasing severity of dr (none, mild, and moderate to severe) for most cognitive measures. participants with moderate - to - severe retinopathy had the worst g and the worst performances on the individual tests. there was a significant interaction between sex and retinopathy for g. in male subjects, the associations of retinopathy with g (and with tests of verbal fluency, mental flexibility, and processing speed but not memory and nonverbal reasoning) persisted (p < 0.05) when further adjusted for vocabulary (to estimate lifetime cognitive decline), depression, sociodemographic characteristics, cardiovascular risk factors, and macrovascular disease.dr was independently associated with estimated lifetime cognitive decline in older men with type 2 diabetes, supporting the hypothesis that cerebral microvascular disease may contribute to their observed accelerated age - related cognitive decline. a sex interaction with stronger findings in men requires further confirmation.

optical coherence tomography (oct) enables high - resolution imaging of the anterior segment of the human eye. in a noncontact examination it is possible to analyze layers and shape of cornea in cross - sectional images central corneal thickness itself affects the accuracy of intraocular pressure measurements. in the last decades , correlations between central corneal thickness and intraocular pressure readings have been evaluated in many studies establishing the correlation between these two ocular parameters. many of these studies have used optical or ultrasonic pachymetry for corneal thickness measurements and applanation tonometry for intraocular pressure measurement. nowadays, noncontact tonometry is widely used in clinical daily routine for screening purposes. therefore, the relationship of this measurement technique with central corneal thickness readings as determined by spectral - domain oct (sd - oct) imaging should be further investigated. for clinical evaluation of central corneal thickness as determined by sd - oct, it is essential to know physiological factors that are associated with the dimensions of the tissue. based on this , this study aimed to investigate the associations of distinct central corneal thickness measurements determined by sd - oct with ocular and systemic cardiovascular parameters in a caucasian cohort. our hypothesis is that higher central corneal thickness may be associated with a flatter corneal curvature and with higher intraocular pressure, while systemic cardiovascular parameters are not associated. the study was designed as a cross - sectional, observational study conducted within the scope of a health promotion project conducted within a major industry company. the study population consists of working age subjects (age range from 17 to 64 years) of the miph eye & health study. exclusion criteria were any manifest eye disease, either self - reported or detected on fundus photographs, and insufficient oct scan quality of the anterior segment oct. 1460 healthy eyes of 734 subjects (730 right eyes, 730 left eyes ; 514 men, 220 women) were included in the analysis. the study was conducted according to the tenets of the declaration of helsinki and fully approved by both the ethics approval committee of the university of heidelberg (institutional review board), the company's advisory board, and the board of employees. written informed consent as to the scientific objectives of the study blood pressure was recorded from the dominant arm in the seated position after a standardized 20 min rest period by sphygmomanometry. blood samples were drawn to determine triglycerides (tri), low density lipoproteins (ldl), high density lipoproteins (hdl), and glycosylated hemoglobin (hba1c) using routine laboratory analyzers. the cornea was imaged with the anterior segment mode of the 3d oct-2000 (topcon corp ., automated calculation of corneal curvature and central corneal thickness ( cct) with the integrated software was performed and all oct images were checked for correct identification of the corneal surface. quality of oct scans were graded in a four categories: high, , tokyo, japan ) and noncontact tonometry was performed (ct-80 tonometer, topcon corp ., fundus photographs of the macula and optic nerve head were obtained from all participants and images were evaluated by two independent ophthalmologists . eyes with previous intraocular or refractive surgery were excluded from the study, as were eyes with known and confirmed ocular disease . raw data were processed by statistical analysis software ( spss 21.0, chicago, il) and plots were performed with stata software (version 13.1 se, college station, tx : statacorp lp). associations with central corneal thickness were analyzed by multivariable linear regression algorithms with parameter estimation performed with generalized estimation equations. this statistical model takes the relationship between the right and left eye into account by linking the individual pair of eyes. as dependent variable central corneal thickness was included in the statistical model, mean corneal radius, intraocular pressure, refraction, age, gender, body mass index, mean arterial blood pressure, hba1c, hdl, ldl, and triglyceride values were evaluated as associated factors. association analysis between central corneal thickness and intraocular pressure, respectively, means corneal curvature was performed with univariate linear regression as well to determine comparable parameter estimates with literature reports. in addition, a reverse analysis with intraocular pressure as dependent and central corneal thickness as explanatory variable was computed. spearman correlation coefficient was computed to determine the relationship between central corneal thickness readings and oct image quality. , we corrected this parameter to 0.004 using the bonferroni method. for further interpretation of the , all p values above 0.001 considering all eyes together, mean cct was 561.1 32.3 m (mean standard deviation ; right eyes : 560.1 31.8 m ; and left eyes : 562.2 32.8 m). association analysis of cct measurements found a statistically significant relationship between corneal curvature and intraocular pressure: cct was found to be negatively associated with intraocular pressure readings (p < 0.001 ; figure 1) and positively associated with a flatter corneal curvature (p < 0.001 ; figure 2). reversely, univariable analysis between intraocular pressure as dependent variable and central corneal thickness as explanatory variable revealed that an increase of 22 m in central corneal thickness is linked to an increased measurement of intraocular pressure of 1 mmhg. in the multivariable model, an increase of 10 m in central corneal thickness was associated with an increase of 3 mmhg in intraocular pressure reading. refraction of the eye did not show a significant association with cct (table 2). regarding anthropometric characteristics of the study sample , age was independently associated with cct: older subjects had a higher central corneal thickness (table 2). central corneal thickness values from oct measurements increase by 0.34 m per year of age. regarding cardiovascular parameters, none of the investigated parameters (gender and bmi and mean arterial blood pressure and biochemical parameters : hba1c and hdl and ldl and triglycerides) was significantly associated with cct after bonferroni correction for multiple testing (p < 0.004) (table 2). a weak association between lower scan quality and thinner central corneal thickness measurement was determined (spearman correlation coefficient = 0.09, p < 0.001). nevertheless, upon incorporating this parameter as sensitivity analysis in the multivariable model, our findings remained unaltered. our study in a caucasian cohort demonstrates that central corneal thickness readings as measured with the topcon 3d oct-2000 are independently associated with corneal curvature and with intraocular pressure readings. previously published studies have yielded congruent findings using optical or ultrasound pachymetry and association with intraocular pressure. however, we used sd - oct for determining central corneal thickness which makes a technical difference to other studies where analyses were based on ultrasound pachymetry or scheimpflug imaging. sd - oct accurately measures central corneal thickness as reported by prior publications within the physical limitations of the method itself as determined by wavelength, optical system, and sensor characteristics. furthermore, our finding of a relationship of corneal curvature with central corneal thickness as measured by oct is new. nevertheless, there are several studies reporting such a relationship using other methods. also, our study may contribute to deepening the understanding of associations between cct readings obtained from oct and intraocular pressure readings obtained from noncontact tonometry and add to knowledge being generated with different methods. in the range between 11 and 21 mmhg intraocular pressure and 500 to 625 m central corneal thickness, we found an approximately linear relationship between these two parameters, but this relationship can not be extrapolated to higher or lower values as measurements were not sufficiently available there. with an increase of 22 m in cct, intraocular pressure (as dependent variable) increased by 1 mmhg, and reported associations of central corneal thickness with male gender, higher body height, and body mass index in a german population. the differences with our study might be explained by the underlying study population: we included far more male than female participants, a different age span (17 to 64 years versus 35 to 74 years by elflein et al .), and a different mean body mass index (22.9 versus 27.2), while both studies included mainly caucasians. in a similar age span to elflein et al., nangia and coworkers found associations of central corneal thickness with male gender, younger age, and higher body mass index in an indian population. in chinese people, central corneal thickness is associated with male gender and urban region, while it is not associated with age, body mass index, and body height. a study in a japanese population found similar findings for asian people reporting an association of central corneal thickness with male gender but none for age, body weight, and body height. the ambiguities to our study may arise from the use of different cct assessment methods and variations in age, body measurements, and genetic of the study populations. in our study population consisting of a cohort of caucasians with an age range from 17 to 64 years , central corneal thickness was associated with age: each decade of age went along with an increase of 3.4 m in central corneal thickness over the entire age span. upon comparing our findings to those published by other groups , it must be considered that our age range corresponds to the working age and that we have only included healthy eyes with the purpose of reporting physiological conditions. this means that our study group starts at a juvenile age and terminates at retirement, whereas many other studies have appeared with a much older starting age reaching into senility. there are some studies in european cohorts with different age ranges that did not find an association between central corneal thickness and age. in contrast, we found an association with age: central corneal thickness increased slightly up to the age of 64 years. in other ethnicities and especially in persons aged 70 years and older, chua et al. reported a decrease of central corneal thickness with age; similar were reported by other research groups. this indicates that central corneal thickness may gradually increase in the decades covered by our cohort, while over 70 years a decrease may be observed. in contrast to earlier studies, gender and body mass index were not related to central corneal thickness, nor was any other cardiovascular parameter. interestingly, mean arterial blood pressure showed some association with cct. however, this association was only small and failed to reach the level of significance after bonferroni correction. sensitivity analysis revealed that systolic blood pressure is rather associated with central corneal thickness compared to diastolic blood pressure. based on these negative findings for associations between central corneal thickness and cardiovascular parameters, the underlying hypothesis seems to be true that central corneal thickness itself is independent of cardiovascular changes. our study has several limitations: first, we did not examine axial length and consequently could not control for it as influencing factor, as previously reported by nangia et al.. as refractive power of the cornea is known to be associated with central corneal thickness, we included both corneal curvature and overall manifest refraction into our analysis model: we found an association of central corneal thickness with corneal curvature, but not with refraction after having corrected for corneal curvature. though not expected, this finding is similar to the study report by zhang et al. , who also did not find an association of central corneal thickness with refraction. in addition, due to the fact that our study cohort is a cross section of a working population, this may not strictly reflect the entire caucasian population. nevertheless, we examined a cohort of over 700 subjects with a broad range of age (from 17 to 64 years) and refraction (from 11.75 d to + 7.625 d) which may come close to this criterion. oct scan quality is a critical issue for further data processing. we found that there is a correlation between lower oct scan quality and thinner central corneal thickness measurements; nevertheless, the findings for associated factors did not alter upon including the oct scan quality parameter. in , our study evaluated associations of central corneal thickness readings determined by sd - oct in healthy eyes. as our study was explicitly focused on healthy eyes, this approach may be worthwhile for defining norm values for this specific technology. analysis confirmed intraocular pressure and corneal curvature as ocular factors associated with central corneal thickness. regarding cardiovascular factors, central corneal thickness was not associated with any examined parameters except age. these data suggest that clinical evaluation of central corneal thickness as determined by sd - oct should also be performed with respect to individual corneal curvature and intraocular pressure interpretation merits the knowledge of central corneal thickness.

. optical coherence tomography (oct) allows quantitative analysis of the anterior segment of the eye with a noncontact examination. the aim of this study is to analyze associations of central corneal thickness (cct) as measured by oct with ocular and systemic cardiovascular parameters. methods. a cross - sectional study of 734 persons was performed in a working age population. only healthy eyes were included. a comprehensive ophthalmological examination including refraction, noncontact tonometry, and imaging of the anterior segment by sd - oct was performed. in parallel , a broad range of systemic cardiovascular parameters were measured. associations were analyzed using a generalized estimating equations' model. . cct measurements showed a significant association with corneal curvature and intraocular pressure: a thinner cct was associated with a flatter cornea and with lower intraocular pressure (p < 0.001). age was positively associated with cct (p < 0.001); all other cardiovascular parameters were not associated. . a thinner cornea is associated with a flatter surface and with lower intraocular pressure readings, while there are no independent associations with refraction and systemic cardiovascular parameters. our findings highlight the value of sd - oct cct measurements as a standard tool in anterior segment analysis.

sarcoidosis is a systemic disease characterized by the involvement of multiple tissues and organs with a noncalcified granuloma reaction, which is not yet well understood. although the exact pathogenesis of sarcoidosis is not known, it is currently accepted that, in genetically susceptible individuals, it is caused through alteration of the cellular immune response after exposure to an environmental, occupational, or infectious agent. accumulations of th1 and macrophages with increased production of proinflammatory cytokines induce the inflammatory cascade and consecutive impairment in tissue permeability; increase in cellular influx and local cellular proliferation cause the formation of granulomas. the disease most frequently presents with bilateral hilar lymphadenopathy and infiltrations in the lungs and skin, as well as with eye lesions. acute disease is the most common form and the patient presents with arthralgia and signs of arthritis and/or periarthritis as the first sign of sarcoidosis. chronic sarcoid arthritis usually coexists with pulmonary parenchymal disease or other organ involvement and is rare. spondyloarthropathies are a group of chronic inflammatory diseases primarily characterized by the involvement of axial and peripheral joints. common characteristics of these diseases are inflammatory back pain, enthesitis, uveitis, psoriasis, inflammatory bowel disease, and rf negativity. coexistence of sarcoidosis and sacroiliitis has been published in numerous case reports; however the prevalence of sacroiliitis and spondyloarthritis has not been reported in sarcoidosis. evidence is collected for the association of sarcoid - like granulomatous disease developing after the initiation of anti - tnf- therapy, with disease reversal after discontinuation. therefore, this study is designed to determine the prevalence of sacroiliitis and spondyloarthritis in patients diagnosed with sarcoidosis and to establish any possible correlation with the findings of the disease. forty - two consecutive sarcoidosis patients followed by our rheumatology outpatients clinic between december 2011 and june 2013 were enrolled in this study. the initial diagnosis of sarcoidosis had been made by the rheumatologists with reference to clinical signs and symptoms and histopathological confirmation of noncalcified granulomas in the biopsies of various organs and tissues. conditions that might cause granulomatous disease (such as bacterial and fungal infections) had been ruled out. after given consents were taken, laboratory tests were performed in all patients, including routine biochemistry, acute phase reactants (esr, crp), serum angiotensin converting enzyme (ace), and calcium and hydroxyvitamin d3 levels. detailed questioning in regard to spondyloarthritis (the existence of inflammatory back pain, gluteal area pain, uveitis, enthesitis, peripheral arthritis, dactylitis, psoriasis, inflammatory bowel disease, the presence of a preceding infection, and family history for spondyloarthritis) was performed. the diagnoses of spondyloarthritis were made based on the european spondyloarthritis study group (essg) and the assessment of spondyloarthritis international society (asas) classification criteria. standard pelvic radiographs were obtained in all patients to assess the sacroiliac joints (sijs). each sij was scored on radiographs according to the mny criteria as follows: grade 0: normal; grade 1: suspicious; grade 2: minimal abnormality with small localized erosions, sclerosis without joint spondyloarthritis alteration; grade 3: definite abnormality with erosion, sclerosis, and joint spondyloarthritis widening or narrowing or partial ankylosis; grade 4: total ankylosis of joint. when / if sacroiliitis was determined in radiography, magnetic resonance imaging of sij was performed using the short time inversion recovery (stir) method for the confirmation of the existence of active sacroiliitis. crosstabs were produced to analyse the data, and chi - square analysis or mann - whitney u testing was performed as appropriate. forty - two sarcoidosis patients (ten males, thirty - two females) were included in the study. the mean age was 45.2 years (from 20 to 70 years), and mean duration of the illness was 3.5 years. when the manifestations of sarcoidosis were reviewed, it has been observed that twenty patients (47.6%) had erythema nodosum, three patients (7.1%) had acute, unilateral, anterior, and nongranulomatous uveitis, one patient (2.3%) had myositis, one patient (2.3%) had neurosarcoidosis, and thirty - two patients (76.2%) had arthritis. twenty - eight of the patients with arthritis (87.5%) had ankle involvement, three patients (9.4%) had knee joint involvement, and one patient (3.1%) had wrist involvement. the thoracic computerized tomography scans revealed that twelve patients (28.5%) had stage 1 sarcoidosis, twenty - two patients (52.4%) had stage 2 sarcoidosis, four patients (9.5%) had stage 3 sarcoidosis, and four patients (9.5%) had stage 4 sarcoidosis. laboratory assessments revealed that fifteen patients (35.7%) had elevated serum ace, six patients (14.3%) had elevated serum calcium, two patients (4.7%) had elevated serum d3, twenty - nine patients (69%) had elevated esr, and thirty patients (71.4%) had elevated crp. sacroiliitis was diagnosed in six of the forty - two (14.2%) sarcoidosis patients. all the patients with sacroiliitis were females. while the average age of the cases with sacroiliitis was 55 year, the average duration of disease was 17.8 months. when the first application symptoms of the patients with sacroiliitis were evaluated, it was observed that two patients applied with erythema nodosum, two patients applied with respiration symptoms, and two patients applied with locomotor system complaints. different stages of sarcoidosis were diagnosed in each of the six patients: a stage 1 diagnosis was given in two patients, a stage 2 diagnosis in two patients, and a stage 4 diagnosis in two patients. in radiological staging of sacroiliitis, stage 2 sacroiliitis was found in 6 patients (figure 1). when the patients were evaluated for hla b-27, it was found negative in six patients. comparison between patients with and without sacroiliitis revealed statistically significant differences in terms of some of the parameters (age, inflammatory back pain, enthesitis, and crp levels) (table 1). all the six patients with sacroiliitis were diagnosed with spondyloarthritis according to the criteria of asas (assessment of spondyloarthritis international society) and of essg (european spondyloarthropathy study group). involvement of the joints can be in 2 different ways; while acute, migratory, symmetric arthritis is more common, chronic recurrent erosive form can be especially rare. sacroiliitis is an important finding of the diseases of spondyloarthritis group, but many different causes (e.g., pyogenic, mycobacterial, and fungal infections and malign infiltrations) should also be eliminated. while the prevalence of spondyloarthritis is from 1% to 1.9% in the general population, erb et al. reported prevalence of spondyloarthritis in patients with sarcoidosis as 6%. in our study, the prevalence of sacroiliitis in cases diagnosed with sarcoidosis has been 14.2%. comparisons revealed significant statistical differences in the patient group with sacroiliitis and the group without sacroiliitis, in terms of some of the parameters (age, inflammatory back pain, enthesitis, and crp level). furthermore, all the patients with sacroiliitis were also diagnosed with spondyloarthritis according to the asas and essg criteria. these findings suggest that both diseases may have a common etiopathogenesis and/or sacroiliitis may be an important clinical finding in patients with sarcoidosis. however, the etiopathogenesis of spondyloarthritis is not clear; it starts with an immunological reaction which develops against an undetermined bacterial antigen. among the findings that support this theory are the dna sequences of the many bacteria (i.e., mycobacteria, yersinia, and salmonella) found in patients with spondyloarthritis. the etiology of sarcoidosis is unknown, but the possibility of chronic bacterial infection similar to spondyloarthritis is under investigation since the dna of mycobacteria strains has been shown. in the light of these discoveries, it is considered that infection - related reactive arthritis and sacroiliitis may develop in patients with sarcoidosis with immune susceptibility. the role of the molecule hla - b27 is obvious in the pathogenesis of spondyloarthritis and the elevated molecule hla - dr5 has been found in patients with sarcoidosis. furthermore, it has been suggested that the hla - b8 and hla - dr3 molecules are related to acute sarcoid arthritis and spontaneous remission. the fact that the two diseases develop on different genetic bases (msc class 1 in spondyloarthritis and mhc class 2 in sarcoidosis) suggests a coincidence rather than a common etiopathogenesis. some agents (e.g., propionibacterium acnes) have been detected in tissue samples in both diseases, but their role in pathogenesis has not yet been determined. another study has discovered that spondyloarthritis finding has been determined in 13.6% of hla - b27-positive patients; the study has reported that there is elevated risk of spondyloarthritis with this allele. furthermore, while bilateral sacroiliitis is seen in hla - b27-positive patients, unilateral sacroiliitis was found in hla - b27-negative patients. a further study has recorded three hla - b27-positive patients among fifteen cases determined to have sarcoidosis spondyloarthritis. in our study, all the cases determined to have sacroiliitis were also hla - b27-negative. this suggests that different mechanisms may be involved in the pathogenesis of sacroiliitis developing in patients with sarcoidosis. firstly, the diagnosis of sacroiliitis was performed using x - radiography in the first instance and further confirmed by mri of sij. however, if mri was performed in all patients, higher prevalence of sacroiliitis could have been detected at earlier stages of the disease. but we are conscious that the mri method is too expensive, and the use of x - radiography for the small number of patients was considered cost - effective. secondly, the number of patients was small in our study and it will be wrong to make a generalization. in , we detected a higher prevalence of sacroiliitis in patients with sarcoidosis when compared to the general population. patients with sarcoidosis should be questioned in terms of inflammatory back pain and should be assessed with sijs imaging and/or mri. trials that involve large series of patients are needed for this.

introduction. sarcoidosis is a chronic granulomatous disease, which can involve different organs and systems. coexistence of sarcoidosis and spondyloarthritis has been reported in numerous case reports. purpose. to determine the prevalence of sacroiliitis and spondyloarthritis in patients previously diagnosed with sarcoidosis and to investigate any possible relation with clinical findings. materials and methods. forty - two patients with sarcoidosis were enrolled in the study. any signs and symptoms in regard to spondyloarthritis (i.e., existence of inflammatory back pain, gluteal pain, uveitis, enthesitis, dactylitis, inflammatory bowel disease, and psoriasis) were questioned in detail and biochemical tests were evaluated. sacroiliac joint imaging and lateral heel imaging were performed in all patients. . sacroiliitis was found in 6 of the 42 (14.3%) sarcoidosis patients and all of these patients were female. common features of the disease in these six patients were inflammatory back pain as the major clinical complaint, stage 2 sacroiliitis as revealed by radiological staging, and the negativity of hla b-27 test. these six patients with sacroiliitis were diagnosed with spondyloarthritis according to the criteria of asas and of essg. . we found spondyloarthritis in patients with sarcoidosis at a higher percentage rate than in the general population (11.9%). controlled trials involving large series of patients are required for the confirmation of the data.

stat3 belongs to a family of transcription factors (tfs) comprising stat1, stat2, stat3, stat4, stat5a, stat5b and stat6. like stat5 , stat3 was found to play an important role in cell growth, and its activation has been described in nearly 70% of solid and hematological tumors, giving good reason for a search for specific direct inhibitors, of which there are unfortunately only a few, and none in the clinic to this day. stat3 comprises several distinct functional domains including: an n - terminal domain containing an oligomerization and a coiled - coil domain, a dna binding domain (dbd), a linker domain, a src homology 2 (sh2) domain involved in the interaction of two monomers via phosphotyrosine 705 ing in dimerization and a c - terminal transactivation domain (see fig . 1). stat3 activation occurs following cytokine- or growth factor - receptor activation; it involves phosphorylation within the cytoplasm, dimerization and nuclear transfer (fig . 2). nuclear transfer of stat3 requires nuclear localization signals (nls) which are in the coiled - coil domain (comprising arginines 214 and 215) and in the dimer - dependent dbd (comprising arginines 414 and 417). the nlss interact with importin s, yet which of the five importin s (1, 3, 4, 5 or 7) actually carries stat3 is still debated, the complex interacts with importin and is carried through the nuclear pore complex (npc) (fig . 3). while arginines 214 and 215 appear to be the major importin - binding site, arginines 414 and 417 are thought to be required for stat3 to adopt the proper conformation for importin binding. unphosphorylated forms of stat3 can enter the nucleus and stimulate transcription of a subset of gene targets, apparently via interaction with the tf nfb. however, whether unphosphorylated stat3 interacts on its own with importins for nuclear entry is not entirely clear: tyrosine 705-mutated stat3 can shuttle to the nucleus and phosphotyrosine 705/sh2-independent stat3 dimers were shown to enter the nucleus (but more slowly than phosphorylated stat3 dimers) (fig . 2). interestingly, in the case of stat1, unphosphorylated monomers enter the nucleus through direct interaction with the npc proteins nucleoporins, not with importins and unphosphorylated stat1 dimers bind dna with a 200-fold lower affinity than phosphorylated stat1 dimers; in fact, single - molecule imaging showed that interferon (ifn)--activated stat1 has a reduced mobility and resides longer in the nucleus. in any case , the nucleo - cytoplasmic shuttling of stat3 is a major step of the activation process leading to increased transcriptional activity, suggesting that nuclear transfer of stat3 per se can be a target for inhibition. stat3 monomer showing the n - terminal coiled - coil domain, the dbd (half site), the sh2 domain and the c - terminal domain. the stat3 crystal coordinates were downloaded from the protein data bank (pdb, file : 1bg1) and analyzed using the chimera program. note that the model shown comprises residues 136 to 716; hence, the protein s n - terminal and c - terminal domains (comprising the transactivation domain) are missing; the coordinates corresponding to the cdna strand were missing in the model and had to be reconstructed. figure 2. the transcription factor stat3 is present in a latent inactive non - phosphorylated form in the cytoplasm. activated cytokine receptors activate the kinases jak, which phosphorylate tyrosines located in the cytoplasmic portion of the cytokine receptors creating stat - binding motifs. once bound to these motifs, stat3 becomes in turn phosphorylated by the jaks. the phospho stat3 dimers (colored in pink) enter the nuclei and bind stat3 target genes; note that the dna - bound dimer is drawn differently to indicate the stat3 conformational change suggested by molecular dynamics simulations. stat3 can also be activated by tyrosine kinases of the src family (sfk), the sfks can themselves be activated by g protein - coupled receptors (gpcr) or growth factor receptors, the growth factor receptors (including egf - receptors and vegf - receptors) can also phosphorylate stat3. there are also monomers, and dimers with non - phosphorylated stat3, which can shuttle between the cytoplasm and the nucleus. the tyrosine phosphorylated stat3 dimer interacts with the importins through its two nlss. this complex is carried through the nuclear pore complex (npc) by the ran gdp which is formed in the cytoplasm by hydrolysis of ran - bound gtp by gtpase activating protein (gap). the high level of ran - gtp in the nucleus is the of a high gtp - exchange factor (gef) activity in this compartment. the stat3 dimer interacts with the stat3 dna consensus motif and then is released; once released from dna the dimer is dephosphorylated by a nuclear phosphatase (ptpase). the dephosphorylated stat3 is exported to the cytoplasm in combination with exportins and ran - gtp. constitutive activation of stat3 in tumors can from upstream activated signaling components, including increased cytokines (il-6 and il-10) production, activated receptor (cytokine receptors, vegfr and egfr) and non - receptor tyrosine kinases (including jaks, src and abl). recently, mutated hyperactive forms of stat3 have been detected in tumors, interestingly most of the described mutations are located within stat3 s sh2 (see ref . due to the involvement of tyrosine protein kinases in the stat3 activating pathways, tyrosine kinase inhibitors indirectly inhibit stat3 ing in anti - tumor activity . but stat3 can also be activated when suppressors of the stat signaling pathway are inactivated such as the socs ( suppressor of cytokine signaling), the pias (protein inhibitor of activated stats) and protein tyrosine phosphatases; furthermore, in cells in which proliferation from the inactivation of negative regulators of signaling, such as the inhibitor of the pi3kinase pathway pten, or the inhibitor of the proapoptotic tf p53, mdm2, the inhibition of upstream signaling pathways may have little effect. thus, when upstream stat3 activators are not identified or do not have any known inhibitor, or when stat3 is itself activated it becomes a relevant target for anti - tumor treatments. this reasoning, reinforced by stat3 s previously noted signaling bottleneck situation, has been used by many authors searching for stat3-specific direct inhibitors. the high similarity between stat3 and stat1 is intriguing. both tfs share a 50% amino - acid sequence homology and share similar activating stimuli (types i and ii ifns, cytokines and growth factors); yet, their functions differ: stat1 is mostly involved in immunity, host defense against pathogens and cell death (see refs . 23 and 24), with the notable exception that in certain contexts stat1 exerts proliferative potential, while stat3 is mostly involved in cell growth and proliferation (see ref . their gene targets are mostly distinct : stat3 stimulates the transcription of cell growth - associated genes, including cyclin - d1, survivin, vegf, c - myc, bcl - xl, mcl-1, vascular endothelial growth factor, il-10, transforming growth factor and bcl2 and stat1 stimulates the transcription of pro - inflammatory and anti - proliferative genes, including caspases, inos, mdm2, p21waf / cip1 and p27kip1 ; but there is also an overlap of repertoires . in reality, stat3 and stat1 recognize very similar dna consensus sequences, based on a ttcnnn(t, g)aa motif (see table 1). in this motif, a 3n spacing (n representing any base) is most frequently present for stat1 and stat3, as shown by electrophoretic migration shift assay (emsa). natural binding sites share this consensus with minor variations such as a preference for t at position 5 for stat1, but there is greater diversity outside this consensus (see table 1), suggesting that target recognition in vivo requires additional co - factors. thus, when attempting to inhibit stat3 with the aim to kill tumor cell or block their growth, one must use substances that have no effect on stat1, as has been pointed out and shown in tumor cell lines with sirna - suppressed stat1. indeed, stat1 is required for the antiproliferative effects of interferons and , apoptosis is defective in stat1-null cells and stat1 is the major effector of ifn-, a cytokine with antitumor and cancer immunosurveillance functions. the recognition that activated stat3 is widely present in tumors and that its inhibition is a valuable anti - cancer strategy led to a search for stat3-targeting compounds. among those the dna - alkylating platinum complexes were found to induce the death of tumor cells with activated stat3 and were thought to act directly on stat3; other compounds target the sh2 of stat3, including g quartet oligodeoxynucleotides and small molecules, some of which are highly specific for stat3. the dimerization of stat3 through reciprocal phosphotyrosine 705/sh2 interaction can be impaired by a phosphopeptide with the sequence ppylktk. this phosphopeptide inhibits stat3 activity in tumor cell lines, induces cell death and has a high affinity and specificity for stat3: in particular it had no effect on the sh2-containing tyrosine kinase p56lck, and little effect on stat1 as determined by emsa. despite their efficacy and specificity, the inefficient cell penetration of phosphopeptides led to a search for smaller equivalents using computational docking studies exploring the phosphotyrosine 705/sh2 interaction area (see fig . these studies yielded several small molecules with high affinity and high specificity for stat3, including sta-21, stattic, s31 - 201 and recently bp-1 - 102, a compound with improved bioavailability and anti - cancer properties . the mechanism of action of these compounds is thought to be based on their interaction with stat3 sh2 ( fig . 4a and b), an area where the other monomer s phosphotyrosine 705 docks, thereby impairing the formation of the active dimer, as shown in (see fig . 44 ; the phosphotyrosine peptide is represented together with the inhibitor s31 - 201, both form h - bonds with the same residues, including lysine 591, serine 611, serine 613 and arginine 609). stat3 sh2-targeting compounds are efficacious stat3 inhibitors, they inhibit stat3 dna binding, reduce stat3-dependent cell proliferation and expression of biological targets. yet, experimental demonstration of their interaction with stat3 is missing, the actual data are based on computational studies, not on actual interaction measurements, as noted earlier. this implies that a compound claimed to interact with sh2 might actually interfere with the binding of stat3 to the receptor s phosphotyrosine site leading to biological effects similar to those of jak - inhibitors. the compound might also interact with the dbd with just the same effect in the cell (reduced dna binding, cell death). besides, compounds usually undergo modifications in a biological environment (cells or body fluids), these modifications can occur before the compounds reach their target. for instance, stattic s inhibitory activity of stat3 increases with time and is temperature- and dithiothreitol - sensitive, this suggests that it interacts with a cysteine, such as cysteine 687 which is next to the phosphopeptide motif of stat3 and faces the phosphopeptide - binding area of sh2. while this point does not invalidate stattic s specificity for stat3, it suggests possible limitations for its in vivo utilization. despite considerable progress and clear anti - cancer efficacy the areas in squares are the sh2 region where small molecules interact (b and c) and the sh2/dbd region where g quartets interact (d and e). (b) close up view of the region of the sh2 of stat3 that interacts with the small molecule inhibitors: the key aminoacids involved in interaction are labeled; the inhibitors interact particularly in the groove located between arginine 595 and lysine 591. (c) the same area as in (b) is shown but stat3 and stat1 are superimposed; this shows the overall great similarity between these two regions, yet some differences are present, accounting for the capacity of the inhibitors to discriminate between stat3 and stat1 (arrows); stat1 crystal coordinates used in (c) were from pdb file 1bf5. (d) modeled interaction of inhibitor s31 - 201 with stat3 sh2 domain, note the important role of lysine 591, serine 611 and arginine 609 in the interaction. (e) same as (d), with added phosphotyrosine - peptide, showing its overlap with the stat3 sh2/small molecule inhibitors binding area. (f) detailed view of the region of stat3 interacting with g quartets, this region includes the phosphotyrosine 705-interacting region of sh2 and a neighboring region including part of the dbd, including glutamic acid 638, glutamine 644, aspartic acid 647, glutamine 643 and asparagine 646. (g) same region as in f is shown with the superimposition of stat1, the major differences are indicated by arrows. g quartets are g - rich oligodeoxynucleotides that form potassium - dependent four - stranded intramolecular g - quartet structures. they inhibit stat3 at micromolar concentrations and induce the death of several tumor cell lines, including head and neck cancer lines, they also arrest the development of breast tumor, prostate tumor or non - small cell lung cancer xenografts in nude mice. these reagents are interesting in that their specificity for stat3, demonstrated by emsa showing high affinity binding to stat3 and much lower affinity for stat1, is somewhat unexpected. a computational study of the interaction of the g quartet with stat3 and stat1 showed the following sh2 domain amino - acids to be involved in the binding: glutamic acid 638, glutamine 644, asparagine 647, glutamine 643 and asparagine 646 (fig . we compared stat3 and stat1 surfaces in the area where the g quartet interacts and found that the surfaces differ significantly ( fig . however, the use of g quartets is problematic due to their large size and potassium dependence, which limit cellular delivery . decoy oligodeoxynucleotides ( dodns) are short stretches of double stranded dna containing a tf s consensus binding sequence. once in the cells, dodns inhibit the corresponding tf as shown for nfb in animal models (cardiovascular disease and ischemia - reperfusion injury) and in cancer cells. this property has been used for stat3: in cells in which stat3 is constitutively activated, stat3 dodns (table 1) induce cell death. while some studies suggested that dodns must enter nuclei to exert their inhibitory action, our studies of the subcellular location of stat3 in dodn - treated cells have shown that the dodns prevent nuclear translocation of stat3. the dodns are thought to interact with stat3s dbd within the cytoplasm and to prevent interaction with importins which carry the stat3 dimer within the nucleus. inhibition of importin binding by the dodn is thought to from the masking of the dbd - located nls (arginines 414 and 417) (see figs . 1 and 5a) indirectly confirming its involvement in nuclear entry. in fact, competition between dna binding and importin binding was observed in vitro with stat1 and within cells with stat3. thus, despite the uncertainty regarding the two stat3 nlss relative requirement for nuclear transfer, it seems that the dbd - located nls plays an important role for nuclear translocation since its masking is sufficient to impair it, confirming that it may be a good target for inhibition, as suggested by others. (a) detail of the area of stat3 interacting with the dna consensus sequence. arginines 414 and 417 play a key role in the interaction of stat3 with importins and nuclear translocation, arginine 423 is involved in the interaction of dna with stat3. (b) the same area as in (a) is shown, the corresponding area of stat1 has been superimposed, showing the high level of homology between stat3 and stat1, except for glutamic acid 421 (in italic) of stat1 whose interaction with dna is very different from that of arginine 423 of stat3 (see ref . 65), and other differences indicated by arrows. as discussed above, the opposed cellular functions of stat1 and stat3, in spite of their similarity, is puzzling. stat1 and stat3 can form heterodimers, whose function is not elucidated to this day, they recognize very similar dna motifs (table), and they have targets in common and regulate one another. indeed, in stat3-deficient cells, stat3-activators such as il-6 trigger an ifn--like response through stat1 activation; and in stat1-deficient cells, ifn- and ifn- trigger stat3-dependent proliferative responses. thus the stat1/stat3 cross - regulation suggests that stat3 inhibitors may work best in stat1-expressing cells. it is therefore essential to inhibit stat3 without inhibiting stat1 to keep cell death processes operational. in this regard , it should be noted that stat3-dodns inhibit stat3 but also inhibit activated stat1, thereby abolishing ifn--induced cell death and reducing the dodns anti - stat3 efficacy. computer analysis of the dodn s interaction with stat1 and stat3 dbds showed that within the highly similar dna sequences, there were subtle differences including a t at positions 7 and 5, a dc at position 0, a da at position + 5 (see table 1, dodn # 22), which had been previously noted by computer analysis and dna - binding studies. this allowed designing a dodn that could inhibit stat3 without inhibiting stat1, demonstrating that at this level specific interaction can be achieved. therapeutic use of the dodns not only requires specific target recognition, but also stability in biological fluids. modifications, including phosphothioate end modification and hairpin structures considerably increased intracellular stability and efficacy. a recent improvement consisting of a cyclic stat3 dodns comprising a hairpin at both ends was designed and found to reduce xenograft tumors following intravenous administration. furthermore, a stat3 dodn has been found to have few side effects when administered to primates, suggesting interesting therapeutic perspectives. another possibility is to design smaller molecules mimicking the dodn s interaction with stat3, similarly to what was achieved with the sh2 domain. the stat3 area that interacts with the dna target and the dodn (fig . this area comprises the dbd - located nls including arginines 414 and 417 that are located closely to arginine 423, involved in interaction with the dodn ; these three arginines surround an opening in the surface in which small molecules could interact ( fig . furthermore, superimposition of stat3 and stat1 showed that in spite of their striking overall similarity, these areas comprise in the same location glutamic acid 421 in stat1 and arginine 423 in stat3 ( fig . 5b), a difference that might be exploited for the design of a stat3 small molecule inhibitor with stat3/stat1 discriminating capacity; studies are underway in the laboratory to try and design such a reagent. the dimerization of stat3 through reciprocal phosphotyrosine 705/sh2 interaction can be impaired by a phosphopeptide with the sequence ppylktk. this phosphopeptide inhibits stat3 activity in tumor cell lines, induces cell death and has a high affinity and specificity for stat3: in particular it had no effect on the sh2-containing tyrosine kinase p56lck, and little effect on stat1 as determined by emsa. despite their efficacy and specificity, the inefficient cell penetration of phosphopeptides led to a search for smaller equivalents using computational docking studies exploring the phosphotyrosine 705/sh2 interaction area (see fig . these studies yielded several small molecules with high affinity and high specificity for stat3, including sta-21, stattic, s31 - 201 and recently bp-1 - 102, a compound with improved bioavailability and anti - cancer properties . the mechanism of action of these compounds is thought to be based on their interaction with stat3 sh2 ( fig . 4a and b), an area where the other monomer s phosphotyrosine 705 docks, thereby impairing the formation of the active dimer, as shown in (see fig . 44 ; the phosphotyrosine peptide is represented together with the inhibitor s31 - 201, both form h - bonds with the same residues, including lysine 591, serine 611, serine 613 and arginine 609). stat3 sh2-targeting compounds are efficacious stat3 inhibitors, they inhibit stat3 dna binding, reduce stat3-dependent cell proliferation and expression of biological targets. yet , experimental demonstration of their interaction with stat3 is missing, the actual data are based on computational studies, not on actual interaction measurements, as noted earlier. this implies that a compound claimed to interact with sh2 might actually interfere with the binding of stat3 to the receptor s phosphotyrosine site leading to biological effects similar to those of jak - inhibitors. the compound might also interact with the dbd with just the same effect in the cell (reduced dna binding, cell death). besides, compounds usually undergo modifications in a biological environment (cells or body fluids), these modifications can occur before the compounds reach their target. for instance, stattic s inhibitory activity of stat3 increases with time and is temperature- and dithiothreitol - sensitive, this suggests that it interacts with a cysteine, such as cysteine 687 which is next to the phosphopeptide motif of stat3 and faces the phosphopeptide - binding area of sh2. while this point does not invalidate stattic s specificity for stat3, it suggests possible limitations for its in vivo utilization. despite considerable progress and clear anti - cancer efficacy the areas in squares are the sh2 region where small molecules interact (b and c) and the sh2/dbd region where g quartets interact (d and e). (b) close up view of the region of the sh2 of stat3 that interacts with the small molecule inhibitors: the key aminoacids involved in interaction are labeled; the inhibitors interact particularly in the groove located between arginine 595 and lysine 591. (c) the same area as in (b) is shown but stat3 and stat1 are superimposed; this shows the overall great similarity between these two regions, yet some differences are present, accounting for the capacity of the inhibitors to discriminate between stat3 and stat1 (arrows); stat1 crystal coordinates used in (c) were from pdb file 1bf5. (d) modeled interaction of inhibitor s31 - 201 with stat3 sh2 domain, note the important role of lysine 591, serine 611 and arginine 609 in the interaction. (e) same as (d), with added phosphotyrosine - peptide, showing its overlap with the stat3 sh2/small molecule inhibitors binding area. (f) detailed view of the region of stat3 interacting with g quartets, this region includes the phosphotyrosine 705-interacting region of sh2 and a neighboring region including part of the dbd, including glutamic acid 638, glutamine 644, aspartic acid 647, glutamine 643 and asparagine 646. (g) same region as in f is shown with the superimposition of stat1, the major differences are indicated by arrows. [panels ( d and e) are reprinted from ref. g quartets are g - rich oligodeoxynucleotides that form potassium - dependent four - stranded intramolecular g - quartet structures. they inhibit stat3 at micromolar concentrations and induce the death of several tumor cell lines, including head and neck cancer lines, they also arrest the development of breast tumor, prostate tumor or non - small cell lung cancer xenografts in nude mice. these reagents are interesting in that their specificity for stat3, demonstrated by emsa showing high affinity binding to stat3 and much lower affinity for stat1, is somewhat unexpected. a computational study of the interaction of the g quartet with stat3 and stat1 showed the following sh2 domain amino - acids to be involved in the binding: glutamic acid 638, glutamine 644, asparagine 647, glutamine 643 and asparagine 646 (fig . we compared stat3 and stat1 surfaces in the area where the g quartet interacts and found that the surfaces differ significantly ( fig . . however, the use of g quartets is problematic due to their large size and potassium dependence, which limit cellular delivery . decoy oligodeoxynucleotides ( dodns) are short stretches of double stranded dna containing a tf s consensus binding sequence. once in the cells, dodns inhibit the corresponding tf as shown for nfb in animal models (cardiovascular disease and ischemia - reperfusion injury) and in cancer cells. this property has been used for stat3: in cells in which stat3 is constitutively activated, stat3 dodns (table 1) induce cell death. while some studies suggested that dodns must enter nuclei to exert their inhibitory action, our studies of the subcellular location of stat3 in dodn - treated cells have shown that the dodns prevent nuclear translocation of stat3. the dodns are thought to interact with stat3s dbd within the cytoplasm and to prevent interaction with importins which carry the stat3 dimer within the nucleus. inhibition of importin binding by the dodn is thought to from the masking of the dbd - located nls (arginines 414 and 417) (see figs . 1 and 5a) indirectly confirming its involvement in nuclear entry. in fact, competition between dna binding and importin binding was observed in vitro with stat1 and within cells with stat3. thus, despite the uncertainty regarding the two stat3 nlss relative requirement for nuclear transfer, it seems that the dbd - located nls plays an important role for nuclear translocation since its masking is sufficient to impair it, confirming that it may be a good target for inhibition, as suggested by others. figure 5. detailed view of the area of stat3 interacting with dna and importins. (a) detail of the area of stat3 interacting with the dna consensus sequence. arginines 414 and 417 play a key role in the interaction of stat3 with importins and nuclear translocation, arginine 423 is involved in the interaction of dna with stat3. (b) the same area as in (a) is shown, the corresponding area of stat1 has been superimposed, showing the high level of homology between stat3 and stat1, except for glutamic acid 421 (in italic) of stat1 whose interaction with dna is very different from that of arginine 423 of stat3 (see ref . 65), and other differences indicated by arrows. as discussed above, the opposed cellular functions of stat1 and stat3, in spite of their similarity, is puzzling. stat1 and stat3 can form heterodimers, whose function is not elucidated to this day, they recognize very similar dna motifs (table), and they have targets in common and regulate one another. indeed, in stat3-deficient cells, stat3-activators such as il-6 trigger an ifn--like response through stat1 activation; and in stat1-deficient cells, ifn- and ifn- trigger stat3-dependent proliferative responses. thus the stat1/stat3 cross - regulation suggests that stat3 inhibitors may work best in stat1-expressing cells. it is therefore essential to inhibit stat3 without inhibiting stat1 to keep cell death processes operational. in this regard , it should be noted that stat3-dodns inhibit stat3 but also inhibit activated stat1, thereby abolishing ifn--induced cell death and reducing the dodns anti - stat3 efficacy. computer analysis of the dodn s interaction with stat1 and stat3 dbds showed that within the highly similar dna sequences, there were subtle differences including a t at positions 7 and 5, a dc at position 0, a da at position + 5 (see table 1, dodn # 22), which had been previously noted by computer analysis and dna - binding studies. this allowed designing a dodn that could inhibit stat3 without inhibiting stat1, demonstrating that at this level specific interaction can be achieved. therapeutic use of the dodns not only requires specific target recognition, but also stability in biological fluids. modifications, including phosphothioate end modification and hairpin structures considerably increased intracellular stability and efficacy. a recent improvement consisting of a cyclic stat3 dodns comprising a hairpin at both ends was designed and found to reduce xenograft tumors following intravenous administration. furthermore, a stat3 dodn has been found to have few side effects when administered to primates, suggesting interesting therapeutic perspectives. another possibility is to design smaller molecules mimicking the dodn s interaction with stat3, similarly to what was achieved with the sh2 domain. the stat3 area that interacts with the dna target and the dodn (fig . this area comprises the dbd - located nls including arginines 414 and 417 that are located closely to arginine 423, involved in interaction with the dodn ; these three arginines surround an opening in the surface in which small molecules could interact ( fig . furthermore, superimposition of stat3 and stat1 showed that in spite of their striking overall similarity, these areas comprise in the same location glutamic acid 421 in stat1 and arginine 423 in stat3 ( fig . 5b), a difference that might be exploited for the design of a stat3 small molecule inhibitor with stat3/stat1 discriminating capacity; studies are underway in the laboratory to try and design such a reagent. because stat3 is constitutively activated in nearly 70% of tumors, it provides a valuable target for anti - cancer therapy. the recent findings that tumors harbor activating mutations of stat3 underlines the need for additional stat3 inhibitors, and inhibitors that target other regions of stat3 than its sh2, especially as most identified mutations are within this region. in addition, studies on cancer cell lines indicate that even when stat3 direct targeting is insufficient to induce cell death; it can diminish the resistance to other anti - cancer compounds such as doxorubicin or egfr inhibitors. finally, stat3 direct inhibitors are also probably less toxic than many others because inhibition of stat3 in mature cells has minimal effects.

the signal transducer and activator of transcription stat3 is a transcription factor which plays a key role in normal cell growth and is constitutively activated in about 70% of solid and hematological cancers. activated stat3 is phosphorylated on tyrosine and forms a dimer through phosphotyrosine / src homology 2 (sh2) domain interaction. the dimer enters the nucleus via interaction with importins and binds target genes. inhibition of stat3 in the death of tumor cells, this indicates that it is a valuable target for anticancer strategies; a view that is corroborated by recent findings of activating mutations within the gene. yet , there is still only a small number of stat3 direct inhibitors; in addition, the high similarity of stat3 with stat1, another stat family member mostly oriented toward apoptosis, cell death and defense against pathogens, requires that stat3-inhibitors have no effect on stat1. specific stat3 direct inhibitors consist of sh2 ligands, including g quartet oligodeoxynucleotides (odn) and small molecules, they induce cell death in tumor cells in which stat3 is activated. stat3 can also be inhibited by decoy odns (dodn), which bind stat3 and induce cell death. a specific stat3 dodn which does not interfere with stat1-mediated interferon - induced cell death has been designed pointing to the stat3 dbd as a target for specific inhibition. comprehensive analysis of this region is in progress in the laboratory to design dbd - targeting stat3 inhibitors with stat3/stat1 discriminating ability.

the online version of this article (doi:10.1007/s00270 - 015 - 1060 - 0) contains supplementary material, which is available to authorized users. since the treatment of patients with intermittent claudication (ic) is primarily aimed at improving their walking ability and health - related quality of life (hrql), it is essential that these endpoints are measured when evaluating treatment. walking ability is a part of a patient s functional status (fs), and is frequently assessed using a treadmill test. however, treadmill tests do not correlate well with real - life walking distances, and are not an adequate reflection of the patient s perceived walking impairment. therefore, fs can better be assessed using patient - reported outcome measures (proms), such as the walking impairment questionnaire (wiq). hrql can be defined as the aspects of quality of life that relate specifically to a person s health. the vascular quality of life questionnaire (vascuqol) is an example of a disease - specific hrql prom for patients with peripheral artery disease (pad). the importance of proms to evaluate treatment outcomes has been recognized by the vascular community, and they are used as endpoints in many clinical trials. however, the interpretation of changes in prom scores may be difficult when it is unknown how much change is actually considered relevant by patients. a statistically significant mean change in score after treatment in a sample does nt necessarily imply that an individual patient experiences a clinically meaningful change in his or her hrql or fs. the minimally important difference (mid) represents the smallest change in score in the construct to be measured which patients perceive as important. the mid can aid to better appreciate trial and individual treatment , can be calculated for all available proms and is relevant in all patient populations. a statistically significant change in mean score for the patient sample from 25 to 33 was found, but it is unknown if this change is relevant to an individual patient. if, however, the mid for that prom was known to be + 10 points on the scale, it would be immediately clear that an individual patient would have to improve from a baseline score of 25 to at least 35 for the improvement to be clinically relevant. the current study aims to introduce the concept of the mid for the vascuqol and the wiq in patients with ic. the institutional review board (irb) of the academic medical center decided that this study met the criteria for exemption from irb approval. we used the patient sample of a prospective pilot study to determine the feasibility of proms as indicators of quality of care for patients with pad. patients were enrolled from july 2012 until october 2012 in nine hospitals in the netherlands. patients were eligible if they presented at the vascular surgery outpatient clinic with complaints of ic due to pad and if they had not visited the outpatient clinic for symptomatic pad in the previous year. other inclusion criteria were sufficient knowledge of the dutch language, an independent living situation, absence of psychiatric disorders, and the ability to communicate with the researchers. as recommended by national guidelines, first line treatment was supervised exercise therapy (set) in most patients. depending on physician and patient preferences percutaneous transluminal angioplasty (pta) was sometimes used as primary treatment, and a few patients were treated with surgical revascularization. therefore in some patients treatment consisted of optimal medical therapy (omt) (antiplatelet drug and a statin, advice to walk, and change lifestyle). in each centre, a local investigator was responsible for the execution of the study and data collection at baseline and at 34 months follow - up. patient characteristics and questionnaires were sent to an independent trusted party (itp) for further data linking and processing. data on smoking history, diabetes, pulmonary and cardiac diseases, renal function, previous vascular interventions (pta or surgery), ankle brachial index (abi), and number of affected legs were recorded at first visit in a pre - specified database. at follow - up, it was also recorded if a patient had received omt or set. no data on age and gender were recorded in this database, since these were retrieved when the itp linked treatment codes (conservative, pta or surgery) in the dutch insurance billing system to patients in each hospital s patient administration. however, because unblinding was impossible due to privacy reasons, it was impossible for the itp to retrieve data on age and gender if no treatment code was listed. if no treatment code was listed, we used the data on treatment modality recorded by the local investigator at follow - up. when necessary, patients were contacted by telephone to remind them and help fill in the prom. only patients with available data on age and gender and a resting abi < 0.9 were analysed in the present study to ensure that the patient sample in the mid analysis had a proven diagnosis of ic due to pad. baseline characteristics and prom scores of patients included and excluded from the mid analysis were compared. the vascuqol is a disease - specific hrql prom, developed for patients with ic and critical limb ischemia. it consists of five subscales (pain, symptoms, activities, emotional, and social) with 25 items in total. each item is rated on a 7-point rating scale, with 1 representing the worst and 7 the best score. a total score, also ranging from 1 to 7, is calculated by dividing the sum of all items by 25. the wiq is a prom to rate walking impairment and consists of a speed, distance, and stairclimbing subscale with 14 items in total. for example, patients are asked to assign a degree of difficulty with which they can walk 100 meters, with answers ranging from no problems to subscale scores are calculated by adding the weighted scores, and dividing this by the maximum score so that each score ranges from 0 to 1, with lower scores indicating a higher level of impairment. in addition to the proms, at follow - up patients filled in the following anchor - question: has your condition changed in the past three months? with the following response options: (a) improved, (b) unchanged, and (c) deteriorated. imputation of the vascuqol subscales took place if at least 50 % of the subscale was filled in. missing values were imputed with the mean value of all the filled - in questions if this condition was satisfied, and under the assumption of completely missing at random. when items were missing for the wiq, we calculated a best- and worst - case scenario. we hereby took into account the questions the patients did fill in, and assumed that patients could never score higher on a harder task and never lower on an easier task. if the best- and worst - case scenario scores were no more than 0.25 points apart, we used the mean of these two values as the total wiq score. for the mid analysis anchor - based approaches determine the mid by comparing proms to other measures or phenomena that have clinical relevance. suggested that the mid should be based on an anchor that has a correlation 0.3 with the prom. therefore, pearson correlation coefficients were calculated between the change in prom scores and the anchor - question. the upper and lower limit of the 95 % confidence interval (ci) of the mean change of the group who indicated on the anchor - question that their situation had not changed after treatment represent the mid for improvement and deterioration, respectively. differences in baseline characteristics and prom scores were determined with a student s t - test for continuous variables, and with a chi - square or fisher s exact test where appropriate for categorical variables. all analyses were performed using sas enterprise guide version 5.1; sas institute, cary, nc, usa. the institutional review board (irb) of the academic medical center decided that this study met the criteria for exemption from irb approval. we used the patient sample of a prospective pilot study to determine the feasibility of proms as indicators of quality of care for patients with pad. patients were enrolled from july 2012 until october 2012 in nine hospitals in the netherlands. patients were eligible if they presented at the vascular surgery outpatient clinic with complaints of ic due to pad and if they had not visited the outpatient clinic for symptomatic pad in the previous year. other inclusion criteria were sufficient knowledge of the dutch language, an independent living situation, absence of psychiatric disorders, and the ability to communicate with the researchers. as recommended by national guidelines, first line treatment was supervised exercise therapy (set) in most patients. depending on physician and patient preferences percutaneous transluminal angioplasty (pta) was sometimes used as primary treatment, and a few patients were treated with surgical revascularization. therefore in some patients treatment consisted of optimal medical therapy (omt) (antiplatelet drug and a statin, advice to walk, and change lifestyle). in each centre, a local investigator was responsible for the execution of the study and data collection at baseline and at 34 months follow - up. patient characteristics and questionnaires were sent to an independent trusted party (itp) for further data linking and processing. data on smoking history, diabetes, pulmonary and cardiac diseases, renal function, previous vascular interventions (pta or surgery), ankle brachial index (abi), and number of affected legs were recorded at first visit in a pre - specified database. at follow - up, it was also recorded if a patient had received omt or set. no data on age and gender were recorded in this database, since these were retrieved when the itp linked treatment codes (conservative, pta or surgery) in the dutch insurance billing system to patients in each hospital s patient administration. however, because unblinding was impossible due to privacy reasons, it was impossible for the itp to retrieve data on age and gender if no treatment code was listed. if no treatment code was listed, we used the data on treatment modality recorded by the local investigator at follow - up. when necessary, patients were contacted by telephone to remind them and help fill in the prom. only patients with available data on age and gender and a resting abi < 0.9 were analysed in the present study to ensure that the patient sample in the mid analysis had a proven diagnosis of ic due to pad. baseline characteristics and prom scores of patients included and excluded from the mid analysis were compared. the vascuqol is a disease - specific hrql prom, developed for patients with ic and critical limb ischemia. it consists of five subscales (pain, symptoms, activities, emotional, and social) with 25 items in total. each item is rated on a 7-point rating scale, with 1 representing the worst and 7 the best score. a total score, also ranging from 1 to 7, is calculated by dividing the sum of all items by 25. the wiq is a prom to rate walking impairment and consists of a speed, distance, and stairclimbing subscale with 14 items in total. for example, patients are asked to assign a degree of difficulty with which they can walk 100 meters, with answers ranging from no problems to subscale scores are calculated by adding the weighted scores, and dividing this by the maximum score so that each score ranges from 0 to 1, with lower scores indicating a higher level of impairment. in addition to the proms, at follow - up patients filled in the following anchor - question: has your condition changed in the past three months? with the following response options: (a) improved, (b) unchanged, and (c) deteriorated. imputation of the vascuqol subscales took place if at least 50 % of the subscale was filled in. missing values were imputed with the mean value of all the filled - in questions if this condition was satisfied, and under the assumption of completely missing at random. when items were missing for the wiq, we calculated a best- and worst - case scenario. we hereby took into account the questions the patients did fill in, and assumed that patients could never score higher on a harder task and never lower on an easier task. if the best- and worst - case scenario scores were no more than 0.25 points apart, we used the mean of these two values as the total wiq score. anchor - based approaches determine the mid by comparing proms to other measures or phenomena that have clinical relevance. suggested that the mid should be based on an anchor that has a correlation 0.3 with the prom. therefore, pearson correlation coefficients were calculated between the change in prom scores and the anchor - question. the upper and lower limit of the 95 % confidence interval (ci) of the mean change of the group who indicated on the anchor - question that their situation had not changed after treatment represent the mid for improvement and deterioration, respectively. differences in baseline characteristics and prom scores were determined with a student s t - test for continuous variables, and with a chi - square or fisher s exact test where appropriate for categorical variables. all analyses were performed using sas enterprise guide version 5.1; sas institute, cary, nc, usa. the vascuqol was sufficiently completed twice by 223 patients, the wiq by 184 patients. after exclusion of patients with unknown age, gender, and resting abi > 0.9 there were 163 patients who were suitable for the mid analysis of the vascuqol, and 134 for the wiq. baseline characteristics of both the patients included and excluded from the analysis are shown in table 1. all baseline characteristics and scores on proms were comparable for included and excluded patients, except for the abi. missing items for both proms are presented in table s2 (online only).table 1baseline characteristicsbaseline characteristicstotal population (n = 294)vascuqol (n = 163)wiq (n = 134)excluded patients vascuqol (n = 131)excluded patients wiq(n = 160)age (years, sd)66.5 (10.4) (n = 271)66.6 (10.3)65.9 (10.1)66.5 (10.6) (n = 108)67.1 (10.7)(n = 137)nsmale / female gender163/108 (n = 271)95/6876/5868/40 (n = 108)87/50 (n = 137)nscurrent smoker134 (45.9 %) (n = 292)72 (44.2 %) (n = 162)54 (40.3 %) (n = 129)60 (48.4 %) (n = 124)78 (48.8 %) (n = 152)nshistory of smoking271 (93.1 %) (n = 291)141 (93.3 %) (n = 150)123 (91.7 %) (n = 129)97 (69.4 %) (n = 107)115 (71.8 %) (n = 128)nsdiabetes74 (25.2 %) (n = 284)43 (26.3 %) (n = 161)33 (24.6 %) 31 (23.6 %) (n = 123)41 (25.6 %) (n = 150)nscardiac disease74 (26 %) (n = 285)43 (26.3 %) (n = 162)35 (26.1 %) 31 (25.2 %) (n = 123)39 (25.8 %) (n = 151)nslung disease30 (10.2 %) (n = 84)17 (10.4 %) (n = 162)13 (9.7 %) (n = 134)13 (9.9 %) (n = 122)17 (10.6 %) (n = 150)nsegfrns<6055 (19.3 %) 30 (18.4 %) 25 (18.6 %) 25 (19.1 %) 30 (18.6 %) > 60162 (55.1 %) 114 (69.9 %) 98 (73.1 %) 48 (36.6 %) 64 (40 %) unknown7719115866previous vascular intervention97 (34.2 %) (n = 284)51 (31.3 %) (n = 160)40 (29.9 %) (n = 133)46 (35.1 %) (n = 124)57 (35.6 %) (n = 151)abi at rest p < 0.0001<0.540 (13.6 %) 30 (18.4 %) 28 (20.9 %) 10 (7.6 %) 12 (7.5 %) 0.50.75112 (38.1 %) 83 (50.9 %) 71 (53 %) 29 (22.1 %) 41 (25.6 %) 0.750.963 (21.4 %) 50 (30.7 %) 35 (26.1 %) 13 (9.9 %) 28 (17.5 %) 0.91.118 (6.1 %) 0018 (13.7 %) 18 (11.3 %) 1.11.34 (1.4 %) 004 (3.1 %) 4 (2.5 %) unknown57005757affected legsnsunilateral125 (42.5 %) 78 (47.9 %) 68 (50.7 %) 47 (35.9 %) 57 (35.6 %) bilateral130 (44.2 %) 84 (51.5 %) 65 (48.5 %) 46 (35.1 %) 65 (40.6 %) unknown39113838received treatmentconservative / optimal medical treatment11347406673set14193734868endovascular1710978surgical treatment2313121011questionnairesfollow - up time (days, sd)123126baseline vascuqol score4.25 (1.2)4.26 (n = 119) nsbaseline wiq score0.39 (0.1)0.42 (n = 120) nsdata displayed as number (percentage) or mean (standard deviation) score was calculated in the number of patients that did sufficiently fill in the baseline questionnaire, but were excluded for not sufficiently filling follow - up questionnaire ns not significant baseline characteristics data displayed as number (percentage) or mean (standard deviation) score was calculated in the number of patients that did sufficiently fill in the baseline questionnaire, but were excluded for not sufficiently filling follow - up questionnaire table 2 shows that the mean improvement in vascuqol summary score was 0.83. the correlation between the anchor - question and the vascuqol was 0.47, thus meeting the criteria of revicki.table 2distribution of scores and mid vascuqolvascuqol (n = 163)baselinefollow upmean change scorecorrelation with anchor - question4.25 (1.20)5.08 (1.28)0.830.47anchor - question n mean change on vascuqol95 % ciimproved971.23(1.011.46)unchanged430.55(0.230.87)deteriorated230.36(0.74 to 0.01)mid vascuqolimprovement0.87deterioration0.23 distribution of scores and mid vascuqol the mids calculated by the anchor - based approach were 0.23 and 0.87, for deterioration and improvement, respectively (table 2). this means that patients with an increase of 0.87 compared to their baseline score have improved in a clinically relevant way. for deterioration while one might expect a negative mid value for deterioration, the mid value found here indicates that an increase in vascuqol summary score of less than 0.23 points is actually experienced as deterioration by patients. figure 1 shows the proportion of patients with a clinically relevant improvement or deterioration of their hrql on the vascuqol. this figure shows that 44 % of the patients achieved a clinically meaningful improvement at follow - up. 1proportion of patients that show a clinically relevant improvement and deterioration per prom proportion of patients that show a clinically relevant improvement and deterioration per prom distribution of scores and details on mid calculation for the wiq are presented in table 3. the correlation between the anchor - question and the wiq was 0.41, also meeting the criteria of revicki.table 3distribution of scores and mid wiqwiq (n = 134)baselinefollow upmean change scorecorrelation with anchor - question0.39 (0.24)0.55 (0.28)0.160.41anchor - questionnmean change on wiq95 % ciimproved790.25(0.190.3)unchanged370.04(0.03 to 0.11)deteriorated180.01(0.05 to 0.06)mid wiqimprovement0.11deterioration0.03 distribution of scores and mid wiq the mid values found were 0.03 and 0.11 for deterioration and improvement, respectively. figure 1 shows the proportion of patients that reached a clinically relevant improvement or deterioration on the wiq. this figure shows that 57 % of the patients achieved a clinically meaningful improvement at follow - up. the correlation between the anchor - question and the vascuqol was 0.47, thus meeting the criteria of revicki.table 2distribution of scores and mid vascuqolvascuqol (n = 163)baselinefollow upmean change scorecorrelation with anchor - question4.25 (1.20)5.08 (1.28)0.830.47anchor - question n mean change on vascuqol95 % ciimproved971.23(1.011.46)unchanged430.55(0.230.87)deteriorated230.36(0.74 to 0.01)mid vascuqolimprovement0.87deterioration0.23 distribution of scores and mid vascuqol the mids calculated by the anchor - based approach were 0.23 and 0.87, for deterioration and improvement, respectively (table 2). this means that patients with an increase of 0.87 compared to their baseline score have improved in a clinically relevant way. for deterioration while one might expect a negative mid value for deterioration, the mid value found here indicates that an increase in vascuqol summary score of less than 0.23 points is actually experienced as deterioration by patients. figure 1 shows the proportion of patients with a clinically relevant improvement or deterioration of their hrql on the vascuqol. this figure shows that 44 % of the patients achieved a clinically meaningful improvement at follow - up. 1proportion of patients that show a clinically relevant improvement and deterioration per prom proportion of patients that show a clinically relevant improvement and deterioration per prom distribution of scores and details on mid calculation for the wiq are presented in table 3. the correlation between the anchor - question and the wiq was 0.41, also meeting the criteria of revicki.table 3distribution of scores and mid wiqwiq (n = 134)baselinefollow upmean change scorecorrelation with anchor - question0.39 (0.24)0.55 (0.28)0.160.41anchor - questionnmean change on wiq95 % ciimproved790.25(0.190.3)unchanged370.04(0.03 to 0.11)deteriorated180.01(0.05 to 0.06)mid wiqimprovement0.11deterioration0.03 distribution of scores and mid wiq the mid values found were 0.03 and 0.11 for deterioration and improvement, respectively. figure 1 shows the proportion of patients that reached a clinically relevant improvement or deterioration on the wiq. this figure shows that 57 % of the patients achieved a clinically meaningful improvement at follow - up. these can be assessed using proms, which are common endpoints in trials, have the potential to support clinical management of patients and can help assess provider performance. when interpreting changes in prom scores there are some important points to consider. while physicians have a distinct idea which amount of change in clinical measures such as blood pressure is relevant, interpretation of prom scores is less apparent. this is hampered even more by the fact that many proms have different rating scales (e.g., 01, 17, 1100), making score changes incomparable. furthermore, it is important to realize that in larger sample sizes the standard deviations of scores become smaller, ing in earlier significant findings than in a small sample sizes. they can be applied independent of sample size, and are thus useful in both individual care and research. in individual care , caregivers may decide to alter treatment strategy when after a certain period a patient does nt meet a relevant improvement. in research , a big advantage of applying mid values is that it helps display the proportion of patients in a sample that reaches a clinically relevant improvement. concurrently, it can display how many patients show a clinically relevant deterioration despite treatment, as shown in fig. 1. this would have been missed when only comparing the mean baseline score of the sample with the mean score after treatment, since this would have probably ed in a positive mean change score, falsely indicating improvement for all patients in the sample. while it was beyond the scope of this paper, in future studies that compare treatment modalities it may be insightful to compare the proportion of patients that reach a clinically relevant improvement and deterioration per treatment group. it may be attributed to a learning effect, i.e., patients who do not improve (unchanged group) may still learn to fill in a prom more accurately by repetition, ing in a higher follow - up score, and thus a positive mid for deterioration. furthermore, the vascuqol is a disease - specific prom, in contrast to the anchor - question. therefore, the mean prom score may increase, while the anchor - question is rated as unchanged. generally, calculation methods are divided into anchor - based approaches and distribution - based approaches. anchor - based approaches determine the mid by comparing proms to other measures or phenomena that have clinical relevance. this can for example be an anchor - question, as we have shown in this study. distribution - based approaches are based on statistical characteristics of the prom scores in a patient sample. while studies have shown that values found in anchor - based and distribution - based approaches are often comparable, in calculations based on distribution - based approaches it is still not taken into account which amount of change is considered relevant by patients. first, the proportion of patients that did not sufficiently complete the proms twice was substantial. this is a well - known problem and not exclusive to our study, but it should be considered when applying proms, since it limits their overall use. second, to ensure that the study population was representative for all ic patients, we intentionally excluded patients of unknown age, gender, and/or abi, which may have induced bias. yet, the included and excluded patients did not differ in terms of baseline characteristics and prom scores, and despite excluding many patients an acceptable sample was left for the mid analysis. finally, we do not know how many patients refused to participate in the pilot study, and how this may have influenced mid values. we have calculated the mid values for two frequently used proms for patients with ic. as demonstrated in this study, the mid is a helpful tool to interpret the clinical relevance of changes in prom scores, which may be used in research and individual care. below is the link to the electronic supplementary material. supplementary material 1 (doc 46 kb) supplementary material 1 (doc 46 kb) wilma jonkers, ellen rouwet, anco vahl, jim reekers, mark koelemay have no conflict of interest.

purposethe minimally important difference (mid) represents the smallest change in score on patient - reported outcome measures that is relevant to patients. the aim of this study was to introduce the mid for the vascular quality of life questionnaire (vascuqol) and the walking impairment questionnaire (wiq) for patients with intermittent claudication (ic).methodsin this multicenter study, we recruited 294 patients with ic between july and october 2012. patients completed the vascuqol, with scores ranging from 1 to 7 (worst to best), and the wiq, with scores ranging from 0 to 1 (worst to best) at first visit and after 4 months follow - up. in addition, patients answered an anchor - question rating their health status compared to baseline, as being improved, unchanged, or deteriorated. the mid for improvement and deterioration was calculated by an anchor - based approach, and determined with the upper and lower limits of the 95 % confidence interval of the mean change of the group who had not changed according to the anchor-question.for the mid analyses of the vascuqol and wiq, 163 and 134 patients were included, respectively. the mid values for the vascuqol (mean baseline score 4.25) were 0.87 for improvement and 0.23 for deterioration. for the wiq (mean baseline score 0.39), we found mid values of 0.11 and 0.03 for improvement and deterioration, respectively.in this study, we calculated the mid for the vascuqol and the wiq. applying these mid facilitates better interpretation of treatment outcomes and can help to set treatment goals for individual care.electronic supplementary materialthe online version of this article (doi:10.1007/s00270 - 015 - 1060 - 0) contains supplementary material, which is available to authorized users.

participants (51 obese men and women) aged 3575 years who were diagnosed with type 2 diabetes were enrolled. the study was approved by the institutional review board, and participants provided written informed consent. participants had not been previously treated with tzds and were not using drugs that affect metabolism or body weight (e.g., sibutramine). participants were randomly assigned to one of three treatment groups for the 16-week study: 1 ) pioglitazone plus standard dietary advice from the american diabetes association (ada) (pio+ada); 2 ) pioglitazone plus a portion - controlled diet (pio+pc); and 3 ) an active control group, metformin plus ada advice (met+ada). briefly, all participants were prescribed a diet that was 500 kcal / day less than their energy requirements. the pio+pc group drank one glucerna (290 kcal) for breakfast and one for lunch, with a planned evening meal. change from baseline to week 16 (week 16 minus week 0) on the following variables was quantified. four hours after a 371-kcal breakfast, energy intake was measured objectively at lunch in the laboratory using methods that produce repeatable / reliable energy intake measurements. serum ghrelin levels were measured before and 2 h after the start of lunch to quantify ghrelin response to a meal (postmeal minus premeal). ratings of hunger, desire to eat, fullness, and prospective food consumption were measured with visual analog scales (vass) before and after lunch. the eating inventory quantified dietary restraint (the intent to restrict energy intake) and disinhibition (the tendency to overeat). the food - craving inventory (fci) measured general cravings (total score) and cravings for the following specific types of foods: sweets, high fats, carbohydrates / starches, fruits / vegetables, and fast - food fats. analyses were conducted with = 0.05 using sas version 9.0 (cary, nc). mixed models tested if change on the outcome variables differed by group (baseline values were covariates). regression methods were used to test for mediators of differential body weight change between the pio+pc and pio+ada groups. the following possible mediators were tested: ghrelin, energy intake, dietary restraint, and disinhibition. pearson correlation coefficients were calculated to determine the amount of variance in body weight (and energy intake) change that was accounted for by change in restraint and disinhibition. participants were randomly assigned to one of three treatment groups for the 16-week study: 1 ) pioglitazone plus standard dietary advice from the american diabetes association (ada) (pio+ada); 2 ) pioglitazone plus a portion - controlled diet (pio+pc); and 3 ) an active control group, metformin plus ada advice (met+ada). briefly, all participants were prescribed a diet that was 500 kcal / day less than their energy requirements. the pio+pc group drank one glucerna (290 kcal) for breakfast and one for lunch, with a planned evening meal. change from baseline to week 16 (week 16 minus week 0) on the following variables was quantified. four hours after a 371-kcal breakfast, energy intake was measured objectively at lunch in the laboratory using methods that produce repeatable / reliable energy intake measurements. serum ghrelin levels were measured before and 2 h after the start of lunch to quantify ghrelin response to a meal (postmeal minus premeal). ratings of hunger, desire to eat, fullness, and prospective food consumption were measured with visual analog scales (vass) before and after lunch. the eating inventory quantified dietary restraint (the intent to restrict energy intake) and disinhibition (the tendency to overeat). the food - craving inventory (fci) measured general cravings (total score) and cravings for the following specific types of foods: sweets, high fats, carbohydrates / starches, fruits / vegetables, and fast - food fats. analyses were conducted with = 0.05 using sas version 9.0 (cary, nc). mixed models tested if change on the outcome variables differed by group (baseline values were covariates). regression methods were used to test for mediators of differential body weight change between the pio+pc and pio+ada groups. the following possible mediators were tested: ghrelin, energy intake, dietary restraint, and disinhibition. pearson correlation coefficients were calculated to determine the amount of variance in body weight (and energy intake) change that was accounted for by change in restraint and disinhibition. forty - eight of 51 participants completed the trial (2 subjects dropped out from the pio+ada and 1 from the met+ada group). as previously reported, pio+ada gained (means sd) 2.15 1.09 kg, met+ada lost 3.21 0.7 kg, and pio+pc lost 2.59 1.25 kg. the pio+ada group had a smaller decrease in visceral fat compared with pio+pc group but a larger increase in deep subcutaneous fat compared with the met+ada group. energy intake increased significantly in the pio+ada (p < 0.001) but not the met+ada (p = 0.30) or pio+pc (p = 0.28) groups. increased energy intake in the pio+ada group was significantly larger than the met+ada and pio+pc groups (p < 0.05). the difference in least squares (ls) means se between the pio+ada and met+ada and pio+ada and pio+pc groups was 155 73 and 157 70 kcal, respectively. baseline (week 0) participant characteristics and change on outcome variables from week 0 to 16 data are means se. ls means se, which are adjusted for baseline values, depict change on the outcome variables from week 0 to 16 and are included below the week 0 values. * lettered superscripts that differ indicate that those groups' change scores differed significantly from each other (p < 0.05). the suppression of ghrelin after a meal at week 0 and week 16 is included in the table. the pio+ada group had a significantly larger meal - induced suppression of ghrelin at week 16 compared with week 0, which was significantly larger than the nonsignificant changes in the met+ada and pio+pc groups. change in appetite ratings did not differ significantly among the groups (data not shown) (p > 0.25). the pio+pc group had a significant increase in dietary restraint and a decrease in disinhibition and hunger. change in these end points differed significantly between the pio+pc and pio+ada groups (p < 0.01). the difference in ls means se between the pio+pc and pio+ada groups on restraint and disinhibition was 4.8 1.4 and 2.5 0.9, respectively. the met+ada group experienced a significant decrease in general cravings and cravings for high - fat foods. change in restraint and disinhibition mediated differential weight change between the pio+pc and pio+ada groups. change in restraint and disinhibition were negatively (r = 0.53, p < 0.001) and positively (r = 0.39, p < 0.01) associated with change in body weight, accounting for 28.4 and 15.2% of body weight change variance, respectively. change in restraint and disinhibition were negatively (r = 0.44, p < 0.01) and positively (r = 0.31, p < 0.05) associated with change in energy intake, accounting for 19 and 9.3% of energy intake change variance, respectively. this is the first study to demonstrate that pairing pioglitazone treatment with a portion - controlled diet (pio+pc) attenuates pioglitazone - associated increases in energy intake. suppression of ghrelin in response to a meal increased only in the group who gained weight (pio+ada), indicating that ghrelin suppression is dependent on body weight change and not pioglitazone treatment. the indicate that pioglitazone - associated weight gain is secondary to increased energy intake. larger increases in restraint and decreases in disinhibition were observed in the pio+pc group, with restraint accounting for 28.4% of the variance in body weight change. strengths of the study include the objective measurement of energy intake in a controlled study design. limitations include measuring energy intake during only one meal before and after short - term treatment. further research is warranted to examine the long - term effect of pioglitazone treatment on energy and macronutrient intake.

objectiveto measure ghrelin and energy intake in the laboratory after pioglitazone treatment.research design and methodsthis was a parallel, three - arm study with 51 obese diabetic subjects randomized to either 1 ) pioglitazone plus a portion - controlled diet (pio+pc), 2 ) pioglitazone plus american diabetes association (ada) dietary advice (pio+ada), or 3 ) metformin plus ada advice (met+ada). energy intake and the suppressive response of a meal on ghrelin were measured at weeks 0 and 16. mixed models tested if changes from week 0 to 16 differed by group.the pio+ada group had a significantly larger increase (p < 0.05) in energy intake (207 53 kcal) compared with the pio+pc (50 46 kcal) and met+ada (52 49 kcal) groups. change in restraint and disinhibition (variables associated with eating behavior) mediated weight change. ghrelin suppression increased in the pio+ada group, which gained weight.a portion - controlled diet attenuated the increase in energy intake after pioglitazone. ghrelin responded to weight change not pioglitazone exposure.

the buccal fat pad also called as buccal fat pad of bichat or suctorial pad is relatively large and prominent in neonates, infants and young children. it has great relevance to this area giving fullness to face, aids in cushioning and sucking in neonates and children. it serves to line the masticatory space, separating the muscles of mastication from the zygomatic arch, ramus of the mandible and other muscles. the buccal fat pad consists of a central body and four extensions: buccal, pterygoid, superficial and deep temporal. the buccal extension is encapsulated by a parotidomasseteric fascia and enters the cheek below the parotid duct. the term traumatic pseudolipoma was proposed by brooke and macgregor in their case report involving adipose tissue in a swelling of the buccal mucosa distinguishing it from other benign fatty tumors of the oral cavity. two etiologic factors and pathogenetic links between soft - tissue trauma and adipose tissues growth had been discussed in the literature. the first possibility is the herniation of the buccal fat pad with subsequent epithelialization, termed this usually from direct impact, has a relatively short natural history of presentation and commonly occurs in young children. the second possibility suggests lipoma formation is due to deposition and differentiation of adipocytes, mediated by cytokine release, secondary to trauma and hematoma formation. the average time between soft - tissue trauma and lipoma formation is almost 3 years and occurs more commonly in the fourth to sixth decade. all the reported cases of traumatic pseudolipoma except one 12-year - old boy, were in infants or young children, with an age range from 5 months to 5 years. in every case, two contributory factors were proposed: the buccal fat pad is relatively prominent in infants and young children, who frequently investigate foreign objects by placing them in their mouths. further sucking action in fat pad being pulled out. a 3 year old female patient was referred from a pediatrician to the department of pedodontic and preventive dentistry with a chief complaint of extra- and intra - oral swelling and pain on the right side of face. child's father gave history of appearance of extra - oral swelling 5 days back with pain, discomfort, drooling of saliva from mouth and inability to eat. the intra - oral swelling was first noticed approximately 1 month ago as a small reddish pink growth which increased gradually the patient reported to the department after 3 days with complain of intense pain and increased extra - oral swelling and compressed intra - oral swelling. gross clinical intra - oral examination of the lesion revealed the presence of a reddish pink, ovoid, smooth, pedunculated, non - ulcerated, freely mobile, tender swelling of approximately 3.0 2.0 1.5 cm size, located between the maxillary and mandibular primary second molar regions. the lesion was found to be soft, fluctuant in consistency and freely mobile on palpation. the child was having the habit of placing toothbrush for more than 1h daily while brushing teeth. she used to bite on it while keeping it in the right buccal vestibule from last 6 months. intra - oral swelling noticed 1 month ago compressed intra - oral swelling seen after 3 days differential diagnosis of the mass was given as a pyogenic granuloma, traumatic fibroma, haemangioma, infected lipoma, inflammatory pseudotumour, infected benign minor salivary gland tumor, foreign body granuloma and traumatic neuroma. the child was prescribed antibiotics, analgesic and antiseptic mouthwash for 3 days to reduce infection, inflammation, pain as well as post - operative complications while healing. the intra - oral growth was seen pale yellow in color and shrunken after 3 days. as the patient was highly uncooperative (frankl rating i), after complete hematological investigations, surgical excision of the mass was done under local anesthesia with soft - tissue diode laser (doctor smile wiser laser) at 2w continuous pulse. tissue mass was excised in - toto from the base of buccal mucosa and sent for histopathological examination to the department of oral pathology. an attempt was made to express saliva from the parotid duct to identify the location of the parotid papilla and confirm that it is not severed or damaged. patient was recalled after 1 week and 3 months for follow - up of wound healing. manoeuvre to express saliva from stensen's duct was repeated to make sure that it was not damaged. excised tissue mass from buccal mucosa buccal mucosa after excision of lesion and identification of parotid papilla macroscopic examination revealed one soft - tissue of a reddish pink, ovoid, firm in consistency, smooth surfaced, pedunculated, non - ulcerated mass of approximately 3.0 2.0 1.5 cm size. histological examination of the tissue revealed the presence of adipose tissues with non - ulcerated overlying epithelium. presence of marked edema and diffuse proliferation of capillaries along with formation of granulation tissue, composed of moderately dense polymorphonuclear neutrophillic and lymphoplasmocytic infiltrate. the connective tissue stroma consists of dense collagen bundles and lobules of mature adipocytes with no cellular atypia. compressed blood vessels engorged with red blood cells were also evident in the connective tissue stroma. the overall histo - pathological features were confirmatory of traumatic pseudolipoma (traumatic herniation of buccal fat pad). it is closely associated with the muscles of mastication, the parotid duct and the facial nerve. its importance in masticatory function is best illustrated in infants where it acts as sucking pad. it contributes to the bulging of the infant's cheeks and usually persists in adults, the body and the buccal extensions of which are largely responsible for cheek contour in adults. literature suggests that traumatic intra - oral herniation of the buccal fat pad is a rare phenomenon. most of the reported cases were observed in infants and young children ranging from age of 5 months to 5 years. relatively increased incidence of traumatic herniation of buccal fat pad in infants and young children is attributed to habit of investigating or frequently placing foreign objects like pencil, toothbrush, chopstick in their mouths. the most characteristic aspect of such a lesion is that the mucosal injury or perforation is very small compared to the size of the extruded mass. as to the anatomic location of this lesion , matarasso suggested that a defect or weakness in the parotidomasseteric fascia of the region contribute to the occurrence. sucking action of an infant might encourage the herniation of fat pad through the wound into the mouth, and may also pose the risk of respiratory embarrassment. majority of cases documented involved a foreign object in the mouth, which subsequently caused the penetrating injury through buccal mucosa and buccinator muscle. in this case , traumatic herniation of buccal fat pad can be thought to be caused by primarily because of improper brushing technique. gradual increase in the size of the mass can be because of prolonged placement and sucking of brush for more than 1 h daily. cases in which there is early evaluation and the protruded mass is small, the tissues are able to be repositioned immediately back to its place, followed by suturing of the mucosal laceration. the approximate period between the injury and the first visit should be less than 4 h as tissues start necrotizing after this duration. if the mass is too large to be replaced in the limited laceration or infected because of prolonged exposure to oral cavity, it is recommended to excise the mass at the base with no recurrence. in this case , it was decided to excise the mass from its base under local anesthesia using diode laser in a continuous wave in a contact mode. diode laser cuts soft - tissues and reduces bacterial counts in at the wound surface. there is evidence that this wavelength may cause a reduction in tissue inflammation and a reduced need for local anesthetic during surgical procedures. thermal necrosis zones of less than 1 mm can be achieved, which provides adequate surgical precision and hemostasis for many soft - tissue procedures. the laser when used in a non - contact mode; coagulates soft tissues or provides hemostasis over the surgical area. it can be concluded from above discussion that the penetrating injuries of soft tissues of oral cavity or lesions occurring from it should be considered potentially serious. proper evaluation of such lesions or injuries is important before coming to final diagnosis and treatment modality. while excising such a traumatic pseudolipoma and closing the wound care

the buccal fat pad is relatively large and prominent in neonates, infants and young children. the main function of this fat pad is considered as a cushioning tissue and sucking pad. a minor tear of buccal mucosa and buccinator muscle can in herniation of large volume of fat into oral cavity that is termed as pseudolipoma. the young children tend to be very playful while brushing their teeth. improper brushing technique ed in severe trauma to the buccal fat, including soft - tissue between buccinator and retromolar area. this article presents a case - report of a female child who developed traumatic pseudolipoma after faulty tooth brushing for long duration and its management along with its detail review of literature.

the online version of this article (doi:10.1007/s13300 - 014 - 0093 - 8) contains supplementary material, which is available to authorized users. lifestyle modification and oral antidiabetic drugs are first line treatment measures in patients with type 2 diabetes mellitus (t2 dm). over time, progressive beta cell dysfunction in deteriorating glycemic control requiring insulin therapy. however, insulin is often associated with weight gain and increased risk of hypoglycemia. together with the need for (multiple) injections, insulin therapy frequently meets reluctance in both patients and physicians, and initiation of therapy is often delayed. several trials showed that glucagon - like peptide-1 (glp-1) receptor agonists as monotherapy or in combination with one or more oral antidiabetic agent have beneficial metabolic effects leading to improved glycemic control with a low risk of hypoglycemia, and weight loss. they are now recommended as one of several two - drug combination therapies, if metformin monotherapy does not achieve / maintain the defined glycated hemoglobin (hba1c) target over 3 months. several reports demonstrated favorable effects of a combination therapy of a glp-1 receptor agonist with insulin. however, only few studies included the use of liraglutide , and follow - up time was short. despite these limited data, liraglutide is widely used (off - label) as add - on therapy in patients on various insulin regimens and oral antidiabetic drugs. in 2013, liraglutide was officially approved for use in combination with basal insulin in europe (and the us). the aim of this observational study was to retrospectively assess the efficacy and safety of liraglutide as add - on therapy to insulin in a daily clinical practice setting for a duration up to 2428 months. eligible participants were adults (1880 years) with t2 dm, bmi 28 kg / m and hba1c values of 6.510% treated with metformin another oral antidiabetic drug and insulin. exclusion criteria were pregnancy and intolerance for liraglutide. the authors reviewed the charts of all patients with t2 dm with liraglutide being added - on to insulin from october 2009 until december 2011 at their outpatient clinic in a tertiary referral hospital. weight, body mass index (bmi), hba1c and insulin dose (units / day, units / kg / day) were assessed 58 months prior to liraglutide, at baseline, after 3, 6, 1216 and 2428 months. starting dose of liraglutide was 0.6 mg once daily, with an increase to 1.2 mg after 2 weeks. during the treatment period, oral drugs and insulin dosage were individually reduced by the attending physician according to current glucose control. as the outpatient clinic is part of a teaching hospital , all physicians followed the same treatment strategies according to the guidelines of the swiss society of endocrinology and diabetes. hypoglycemic events were assessed by patient interview and patients blood glucose meter readings were imported into the diabass pro software, mediaspects gmbh, konstanz, germany. severe hypoglycemia was defined as an event requiring assistance of another person to actively administer carbohydrate, glucagon or other resuscitative actions. all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the helsinki declaration of 1975, as revised in 2000 and 2008. informed consent was obtained from all patients for being included in the study. for statistical analysis linear mixed models with random intercept were used to examine changes in endpoints over time from baseline, after 3, 6, 1216 and 2428 months. such regression models do not require complete data (unlike in analysis of variance), so all available data was analyzed. all analysis was performed in the r programming language (version 3.0.1, r core team 2013 center for statistics, copenhagen, denmark). the package lme4 was used to estimate the mixed models, while the multcomp was used to compute the p values and confidence intervals. a total of 36 patients was on this combined therapy regimen. after the baseline visit, four patients preferred to be followed up by their family physician and were therefore not included in this follow - up. three patients stopped liraglutide due to exanthema at the injection site, nausea and abdominal pain, respectively. among the remaining 29 patients, median age was 59 years , disease duration 11 years (iqr 513), hba1c 7.7% (iqr 7.08.6), weight 99.8 kg (iqr 81110) and bmi 33.5 kg / m (iqr 29.437.6). at baseline, 17 were on basal insulin, 2 on prandial insulin and 10 on multiple insulin injections. in addition, 24 patients were on metformin, 12 on sulfonylureas, and 6 on other oral antidiabetic agents. median hba1c decreased significantly from 7.7% (iqr 7.08.6) at baseline to 6.8% (iqr 6.57.7, p = 0.001) at 3 months and 6.9%, (iqr 6.37.6, p = 0.0001) at 6 months, but re - increased at 1216 months (median 7.2%, iqr 6.77.9, p = 0.32) and 2428 months (median 7.5%, iqr 7.18.2, prior to intervention was lower ( median 7.3%, iqr 7.17.9) than at baseline. weight and bmi decreased significantly from 99.8 kg and 33.5 kg / m (iqr 81110 and 29.437.6) respectively at baseline to 97.7 kg and 31.9 kg / m (iqr 81.2108.2 and 29.436), (p = 0.023 and 0.027) at 3 months. however weight and bmi increased again after 6 months (median 97.5 kg and 31.5 kg / m, iqr 84.5.5111.5, p = 0.16 and 29.435.1, p = 0.15), at 1216 months (100.5 kg and 34.1 kg / m, iqr 100.5109.5, p = 0.17 and 30.736.7, p = 0.16) and at 2428 months (106.4 kg and 36.4 kg / m, iqr 96.9118, p = 1.0 and 30.938.4, however, during follow - up, three patients ( 10.3%) were able to completely stop insulin therapy, and two patients (6.9%) could simplify their multiple daily insulin injection regimens to basal insulin at bedtime only. furthermore, sulfonylureas were stopped in 5 of 12 patients (41.7%) and all other antidiabetic drugs beside metformin were stopped (e.g. glitazones, glinides). overall, 5 patients (15.6%) out of 32 followed - up patients discontinued liraglutide due to side effects. three patients stopped liraglutide due to exanthema at the injection site, nausea or abdominal pain at the beginning and two patients discontinued liraglutide due to nausea / vomiting and pain at the injection site after 3.5 and 12 months.fig. 1course of glycated hemoglobin (hba1c), insulin (total units and units per kg body weight), weight and body mass index (bmi) prior (t-1), at baseline (t0) and during follow - up after 3 (t1), 6 (t2), 1216 (t3) and 2428 months (t4) course of glycated hemoglobin (hba1c), insulin (total units and units per kg body weight), weight and body mass index (bmi) prior (t-1), at baseline (t0) and during follow - up after 3 (t1), 6 (t2), 1216 (t3) and 2428 months (t4) this retrospective study shows that liraglutide as add - on therapy to insulin therapy decreases hba1c and weight during the first 6 months with a fading effect thereafter. the most frequently reported side effects were nausea / vomiting and pain / exanthema at the injection site, leading to discontinuation of liraglutide in 15.6% of the patients. the short - term findings on hba1c and weight reduction are in accordance with earlier randomized and observational studies over 12 weeks on liraglutide added to insulin, which found a reduction of hba1c by 1.41.9% and body weight by 5.15.6 kg, respectively. a recent report showed a decrease in hba1c levels of 0.77% and 4.5 kg weight reduction over 26 weeks. a most recent meta - analysis of 15 studies on glp-1 agonists in combination with basal insulin with a maximum observation time of 36 weeks (mean 24.8 weeks) yielded an hba1c reduction of 0.44% and weight loss of 3.22 kg. the longest follow - up was in a retrospective study until 38 weeks, where body weight also re - increased in 20 patients on liraglutide or exenatide in combination with insulin, similar to the patient population in the current study. with longer follow - up time are only available in the reverse situation of basal insulin added on to metformin and liraglutide in a prospective study, showing sustained hba1c and weight reduction even after 52 weeks. several reasons may account for the unsustained effect of liraglutide on body weight and hba1c beyond 6 months in the patients presented here. firstly, reduction of insulin dose or termination of oral antidiabetic drugs may have led to the re - increase in hba1c. secondly, no antidiabetic treatment has been shown to prevent deterioration of -cell function, which drives the natural history of the disease. thirdly, the authors often observe that patient adherence, especially regarding dietary restrictions, fades after a few months of starting liraglutide, which may be explained by the disappearing gastrointestinal side effects. subjects willing to participate in a prospective study are supposedly more motivated and adherent to improve their glycemic control in general. finally, subjects early achieving adequate glycemic control within a few weeks or months usually return to their referring physicians, whereas the more challenging patients remained in the tertiary clinic and were eligible for this study. the clinical setting reflects a real world situation and shows the difficulties which diabetologists face in their daily work. in contrast to other studies with glp-1 receptor agonists and insulin, there was no statistically significant reduction in daily insulin dose in the present study. however, a remarkable proportion of the patients either completely ceased insulin or at least were able to omit prandial insulin injections. moreover, in many of the patients, oral antidiabetic drugs (beside metformin) could be stopped, which is especially beneficial for patients with sulfonylureas bearing an increased risk of hypoglycemia. in accordance with earlier reports the dropout rate of 15.6% due to side effects was comparable to other reports. limitations of this study are the small patient number and potentially heterogeneous patient characteristics, including patients with long - standing diabetes as well as the retrospective study design. as this was an observational study, there was no control group. the fact that hba1c was increasing before baseline may point to a regression towards the mean. the setting reflects the real life situation in an outpatient clinic of a tertiary referral hospital covering around 500,000 inhabitants. during the observational period, combination therapy of liraglutide and insulin was not yet approved by swissmedic (swiss agency for the authorisation and supervision of therapeutic products), which explains the small number of patients. however, a major strength of this study is the long observational period of up to 2428 months, as compared to most earlier studies with a follow - up time of usually 24 weeks with a few lasting up to 38 weeks. in the meantime, add - on therapy of liraglutide to basal insulin was approved due to its unequivocal positive effect. the combination of liraglutide with multiple insulin injections, however, is still awaiting approval. adding liraglutide to pre - existing insulin therapy in t2 dm reduces hba1c and body weight significantly during the initial 6 months of treatment, but there may be a non - sustainable effect during long - term treatment. lisa sze, ina krull, michael brndle declare they have no conflicts of interest. all procedures followed were in accordance with the ethical standards of the committee on human experimentation (institutional and national) and with the helsinki declaration of 1975, as revised in 2000 and 2008. the study was approved by the local ethics committee and informed consent was obtained from all patients for being included in the study. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

introductionin most patients with type 2 diabetes mellitus (t2 dm) and progressive beta - cell insufficiency, insulin therapy is required to achieve sufficient glycemic control. however, insulin therapy may lead to weight gain and increasing risk of hypoglycemia. glucagon - like peptide-1 receptor agonists are being used as add - on therapy to insulin with favorable metabolic effects. nonetheless, to date only few studies exist reporting on the combination of liraglutide and insulin with a short follow - up period. the aim of this study was to evaluate the efficacy and safety of liraglutide as add - on to insulin in patients with t2 dm over a time period of up to 2428 months.methodsdata of patients with t2 dm, treated with insulin and liraglutide at an outpatient clinic in a tertiary referral hospital from october 2009 until december 2011 were retrospectively examined (n = 36). glycated hemoglobin (hba1c), weight, total daily insulin dose and side effects were assessed 58 months prior to liraglutide, at baseline and at follow - up visits after 3, 6, 1216 and 2428 months.median hba1c decreased significantly from 7.7% at baseline to 6.8% (iqr 6.57.7, p = 0.001) at 3 months and 6.9% (iqr 6.37.6, p = 0.0001) at 6 months, but re - increased thereafter (at 2428 months, median 7.5%, iqr 7.18.2, p = 1.0). median weight decreased significantly from 99.8 kg (iqr 81110) at baseline to 97.7 kg (iqr 81.2108.2, p = 0.027) at 3 months, but rose again thereafter. insulin dosage did not change significantly over time. no severe hypoglycemia or major side effects occurred.in this observational study, adding liraglutide to insulin in daily clinical practice reduced hba1c significantly within 6 months, but there may be a non - sustainable effect during long - term treatment.electronic supplementary materialthe online version of this article (doi:10.1007/s13300 - 014 - 0093 - 8) contains supplementary material, which is available to authorized users.

this study provides class iii evidence that serum gdf-15 accurately distinguishes patients with mitochondrial diseases from those without them. this case - control study provides class iii evidence that gdf-15 is a better diagnostic indicator of mitochondrial diseases when compared with fgf-21 (diagnostic sensitivity, 77.8% vs 68.5% ; diagnostic odds ratio , 73.5 vs 45.7 ; area under the receiver operating characteristic curve, 0.941 vs 0.911). briefly, the adult cohort consisted of 66 controls, 20 nonmitochondrial neuromuscular disease controls, and 54 consecutive patients with mitochondrial disease. mitochondrial disease patients were recruited between november 2011 and october 2012 from the adult mitochondrial disease clinic at royal north shore hospital, sydney, australia, if they met the walker criteria for mitochondrial disease and had a confirmed genetic diagnosis or changes on muscle biopsy indicative of mitochondrial myopathy. participants ranged in age from 20 to 84 years and 57.9% of participants were female. of the 54 patients with mitochondrial disease, 27 had a genetic diagnosis and 27 had muscle biopsy changes indicative of a mitochondrial myopathy (9 patients with a genetic diagnosis also had a muscle biopsy showing changes consistent with mitochondrial myopathy). we refer to the standards for reporting of diagnostic accuracy diagram published previously for this cohort for inclusion and exclusion details. when possible, the newcastle mitochondrial disease scale for adults in addition, patients were routinely assessed for proximal muscle weakness according to medical research council (mrc) criteria. written informed consent was obtained from all participants at the time of sample collection and ethical approval was granted by the northern sydney local health district human research ethics committee. serum creatine kinase, lactate, and pyruvate levels (along with other biochemical parameters) measured previously were used here for analysis with serum gdf-15 levels. gdf-15 levels were measured in archived serum samples by quantitative sandwich elisa (biovendor, laboratorni medicina a.s ., brno, czech republic), according to the manufacturer's instructions. absorbance readings at 570 nm were subtracted from absorbance readings at 450 nm before normalizing to the mean absorbance of blank wells. triplicate measurements for each sample were averaged and the concentration determined from a 4-parameter logistic standard curve (www.myassays.com). elisa measurements were performed in triplicate for each assay and repeated in at least duplicate assays. the scientist who performed the assays (r.l.d .) statistical analyses were performed, as previously described, using spss version 20 (ibm corporation, armonk, ny) and clinical research calculators on the vassar university statistics web interface (http://vassarstats.net/). differences between groups were considered statistically significant when 2-tailed p values were less than 0.05. all p values below 0.0001 the reference cutoff concentration for gdf-15 was set at the 95th percentile of control group serum concentrations, as for fgf-21. we used mann - whitney u and kruskal - wallis nonparametric testing to determine statistical differences between groups. we calculated the diagnostic sensitivity and the corresponding 95% confidence intervals. an unadjusted diagnostic or was determined for comparison with fgf-21. receiver operating characteristic (roc) curves were plotted using sensitivity vs 100 specificity on a continuous scale and the area under the curve (auc) was used to determine the relative diagnostic capacity. biomarker levels were correlated with other biochemical measurements and clinical assessment tools using nonparametric spearman correlation testing. linear regression analysis was used to determine if statistically correlated parameters could predict gdf-15 concentration. multivariate linear regression analysis was performed to determine factors capable of predicting disease while controlling for confounders that may influence serum gdf-15 concentration. this case - control study provides class iii evidence that gdf-15 is a better diagnostic indicator of mitochondrial diseases when compared with fgf-21 (diagnostic sensitivity, 77.8% vs 68.5% ; diagnostic odds ratio , 73.5 vs 45.7 ; area under the receiver operating characteristic curve, 0.941 vs 0.911). briefly, the adult cohort consisted of 66 controls, 20 nonmitochondrial neuromuscular disease controls, and 54 consecutive patients with mitochondrial disease. mitochondrial disease patients were recruited between november 2011 and october 2012 from the adult mitochondrial disease clinic at royal north shore hospital, sydney, australia, if they met the walker criteria for mitochondrial disease and had a confirmed genetic diagnosis or changes on muscle biopsy indicative of mitochondrial myopathy. participants ranged in age from 20 to 84 years and 57.9% of participants were female. of the 54 patients with mitochondrial disease, 27 had a genetic diagnosis and 27 had muscle biopsy changes indicative of a mitochondrial myopathy (9 patients with a genetic diagnosis also had a muscle biopsy showing changes consistent with mitochondrial myopathy). we refer to the standards for reporting of diagnostic accuracy diagram published previously for this cohort for inclusion and exclusion details. when possible, the newcastle mitochondrial disease scale for adults (nmdas) clinical rating scale was performed on patients with mitochondrial disease. in addition, patients were routinely assessed for proximal muscle weakness according to medical research council (mrc) criteria. written informed consent was obtained from all participants at the time of sample collection and ethical approval was granted by the northern sydney local health district human research ethics committee. serum creatine kinase, lactate, and pyruvate levels (along with other biochemical parameters) measured previously were used here for analysis with serum gdf-15 levels. gdf-15 levels were measured in archived serum samples by quantitative sandwich elisa (biovendor, laboratorni medicina a.s ., brno, czech republic), according to the manufacturer's instructions. absorbance readings at 570 nm were subtracted from absorbance readings at 450 nm before normalizing to the mean absorbance of blank wells. triplicate measurements for each sample were averaged and the concentration determined from a 4-parameter logistic standard curve (www.myassays.com). elisa measurements were performed in triplicate for each assay and repeated in at least duplicate assays. the scientist who performed the assays (r.l.d .) statistical analyses were performed, as previously described, using spss version 20 (ibm corporation, armonk, ny) and clinical research calculators on the vassar university statistics web interface (http://vassarstats.net/). differences between groups were considered statistically significant when 2-tailed p values were less than 0.05. all p values below 0.0001 are represented as p < 0.0001. the reference cutoff concentration for gdf-15 was set at the 95th percentile of control group serum concentrations, as for fgf-21. we used mann - whitney u and kruskal - wallis nonparametric testing to determine statistical differences between groups. receiver operating characteristic (roc) curves were plotted using sensitivity vs 100 specificity on a continuous scale and the area under the curve (auc) was used to determine the relative diagnostic capacity. biomarker levels were correlated with other biochemical measurements and clinical assessment tools using nonparametric spearman correlation testing. linear regression analysis was used to determine if statistically correlated parameters could predict gdf-15 concentration. multivariate linear regression analysis was performed to determine factors capable of predicting disease while controlling for confounders that may influence serum gdf-15 concentration. median serum gdf-15 concentrations varied considerably among the 3 cohort groups: the control group had a median serum gdf-15 concentration of 1,183.5 pg / ml (interquartile range 1,037.51,475.3), disease controls had a median concentration of 1,791.0 pg / ml (iqr 1,148.03,406.8), and patients with mitochondrial disease had a median concentration of 3,956.0 pg / ml (iqr 2,516.58,676.8). the interassay coefficient of variation was 9.6% for repeated samples (40 samples repeated in duplicate and 42 samples repeated in triplicate from across the 3 experimental groups) and 10.7% for assay quality controls. on average, serum gdf-15 concentrations were 2 and 4.6 times higher than control levels for the disease control and mitochondrial disease groups, respectively. when compared to mean fgf-21 levels, disease control and mitochondrial disease groups were 2.2 and 7.3 times higher than control levels, respectively. as with fgf-21 (threshold cutoff 350 pg / ml), the 95th percentile of control subjects was set as the threshold cutoff for gdf-15 (threshold cutoff 2,330 pg / ml) (figure 1). the same relationship in median biomarker concentration between groups was observed for both biomarkers and nonparametric mann - whitney u testing showed serum biomarker concentrations to differ between the groups (figure 1). serum fgf-21 (red) and gdf-15 (green) concentrations are displayed for the 3 experimental groups on a logarithmic scale. the same relationship between median group concentrations can be seen for both biomarkers, with higher levels in the mitochondrial disease group compared to disease controls and controls. threshold cutoffs (determined from the 95th percentile of control group biomarker concentrations) are indicated by blue horizontal lines at the level of 350 pg / ml for fgf-21 and 2,330 pg / ml for gdf-15. in addition, mann - whitney u (muscle biopsy vs genetic diagnosis, mtdna vs ndna mutation, muscle manifesting vs non muscle manifesting, mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes vs non - melas, male vs female, diabetic vs normal, cardiovascular involvement vs no cardiovascular involvement) and kruskal - wallis (walker criteria , diabetic status , body mass index , cardiovascular status) nonparametric analyses revealed only one statistical difference among all the groups and categories compared. the criterion of muscle biopsy diagnosis vs genetic diagnosis showed a marginal difference in serum gdf-15 concentration (3,206 pg / ml vs 6,415 pg / ml , respectively ; p = 0.046). of note, in our previous fgf-21 study, there was a difference between muscle manifesting and non muscle manifesting disease patients, indicative of fgf-21 elevation in myopathic disease states. this difference was not evident when analyzing gdf-15 (p = 0.324), suggesting gdf-15 production may not be related to tissue - specific pathways, but rather pathways common to all tissues. this highlights a potentially broader indication of mitochondrial diseases by gdf-15 that is not evident for fgf-21. the vast majority of literature discussing gdf-15 as a disease biomarker pertains to cardiovascular abnormalities, in particular ischemic heart disease. although cardiac involvement is a relatively common comorbidity of mitochondrial disease, the majority of patients in this study did not have cardiac pathology. when analyzing gdf-15 against all cardiac involvement and also against subcategories of cardiac involvement, there was no apparent statistical difference in serum gdf-15 concentration between those with (n = 11) or without (n = 43) cardiac involvement (p = 0.788), or when subcategorized (kruskal - wallis, p = 0.451). serum biomarker concentration exceeding the threshold cutoff was considered indicative of disease for statistical analyses. as the threshold cutoffs were set at the 95th percentile of control concentrations, the specificity was artificially determined to be 95.5% (95% confidence interval 86.4%98.8%) for fgf-21 and gdf-15 in both disease control and mitochondrial disease groups. the sensitivity for detecting mitochondrial disease if serum gdf-15 concentrations were raised was 77.8% (95% ci 64.1%87.5%), as compared to fgf-21 at 68.5% (95% ci 54.3%80.0%). for the disease control group, the sensitivity for predicting mitochondrial disease when there was none was 30.0% (95% ci 12.8%54.3%) using gdf-15, compared to 35.0% (95% ci 16.3%59.1%) using fgf-21. when subcategorizing the mitochondrial disease group by diagnosis, the sensitivity for patients diagnosed by muscle biopsy was 59.3% (95% ci 39.0%77.0%) using serum fgf-21 concentration and 70.4% (95% ci 49.7%85.5%) using serum gdf-15 concentration. for patients diagnosed with a genetic mutation, the sensitivity was 77.8% (95% ci 57.3%90.6%) using serum fgf-21 concentration and 85.2% (95% ci 65.4%95.1%) using serum gdf-15 concentration. nine of the patients with genetic diagnoses also had a muscle biopsy showing changes consistent with mitochondrial disease. the sensitivity when considering these patients was 66.7% (95% ci 30.9290.96) using serum fgf-21 concentrations and 88.9% (95% ci 50.6799.42) using serum gdf-15 concentration. the likelihood of having disease if fgf-21 levels were raised above the threshold cutoff of 350 pg / ml, known as the diagnostic or, was calculated in our previous study to be 45.7 (95% ci 12.5166.5, p < 0.0001). for gdf-15 in this study, the diagnostic or was calculated to be 73.5 (95% ci 19.6276.3, p < 0.0001). further to this, roc curve analysis of fgf-21 and gdf-15 showed gdf-15 to be slightly better (auc 0.941) than fgf-21 (auc 0.911) at predicting disease across combinations of sensitivity and specificity, although the aucs were not statistically different. combining both biomarkers did not increase the auc (0.944) remarkably over that for gdf-15 alone, indicating that there was little synergistic diagnostic benefit when considering the biomarkers together. statistical dependence between gdf-15 and all other measured parameters from the initial study was assessed for the disease group using spearman rank correlation coefficient test. gdf-15 correlated with lactate (spearman correlation coefficient = 0.583, p < 0.0001), lactate: pyruvate (rs = 0.544, p < 0.0001), fgf-21 (rs = 0.554, p < 0.0001), body mass index (rs = 0.322, p < 0.02), mrc proximal muscle weakness (rs = 0.302, p < 0.05), and the walker criteria (rs = 0.322, p < 0.02). interestingly, unlike in other gdf-15 studies, those patients in the mitochondrial disease group for whom an nmdas had been completed at the time of recruitment (n = 30) showed no correlation between the total nmdas score or scores for subsections (i iii) and serum fgf-21 or gdf-15 concentration. following multivariate linear regression analysis for each of the groups to determine any variables that could predict serum gdf-15 levels, only lactate: pyruvate (p < 0.002) and fgf-21 (p < 0.01) remained associated in the mitochondrial disease group. despite a moderate correlation between gdf-15 and fgf-21 (rs = 0.554), an association that persisted after linear regression, there was no benefit to disease prediction when the 2 biomarkers were combined for roc curve analysis (figure 2), as mentioned above. finally, when considering gdf-15 levels from control and mitochondrial disease groups in a multivariate logistic regression model that included possible confounders (age, lactate : pyruvate, insulin, glucose, cholesterol, triglycerides, and fgf-21), gdf-15 was the best predictor of mitochondrial disease (p < 0.002). gdf-15 (green) outperformed fgf-21 (red) across varying sensitivities at high specificities (area under the curve = 0.941 vs 0.911, respectively ; no difference). combining gdf-15 and fgf-21 (blue) had little benefit over gdf-15 alone (auc = 0.944 vs 0.941, respectively ; no difference). in our cohort of adult patients with mitochondrial disease, gdf-15 presents as a sensitive indicator of mitochondrial diseases and outperforms fgf-21 and classical markers. median serum concentrations of gdf-15 showed the same relationship between experimental groups as was observed for fgf-21 (figure 1), but gdf-15 was a better predictor of disease based on diagnostic sensitivity, diagnostic or, roc curves (figure 2), and multivariate linear regression. despite a moderate correlation between serum gdf-15 and fgf-21 concentration, a relationship that persisted after linear regression analysis, there was no synergistic benefit for considering gdf-15 and fgf-21 together (figure 2). furthermore, gdf-15 was more broadly indicative of mitochondrial diseases, rather than preferentially indicating muscle manifesting mitochondrial disease like fgf-21. first, we surveyed an adult - only cohort of patients with mitochondrial disease, whereas other fgf-21 and gdf-15 diagnostic studies have looked at combined adult and pediatric cohorts. as pediatric patients are known to exhibit higher circulating biomarker levels, analyses combining these populations may artificially skew serum biomarker concentrations and therefore the analysis of . second, our cohort included a combination of patients with genetic and muscle histology diagnoses, representing current clinical diagnostic methods. as such, our study represents a more stringent estimation of biomarker diagnostic validity, compared to other studies that have considered only genetically diagnosed cohorts. given our , we propose that biomarker triaging of patients suspected of a mitochondrial disorder may prove to be a more efficient diagnostic paradigm when performed in combination with exhaustive genetic sequencing analysis using next - generation sequencing. third, our cohort represents a wider range of mitochondrial diseases in comparison to other studies that have used limited genetic mitochondrial disease groups, such as m.3243a > g, kearns - sayre syndrome, mitochondrial dna deletions, or tk2 mutations. finally, we used assay kits from the same manufacturer (fgf-21 and gdf-15 ; biovendor, brno, czech republic), as compared to other studies that used assays from different manufacturers (fgf-21 ; biovendor and gdf-15 ; r&d systems, minneapolis, mn), to reduce potential variation in methodology and experimental output. the median gdf-15 concentration for controls in this study (1,183.5 pg / ml) was in good agreement with median gdf-15 concentrations for controls (1,020 pg / ml in men and 1,017 pg / ml in women) from the framingham offspring study, despite differing methods of quantification. however, when age- and sex - matched 97.5th percentile values from the framingham study were used as the threshold cutoff for a genetically diagnosed m.3243a > g european patient cohort study, a diagnostic sensitivity of only 53% was calculated. by comparing the 97.5th percentile values from the framingham study (range 1,0855,006 pg / ml, age- and sex - dependent, using precommercial automated electrochemiluminescence immunoassay) with the threshold cutoff of a recent japanese mitochondrial disease patient gdf-15 diagnostic study (710 pg / ml, using r&d systems quantikine elisa), obtained using the same elisa assay as the european study, it becomes apparent that the use of different assays can give markedly different and the use of data from one assay type in association with a different assay can potentially be misleading. despite this , the correlation between serum gdf-15 and fgf-21 concentrations was in agreement between this study and the european study (rs = 0.554 vs rs = 0.54, respectively) and close to that reported in the japanese study (rs = 0.64). our study demonstrates that elevated gdf-15 levels were indicative of mitochondrial disease regardless of whether there was muscle involvement. this is in contrast to the japanese study, which claimed that gdf-15 may be a better indicator of muscle manifesting disease than fgf-21, even though a measure of muscle involvement was not used in that study and therefore comparisons relating muscle involvement with biomarker concentration were not possible. furthermore, that study used different threshold values for gdf-15: they agreed with our previously determined fgf-21 threshold cutoff value of 350 pg / ml, when using the same elisa from biovendor, but the threshold cutoff values for gdf-15 were set at a lower level (r&d systems = 710 pg / ml vs biovendor = 2,330 pg / ml in this study), possibly owing to the difference in elisa used. in this study and our previous study on fgf-21 , we found no correlation with the total or subcategory nmdas scores for patients who completed this rating scale at the time of recruitment. this is in contrast to other studies that have found moderate to strong correlations between biomarker concentrations and disease severity measured using the nmdas and the japanese mitochondrial disease rating scale. in the european study , a correlation was identified between gdf-15 and nmdas - rated severity for asymptomatic patients, moderately affected patients, or moderately affected patients (rs = 0.54, p < 0.001), but not for severely affected patients. this disparity may be due to the method of analysis, as it was reported that a parametric correlation test (pearson) was used for the severe group and then compared to a nonparametric correlation test (spearman) for asymptomatic, mild and moderate severity groups. a lack of correlation between serum biomarker concentration and disease progression may indicate that the current means of assessing disease progression are inadequate or too narrow for the phenotypic diversity of these disease groups. alternatively, the lack of association may indicate the inability of these biomarkers to infer disease progression on the basis of being surrogate markers and not mediators of disease, a detail that warrants further investigation. finally, the single follow - up sampling of both fgf-21 and gdf-15 in the european m.3243a > g cohort, 2 years after the initial sample, may not provide adequate frequency or time scales for these markers to reliably track disease progression in combination with the nmdas. estimation of progression may therefore be improved by more frequent sampling in addition to more sensitive means of assessing severity. thus, larger studies with more sensitive clinical tools to measure disease progression, performed over longer follow - up periods and with more frequent sampling points, may be required to clarify whether biomarkers such as gdf-15 and fgf-21 are able to indicate disease progression. the clinical diagnostic outlook for mitochondrial disease has been dramatically improved over the past 5 years with the identification of fgf-21 as a useful indicator of muscle manifesting mitochondrial disease and now more so with gdf-15 as a more sensitive and broadly indicative marker of mitochondrial disease. continued investigation into the pathophysiologic relationship of these markers with mitochondrial disease front - line diagnostic indicator tests, such as serum gdf-15 concentration, coupled with exhaustive genetic sequencing methods, afforded by advances in next - generation sequencing technologies, herald a potentially new age for mitochondrial disease diagnosis. it is hoped that the clinical, fiscal, and personal burden of mitochondrial diseases may be improved through better management and treatment ing from improved diagnostic capabilities. statistical analysis was carried out by r.l.d. with assistance from j. patterson ( both from the kolling institute of medical research, st. conceptualized and designed the study, acquired data, analyzed data, carried out statistical analyses, interpreted data, drafted the manuscript, and revised the manuscript. professor c.m.s. designed and funded the study, interpreted data, and revised the manuscript.

objective: to directly compare the diagnostic utility of growth differentiation factor15 (gdf-15) with our previous fibroblast growth factor21 (fgf-21) findings in the same adult mitochondrial disease cohort.methods:serum gdf-15 levels were measured using a quantitative elisa. statistical analyses of gdf-15 data were compared with our published fgf-21 findings.:median serum gdf-15 concentrations were elevated in patients with mitochondrial disease and differed between all experimental groups, mirroring group for fgf-21. there was a difference between patients diagnosed by muscle biopsy and genetic diagnosis, suggesting that serum gdf-15 measurement may be more broadly specific for mitochondrial disease than for muscle manifesting mitochondrial disease, in contrast to fgf-21. gdf-15 showed a markedly higher diagnostic odds ratio when compared with fgf-21 (75.3 vs 45.7), was a better predictor of disease based on diagnostic sensitivity (77.8% vs 68.5%), and outperformed fgf-21 on receiver operating characteristic curve analysis (area under the curve 94.1% vs 91.1%). combining both biomarkers did not improve the area under the curve remarkably over gdf-15 alone. gdf-15 was the best predictor of mitochondrial disease (p < 0.002) following multivariate logistic regression analysis.:gdf-15 outperforms fgf-21 as an indicator of mitochondrial diseases. our data suggest that gdf-15 is generally indicative of inherited mitochondrial disease regardless of clinical phenotype, whereas fgf-21 seems to be more indicative of mitochondrial disease when muscle manifestations are present.classification of evidence: this study provides class iii evidence that serum gdf-15 accurately distinguishes patients with mitochondrial diseases from those without them.

conformational transitions of unstructured proteins into -structure - based oligomeric or amyloid states are crucial processes in the onset and development of a variety of neurodegenerative disorders such as alzheimer's disease (ad) and parkinson's disease. amyloid (a), a major player in ad, is a 40- or 42-amino acid peptide cleaved from its precursor membrane protein by sequential actions of - and -secretases and has a high propensity for toxic aggregation to form cross--fibrils. accumulated evidence indicates that the gm1 ganglioside, a glycosphingolipid abundant in neuronal cell membranes, interacts with a and promotes its assembly, ing in pathogenic amyloid formation. for example, high - density gm1 clustering, which is exclusively observed in synaptosomes, is suggested to accelerate a deposition. in vitro experiments have indicated that the a-gm1 interaction depends on the clustering of gm1, and its carbohydrate moiety alone can not induce conformational changes of a. furthermore, it has been suggested that each of the heredity variants of a reported thus far has its own specificities for gangliosides, which have been supposed to be associated with their ectopic deposition. promotion of amyloid formation in membrane - bound states has also been reported for prion and -synuclein. for example, prion protein has been reported to be localized in the membrane microdomains and caveolae enriched with ganglioside, which interacts with prion protein and thereby promotes its -to- structural conversion. therefore, detailed conformational characterization of a interacting with the ganglioside clusters not only provides structural information as cues for drug development in preventing and treating ad but also offers general insights into the mechanisms underlying the disease - associated amyloid formation facilitated in membrane environments. in previous papers, we have reported nuclear magnetic resonance (nmr) studies of the interactions of a with ganglioside clusters using lyso - gm1 micelles (approximate molecular mass 60 kda) as model systems. our nmr data showed that a is accommodated on the hydrophobic / hydrophilic interface of the ganglioside cluster exhibiting an -helical conformation under ganglioside - excess conditions. in this state, a shows an up - and - down topological mode in which the two -helices at segments his - val and ile - val and the c - terminal val - val dipeptide segment are in contact with the hydrophobic interior of the micelles, whereas the remaining regions are exposed to the aqueous environment. a similar tendency of a has been observed using excess amounts of gm1, which forms micelles with an approximate molecular mass of 140 kda. these findings indicate that ganglioside clusters offer unique platforms at their hydrophobic / hydrophilic interfaces for binding coupled with -helix formation of a molecules. to gain further insights into the underlying mechanisms of the amyloid formation of a, it is necessary to characterize the conformational transition from -helices to -structures on the ganglioside clusters. on the basis of the circular dichroism (cd) data, kakio et al. demonstrated that a/gm1 ratios influence the secondary structure of a on the raft - like lipid bilayers composed of gm1, cholesterol, and sphingomyelin. namely, a adopts an -helical structure at lower a/gm1 ratios (0.025), while it assumes a -sheet - rich structure at higher ratios (0.05). although more detailed structural information on a bound to the gm1 cluster is highly desirable, the small unilamellar vesicles used for the cd measurements are still too large to investigate with solution nmr techniques. in the present study, we attempt to characterize conformational states of a in the presence of varying amounts of gm1 aqueous micelles using stable - isotope - assisted nmr spectroscopy in conjunction with synchrotron - radiation vacuum - ultraviolet cd (vuvcd) spectroscopy. we found that gm1 micelles also induce distinct secondary structures of a depending on the a/gm1 ratios. on the basis of the spectroscopic data , we will discuss a behaviours on the ganglioside clusters from a structural point of view. the plasmid vector encoding a was constructed and cloned as a fusion protein with hexahistidine - tagged ubiquitin (his6-ub) using the pet28a(+) vector (novagene), subsequently transformed into escherichia coli strain bl21-codonplus (stratagene). transformed bacteria were grown at 37c in lb media containing 15 g / ml of kanamycin. for the production of isotopically labelled a protein, cells were grown in m9 minimal media containing nh4cl (1 protein expression was induced by adding 0.5 mm isopropyl--d - thiogalactopyranoside ( iptg) when the absorbance reached 0.8 at 600 nm. after 4 hours, cells were harvested and then suspended into buffer a (50 mm tris - hcl, 150 mm nacl, ph 8.0) containing 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, subsequently disrupted by sonication. after centrifugation, the pellet was dissolved in buffer a containing 8 m urea. his6-ub - a was purified by a ni - nitrilotriacetic acid affinity column (ge healthcare). recombinant glutathione s - transferase- (gst-) tagged yeast ubiquitin hydrolase-1 (yuh-1) was grown until the absorbance reached 0.8 at 600 nm and then induced to express by iptg. cell pellets were dissolved in buffer b (50 mm tris - hcl, 1 mm edta, 1 mm dtt, ph 8.5) and disrupted by sonication. gst - yuh-1 was purified by a glutathione affinity column (ge healthcare). a protein was enzymatically cleaved from his6-ub by incubation with gst - yuh-1 for 1 h at 37c at a molar ratio of his6-ub - a: gst - yuh1 = 10: 1. the cleaved a was purified by reverse - phase chromatography using an octadecylsilane column (tskgel ods-80tm, tosoh) with a linear gradient of acetonitrile. synthetic a labelled with n selectively at val or val was purchased from anygen co. both of recombinant and synthetic a proteins were dissolved at an approximate concentration of 2 mm in 0.1% (v / v) ammonia solution then collected and stored in aliquots at 80c until use. the residual ganglioside was suspended at a concentration of 12 mm in 10 mm potassium phosphate buffer (ph 7.2) and then mixed by vortexing. micelle sizes were determined by dynamic light scattering using a dynapro titan (wyatt technology). a was dissolved at a concentration of 0.2 mm in 10 mm potassium phosphate buffer (ph 7.2) in the absence or presence of 0.49 mm gm1 or lyso - gm1. 980 l of 5 m tht (sigma) solution in 50 mm glycine - naoh buffer (ph 8.5) was added to an aliquot of 20 l of each sample. fluorescence was measured immediately after mixing at the excitation and emission wavelengths of 446 and 490 nm, respectively, using spectrofluorophotometer (hitachi f-4500) at 37c. a was dissolved at a concentration of 0.2 mm in 10 mm potassium phosphate buffer (ph 7.2). the cd spectra of a in the presence or absence of gm1 were measured from 265 to 175 nm under a high vacuum (10 pa) at 37c using the vuvcd spectrophotometer constructed at beamline 15 (0.7 gev) of the hiroshima synchrotron radiation center (hisor). details of the spectrophotometer and optical cell were described previously. the path length of the caf2 cell was adjusted with a teflon spacer to 50 m or 100 m for measurements. the vuvcd spectra were recorded with a 1.0-mm slit, a 16-s time constant, a 4-nm min scan speed, and nine accumulations. the molar ellipticities of a were calculated with the average residue weight of 107.5. the secondary structure contents of a were analysed using the modified selcon3 program and the vuvcd spectra down to 160 nm for 31 reference proteins with known x - ray structures. the secondary structures of these proteins in crystal form were assigned into four classes (-helices, -strandes, turns, and unordered structures) using the dssp program based on the hydrogen bonds between adjacent amide groups. in this analysis, the root - mean - square deviation and the pearson correlation coefficient (r) between the x - ray and vuvcd estimates of the secondary structure contents of the reference proteins were 0.058 and 0.85, respectively, confirming the high accuracy of the vuvcd estimation. nmr spectral measurements were made on a bruker dmx-500 spectrometer equipped with a cryogenic probe as well as a bruker avance iii-400 spectrometer. isotopically labelled a was dissolved at a concentration of 0.2 mm in 10 mm potassium phosphate buffer (ph 7.2) containing 10% (v / v) h2o in the presence or absence of gm1. for h - n heteronuclear single - quantum correlation (hsqc) measurements, the spectra were recorded using a labelled with n uniformly or selectively at the amide group of val or val at a h observation frequency of 500 mhz with 128 (t1) 1024 (t2) complex points and 256 scans per t1 increment. the spectral width was 1720 hz for the n dimension and 6000 hz for the h dimension. one - dimensional carbonyl c spectra were recorded using uniformly c- and n - labelled a at a h observation frequency of 400 mhz with a spectral width of 22,000 hz. in these experiments, we examined whether tht fluorescence is enhanced by a in the presence of varying concentrations of gm1 or lyso - gm1. as shown in figure 1 , gm1 exhibited a bell - shaped dependence on a/gm1 ratios regarding tht fluorescence enhancement, while lyso - gm1 showed virtually no enhancement. maximum enhancement was observed at a 1:15 molar ratio of a to gm1. the dynamic light scattering data confirmed that the gm1 and lyso - gm1 micelles exhibited an approximate hydrodynamic radius of 6 nm and 4 nm, respectively, irrespective of the a/ganglioside ratios. the observed fluorescence intensity remained almost constant up to 12 h. these data indicated that gm1 micelles at appropriate a/gm1 ratios promote some aa interaction with formation of their -sheet - like conformation, which, however, does not in irreversible fibril formation. we characterized the conformational transition of a depending on a/gm1 ratios by cd measurements. the short - wavelength limit of cd spectroscopy can be successfully extended using synchrotron radiation as a high - flux source of photons, which yields much more accurate data than those obtained with a conventional cd spectrophotometer. the spectral data indicated that a undergoes conformational transitions depending on gm1 to a ratios (figure 2). the secondary structure contents of a at a/gm1 molar ratios of 1: 0, 1: 15, and 1: 30 were estimated on the basis of the spectral data (table 1). the -helix content of a in the presence of gm1 at an a/gm1 molar ratio of 1:30 was calculated to be 40.0%, which is consistent with our previous estimation based on the backbone chemical shift data of lyso - gm1, thus confirming close similarity of the binding modes of a between gm1 and lyso - gm1micelles. at an a/gm1 molar ratio of 1:15, where the maximum tht fluorescence enhancement was observed, the cd data consistently indicated a significantly increased content of -strands. the conformation of a in the presence of varying amounts of gm1 micelles was further characterized by c nmr spectroscopy. the carbonyl c nmr spectral data of uniformly c - labelled a indicated that the peaks shifted upfield, roughly corresponding to -structures, are more populated at an a/gm1 molar ration of 1: 15 in comparison with the gm1-excess conditions (figure 3). intriguingly, intensities of these peaks were selectively reduced upon the addition of tht. these nmr data are again consistent with the vuvcd data as well as the of the tht assay. to provide more detailed information on the conformational transition of a on gm1 micelles, we observed h - n hsqc spectral changes of a upon titration with gm1. interestingly, at an a/gm1 molar ratio of 1: 15, a exhibited hsqc peaks that were not observed in the spectra of free or fully micelle - bound forms (supplementary figure 1). by using site - specifically n - labelled a, these extra peaks were assigned to val and val (figure 4 and supplementary figure 1 available online at doi:10.4061/2011/925073). namely, the amide groups of these c - terminal residues of the micelle - bound a species show double hsqc peaks under the condition where a/gm1 ratio is relatively high. more interestingly, these double peaks were perturbed upon the addition of tht, while the corresponding peaks originating from the free and fully micelle - bound forms showed little or no change (figure 4). on the other hand, many of the h - n hsqc peaks from a, including val and val, were not observed at an a/lyso - gm1 molar ratio of 1: 15 due to intermediate chemical exchange between free and micelle - bound states of a (data not shown). accumulating evidence, including our previous reports, indicates that the interaction of a with gm1 involves multiple steps including the initial encounter complex formation and the accommodating process on the hydrophilic / hydrophobic interface of the ganglioside clusters. nmr spectral data of a titrated with gm1 micelles under a-excess conditions indicated that they form a weak complex presumably through an interaction between the n - terminal segment of a and the outer carbohydrate branch of gm1. thus, it is conceivable that the outer - branch structures of the carbohydrate moieties of gangliosides influence the association phase of the interaction and thereby determine the ganglioside specificities of a. nongangliosidic micelles and vesicles are barely or not capable of trapping a effectively. on the other hand, the -helical conformation of a accommodated on sugar - lipid interface of the gm1 and lyso - gm1 micelles have been characterized by nmr under ganglioside - excess conditions (a/ganglioside molar ratio of 1 : 30). because the structure of the inner part is common among the gangliosides, non - gm1 ganglioside, for example, gm2, thus, the spectroscopic characterization of the interactions of a with gangliosidic micelles has so far been performed only under the extreme conditions of the a/ganglioside ratios. the present study attempts to bridge the gap in our understanding of a behavior on gm1 micelles by carrying out spectroscopic analyses of a in the presence of varying amounts of gm1 micelles. the present data all indicated that -structure is more populated in micelle - bound a under the condition where the a/gm1 ratio is higher. although the binding mode of tht to amyloid fibrils has yet to be fully elucidated, it has been suggested that tht is more likely to bind perpendicularly to parallel -strands in a -sheet. in addition, recently reported solid - state nmr data indicate that a tht - reactive, neurotoxic amyloid intermediate of a is composed of parallel -structures. these data suggest that formation of parallel -strands is the minimum prerequisite for tht fluorescence enhancement. with this in mind , the bell - shape dependence of tht fluorescence enhancement (figure 1) can be interpreted as follows. at an extremely low concentration of gm1, most of a exists as a free form, which is an unstructured monomer and therefore is not reactive with tht. fraction of the micelle - bound form of a increases with increase of the gm1 amounts. to some extent, the micelles promote intermolecular interaction of a, giving rise to the tht - reactive a species. under gm1-excess conditions, however, a molecules are presumably relatively isolated from one another and therefore are not capable of forming an intermolecular -structure. the a/gm1 molar ratio, where the maximum enhancement was observed, was 1: 15, which corresponds to average number of a/micelle of 11.2 with the assumption of the micellar gm1 aggregation number of 168 4. thus, the a density on gm1 micelles is a crucial factor determining the occurrence of the tht - reactive a species. under the circumstance where the a density on gm1 micelles is high, the c - terminal dipeptide of a shows, at least, two distinct conformational states that are reactive with tht. in a previous paper, we demonstrated that the c - terminal val - val dipeptide is inserted into the hydrophobic interior of the gangliosidic micelles. this c - terminal segment is involved in the parallel -structure in the amyloid fibril and intermediate. on the basis of these data , we suggest that gm1 clusters promote intermolecular a-a interactions coupled with the conformational transition of their c - terminal hydrophobic anchors into the tht - reactive parallel -structure, in which the local chemical environments of the c - terminal segments are different in different -strands. this may account for the multiple hsqc peaks originating from the c - terminal segments (figure 4). it has been reported that a exhibits tht - reactive -sheet - rich aggregates in the presence of sodium dodecyl sulfate (sds) at submicellar concentrations. under these conditions, all the amide peaks of a disappeared from the h - n hsqc spectrum because of the formation of large aggregates, except for those from the c - terminal residues that should still be mobile in this assembly state. on the basis of the nmr data obtained using paramagnetic probes, the c - terminal segment of a bound to sds micelles taking into account these data in conjunction with our present data, we suggest that different -like structures of a are induced by gm1 aqueous micelles and submicellar concentrations of sds. lyso - gm1 micelles could not induce the formation of the tht - reactive -structure of a although the micelle - interacting modes of a are almost identical between gm1 and lyso - gm1 micelles under ganglioside - excess conditions. by inspection of the dynamic light scattering data on an assumption of their globular shapes, the diameters of gm1 and lyso - gm1 micelles it is plausible that the sizes and curvatures of the gangliosidic micelles are determining factors for the number of a molecules that can be accommodated on their hydrophilic / hydrophobic interface and the occurrence of a-a interactions coupled with tht - reactive -structure formation. indeed, gm1 clusters with flatter curvature such as gm1-containing unilamellar vesicles induce enhanced a fibrillogenesis in comparison with gm1 micelles. lipid composition can also be a determining factor for assembly states of gm1 molecules and their interaction with a. most importantly, there is growing evidence that cholesterol and sphingomyelin contribute to gm1 assembly and thereby influence a deposition promoted by its cluster. elucidation of the structural basis of these molecular events is an important subject for the forthcoming stage of the research. in , in the present study, we firstly identified and characterized the tht - reactive -structure of a promoted on gm1 micelles. our findings offer structural insights into the mechanisms underlying the -to- conformational transition of a on gm1 clusters, which is associated with the nucleation process in the a aggregation.

clusters of gm1 gangliosides act as platforms for conformational transition of monomeric, unstructured amyloid (a) to its toxic -structured aggregates. we have previously shown that a accommodated on the hydrophobic / hydrophilic interface of lyso - gm1 or gm1 micelles assumes -helical structures under ganglioside - excess conditions. for better understanding of the mechanisms underlying the -to- conformational transition of a on gm1 clusters, we performed spectroscopic characterization of a titrated with gm1. it was revealed that the thioflavin t- (tht-) reactive -structure is more populated in a under conditions where the a density on gm1 micelles is high. under this circumstance, the c - terminal hydrophobic anchor val39-val40 shows two distinct conformational states that are reactive with tht, while such a species were not generated by smaller lyso - gm1 micelles. these findings suggest that gm1 clusters promote specific a-a interactions through their c - termini coupled with formation of the tht - reactive -structure depending on sizes and curvatures of the clusters.

animals: two hundred and eighty - seven dogs that were presented at osaka prefecture university veterinary medical center (opuvmc), animal eye center and izumi animal hospital for examination and treatment of ophthalmic, medical and surgical diseases were used in this study. all the dogs underwent a general ophthalmologic examination including gonioscopy with a goldmann two - mirror lens and slitlamp biomicroscopy to confirm ocular conditions. laboratory examinations including blood works, urinalysis and diagnostic imagings were also carried out to exclude systemic disorders, if necessary. animals suspected of having secondary glaucoma, which was indicated by their history and ophthalmic conditions, such as the presence of uveitis, cataract, lens luxation, ocular neoplasia and infections including systemic diseases, were excluded from the study. the examined eyes were divided into 5 groups as follows: (i) normal ocular group (nor) including 336 normotensive eyes of 168 dogs (57 beagle, 18 miniature dachshund, 15 shih tzu, 12 pembroke welsh corgi, 11 chihuahua, 9 mongrel and labrador retriever, 8 yorkshire terrier, 6 papillon, 5 pomeranian, 4 shetland sheepdog, 3 cavalier king charles spaniel and miniature schnauzer, 2 bernese mountain dog, doberman pinscher and pug, and 1 afghan hound and french bulldog) with normal open angle (fig . 1afig . 1.typical canine iridocorneal angles in the five ocular groups examined in this study : ( a) wide open angle in a normal normotensive eye; (b1) open angle with goniodysgenesis in a primary closed angle glaucoma (pcag)-predisposed normotensive eye; (b2) narrow angle with severe goniodysgenesis in a pcag - predisposed normotensive eye; (c) very narrow angle in a normotensive fellow eye of unilateral pcag; (d) closed angle in an acute glaucomatous eye; and (e) closed angle in a chronic glaucomatous eye. ), normal intraocular pressure (iop) of 1025 mmhg and no ocular disorders up to the date of ubm examination; typical canine iridocorneal angles in the five ocular groups examined in this study: (a) wide open angle in a normal normotensive eye; (b1) open angle with goniodysgenesis in a primary closed angle glaucoma (pcag)-predisposed normotensive eye; (b2) narrow angle with severe goniodysgenesis in a pcag - predisposed normotensive eye; (c) very narrow angle in a normotensive fellow eye of unilateral pcag; (d) closed angle in an acute glaucomatous eye; and (e) closed angle in a chronic glaucomatous eye. (ii) primary closed angle glaucoma (pcag)-predisposed ocular group (predis) including 116 normotensive eyes of 58 dogs (18 shiba, 14 american cocker spaniel, 13 toy poodle, 7 golden retriever, 2 maltese terrier, and 1 akita, english springer spaniel, flat - coated retriever and newfoundland, which were recognized as breeds with pcag) with some kind of ica abnormalities containing goniodysgenesis and open to narrow angles (figs . 1-b1 and b2), normal iop of 1025 mmhg and no ocular disorders up to the date of ubm examination; (iii) normotensive fellow ocular group in unilateral pcag (uni) including 22 normotensive eyes of 22 dogs suffering from unilateral pcag (6 shiba, 5 shih tzu, 3 american cocker spaniel, 2 flat - coated retriever, miniature dachshund and mongrel, and 1 chihuahua and welsh terrier), which had narrow angle with moderate to severe ica abnormalities (fig . 1c), normal iop of 1025 mmhg and no clinical signs of glaucoma up to the date of ubm examination; (iv) acute glaucomatous eye group (acg) including 19 hypertensive non - buphthalmic eyes of 19 dogs (6 shiba, 3 miniature dachshund and shih tzu, 2 american cocker spaniel, and 1 chihuahua, english setter, flat - coated retriever, mongrel and welsh terrier) with closed angle (fig . 1d), iop of over 25 mmhg (3158 mmhg) and signs of acute glaucoma, such as tenderness about the head and eyes, ocular pain (blepharospasm), serous discharge, episcleral congestion (red eye), corneal edema, dilated pupil and mild depression of optic nerve head, in which their clinical signs were observed less than 24 hr after the onset of symptoms on the date of ubm examination; (v) chronic glaucomatous eye group (crg) including 20 hypertensive buphthalmic eyes of 20 dogs (6 miniature dachshund, 5 american cocker spaniel, 3 shiba, 2 mongrel and shih tzu, and 1 flat - coated retriever and welsh terrier) with closed angle (fig . 1e), iop of over 25 mmhg (4278 mmhg) and signs of chronic glaucoma, such as globe enlargement with or without descemet streaks, mydriasis, tapetal hyperreflectivity with vascular attenuation, optic nerve cupping and blindness on the date of ubm examination. examination procedure: ultrasonographic images of the ica were obtained with ubm by using a ud-1000 with 40-mhz probes (tomey corp ., nagoya, japan) with room illuminance of 1,2001,300 lux in a general examination room. non - anesthetized or non - sedated dogs were examined using manual restraints in sternal recumbency. when sedation was required, it was achieved following a protocol described previously; these animals were assessed in a lateral recumbent position. ubm examination was also performed by a procedure described previously, and the following 7 parameters were evaluated with the measurement software of the ubm: (i) sld (fig . 2.typical canine images of ultrasound biomicroscopy ( ubm) on the iridocorneal angle (ica, a) and the same images of the ica with indication of ubm parameters evaluated in this study (b to d). the following 7 parameters were evaluated with the measurement software included in the ud-1000: (i) sld; (ii) the width of the ciliary cleft (ccw), measured from the superior surface of the root of the iris (ssri) to the inner surface of the sclera (iss) on a perpendicular line; (iii) the area of the ciliary cleft (cca), measured as the area surrounded by the width of the ciliary cleft, the line of the inner scleral side of the ciliary cleft from the inner surface of the sclera to the angle recess (ar) and the line of the superior side of the root of the iris from the angle recess to the superior surface of the root of the iris; (iv) the angle of the ciliary cleft (acc), the angle of the inner surface of the sclera to the angle recess and the superior surface of the root of the iris to the angle recess; (v) the scleral thickness at the position of the width of the ciliary cleft (st), as measured from the inner surface of the sclera on a perpendicular line to the outer surface of the sclera; (vi) the minimum distance between the angle recess and the scleral venous plexus (svp); and (vii) the total area of the scleral venous plexus (svpa) on the ubm image of the ica. ); (ii) the width of the ciliary cleft (ccw), measured from the superior surface of the root of the iris to the inner surface of the sclera on a perpendicular line (fig . 2b); (iii) the area of the ciliary cleft (cca), measured as the area surrounded by the width of the ciliary cleft, the line of the inner scleral side of the ciliary cleft from the inner surface of the sclera to the angle recess and the line of the superior side of the root of the iris from the angle recess to the superior surface of the root of the iris (fig . 2b); (iv) the angle of the ciliary cleft (acc), the angle of the inner surface of the sclera to the angle recess and the superior surface of the root of the iris to the angle recess (fig . 2c); (v) the scleral thickness at the position of the width of the ciliary cleft (st), as measured from the inner surface of the sclera on a perpendicular line to the outer surface of the sclera (fig . 2c); (vi) the minimum distance between the angle recess and the scleral venous plexus (asd, fig . 2c); and (vii) the total area of the scleral venous plexus (svpa) on the ubm image of the ica (fig . raw ubm measurements were detected from all obtained images in the examined dogs . in order to exclude differences of raw ubm values caused by taking different breeds with varied ocular size, the ubm measurements except for the angle of the ciliary cleft were corrected by using the ratio with sld on the distance / length, that is, ccw / sld, st / sld and asd / sld, or sld on the area, that is, cca / sld and svpa / sld . the mean of ubm values was calculated to obtain a ubm measurement of each parameter of the ica in an individual eye . typical canine images of ultrasound biomicroscopy ( ubm) on the iridocorneal angle (ica, a) and the same images of the ica with indication of ubm parameters evaluated in this study (b to d). the following 7 parameters were evaluated with the measurement software included in the ud-1000: (i) sld; (ii) the width of the ciliary cleft (ccw), measured from the superior surface of the root of the iris (ssri) to the inner surface of the sclera (iss) on a perpendicular line; (iii) the area of the ciliary cleft (cca), measured as the area surrounded by the width of the ciliary cleft, the line of the inner scleral side of the ciliary cleft from the inner surface of the sclera to the angle recess (ar) and the line of the superior side of the root of the iris from the angle recess to the superior surface of the root of the iris; (iv) the angle of the ciliary cleft (acc), the angle of the inner surface of the sclera to the angle recess and the superior surface of the root of the iris to the angle recess; (v) the scleral thickness at the position of the width of the ciliary cleft (st), as measured from the inner surface of the sclera on a perpendicular line to the outer surface of the sclera; (vi) the minimum distance between the angle recess and the scleral venous plexus (svp); and (vii) the total area of the scleral venous plexus (svpa) on the ubm image of the ica. nineteen acg cases received first medical therapy including an intravenous injection of 20% mannitol (yoshindo, toyama, japan) at a dose of 1.5 g / kg on the first admission and administrations of 0.005% latanoprost (xalatan, pfizer, tokyo, japan), 1% dorzolamide hydrochloride (trusopt, santen pharmaceutical co., ltd ., osaka, japan) and 0.5% timolol maleate (timoptol, santen pharmaceutical co., ltd . then, the responsiveness to the therapy, which means keeping reduction in normal iop of under 25 mmhg, and the seven parameters of the ica structures described above were evaluated simultaneously . statistical analysis : to exclude unexpected bias effects of pooling ubm measurements of right and left eyes, the average ubm values calculated from both eyes were used as ubm measurements in nor and predis . comparison between ocular groups was performed using non - repeated measurement one - way analysis of variance ( anova) or kruskal - wallis h test followed by scheffe s test (statcel 2nd ed . ; oms publishing co., tokyo, japan). a p - value reference ranges of ubm measurements in nor and predis were calculated by interative truncation method with correction (usui s method) using stss / excel ver. 3.typical images of the iridocorneal angle (ica) detected by ultrasound biomicroscopy (ubm) in (a) normotensive eyes with no ocular disorders, intraocular pressure (iop) of 1025 mmhg and normal open angle (nor), (b) normotensive eyes of primary closed angle glaucoma (pcag)-predisposed dogs with no ocular disorders, iop of 1025 mmhg, open to narrow angle and some kind of abnormalities of the ica (predis), (c) normotensive fellow eyes of dogs suffering from unilateral pcag, which had iop of 1025 mmhg, narrow angle with moderate to severe abnormalities of the ica and no clinical signs of glaucoma (uni), (d) hypertensive non - buphthalmic eyes with acute pcag having iop of over 25 mmhg, closed angle and clinical signs of acute glaucoma (acg) and (e) hypertensive buphthalmic eyes with chronic pcag having iop of over 25 mmhg, closed angle and clinical signs of chronic glaucoma (crg). the width of the ciliary cleft (ccw) of predis was narrower than that of nor (an arrow in b). the area of the ciliary cleft (cca) of uni was smaller than those of nor and predis, and significantly narrow ccw was observed in the case of uni (an arrow in c). the ciliary cleft was almost or completely collapsed, and it was difficult or impossible to detect ccw and cca in acg and crg (arrows in d and e). although the scleral venous plexus could be observed in ubm images of acg (asterisks in d), it was not found in a dog with crg. the basal iris of the anterior part of the ciliary cleft would be slightly inserted into the sclera of acg (an arrowhead in d). the sclera became thin, and the basal iris was inserted into the sclera in crg (arrowheads in e). , cross - sectional microstructures of the entire ica could be observed clearly by ubm examination in all ocular groups. the width of the ciliary cleft of predis was narrower than that of nor. it was difficult to identify the width of the ciliary cleft of uni, and the area of the ciliary cleft of uni would be smaller than those of nor and predis. the ciliary cleft was almost collapsed, and it was very difficult to detect the width and area of the ciliary cleft in acg. the scleral venous plexus could be observed in ubm images of most acg, although it was not found in crg. the basal iris of the anterior part of the ciliary cleft was slightly inserted into the sclera of acg, whereas it was inserted into the sclera in crg. typical images of the iridocorneal angle (ica) detected by ultrasound biomicroscopy (ubm) in (a) normotensive eyes with no ocular disorders, intraocular pressure (iop) of 1025 mmhg and normal open angle (nor), (b) normotensive eyes of primary closed angle glaucoma (pcag)-predisposed dogs with no ocular disorders, iop of 1025 mmhg, open to narrow angle and some kind of abnormalities of the ica (predis), (c) normotensive fellow eyes of dogs suffering from unilateral pcag, which had iop of 1025 mmhg, narrow angle with moderate to severe abnormalities of the ica and no clinical signs of glaucoma (uni), (d) hypertensive non - buphthalmic eyes with acute pcag having iop of over 25 mmhg, closed angle and clinical signs of acute glaucoma (acg) and (e) hypertensive buphthalmic eyes with chronic pcag having iop of over 25 mmhg, closed angle and clinical signs of chronic glaucoma (crg). the width of the ciliary cleft (ccw) of predis was narrower than that of nor (an arrow in b). the area of the ciliary cleft (cca) of uni was smaller than those of nor and predis, and significantly narrow ccw was observed in the case of uni (an arrow in c). the ciliary cleft was almost or completely collapsed, and it was difficult or impossible to detect ccw and cca in acg and crg (arrows in d and e). although the scleral venous plexus could be observed in ubm images of acg (asterisks in d), it was not found in a dog with crg. the basal iris of the anterior part of the ciliary cleft would be slightly inserted into the sclera of acg (an arrowhead in d). the sclera became thin, and the basal iris was inserted into the sclera in crg (arrowheads in e). 4.box-plots of each ultrasound biomicrosopic measurement in the five ocular groups: (a) corrected width of the ciliary cleft (ccw); (b) the angle of the ciliary cleft (acc); (c) corrected area of the ciliary cleft (cca); (d) corrected minimum distance / length from the angle recess to the scleral venous plexus (asd); (e) corrected total area of the scleral venous plexus on the ultrasound biomicrosopic image of the iridocorneal angle (svpa); (f) corrected scleral thickness (st). the boxes define the 25th and 75th percentiles, with the median value indicated by a horizontal bar. the whiskers delineate the 5th and 95th percentiles, with outliers indicated as open circles. not available means no detectable data due to complete collapse of the ciliary cleft and/or the scleral venous plexus. different letters / alphabets indicate statistically significant differences between groups (p<0.05)., and table 1table 1.reference ranges of corrected ultrasound biomicroscopic values in healthy dogscorrected ccw (ccw / sld)acc corrected cca (cca / sld)corrected asd (asd / sld)corrected svpa (svpa / sld)corrected st (st / sld)nor (n=168)0.0640.1498.322.20.0190.0420.1100.3170.0140.0580.2530.384predis (n=58)0.0280.1314.819.00.0060.0430.1180.3020.0120.0520.2240.339nor; normotensive eyes in normal open angle, predis; normotensive eyes in predisposition to primary closed angle glaucoma. ccw; the width of the ciliary cleft, sld; the distance between schwalbe s line (the borderline of the cornea and sclera) and the anterior lens capsule, acc; the angle of the ciliary cleft, cca; the area of the ciliary cleft, asd; the minimum distance between the angle recess and the scleral venous plexus, svpa; the total area of the scleral venous plexus on the ultrasound biomicroscopic image of the iridocorneal angle, st; the scleral thickness at the ccw. shows reference ranges of each ubm measurement in nor and predis. in predis, ubm measurements of the corrected width of the ciliary cleft and the angle of the ciliary cleft were significantly lower than those of nor. in uni, significantly low values were found for the corrected width and area of the ciliary cleft, and the angle of the ciliary cleft, when compared with those in nor and predis. in acg, the corrected width and area of the ciliary cleft, the total area of the scleral venous plexus and the scleral thickness, and the angle of the ciliary cleft were significantly smaller than those of nor and predis. the corrected width and area of the ciliary cleft, the total area of the scleral venous plexus and the scleral thickness in acg were also significantly decreased compared with those of uni. in crg, the anatomical structures of the sclerociliary cleft including the scleral venous plexus completely collapsed, and all ubm values except the corrected scleral thickness were zero or undetectable. there were significant differences for the corrected width and area of the ciliary cleft, and the total area of the scleral venous plexus between crg and nor, predis and uni. the angle of the ciliary cleft in crg was significantly lower than those of nor and predis. the corrected scleral thickness of crg was significantly smaller than those of nor, predis, uni and acg, as a of the buphthalmos. box - plots of each ultrasound biomicrosopic measurement in the five ocular groups: (a) corrected width of the ciliary cleft (ccw); (b) the angle of the ciliary cleft (acc); (c) corrected area of the ciliary cleft (cca); (d) corrected minimum distance / length from the angle recess to the scleral venous plexus (asd); (e) corrected total area of the scleral venous plexus on the ultrasound biomicrosopic image of the iridocorneal angle (svpa); (f) corrected scleral thickness (st). the boxes define the 25th and 75th percentiles, with the median value indicated by a horizontal bar. the whiskers delineate the 5th and 95th percentiles, with outliers indicated as open circles. not available means no detectable data due to complete collapse of the ciliary cleft and/or the scleral venous plexus. different letters / alphabets indicate statistically significant differences between groups (p<0.05). nor; normotensive eyes in normal open angle, predis; normotensive eyes in predisposition to primary closed angle glaucoma. ccw; the width of the ciliary cleft, sld; the distance between schwalbe s line (the borderline of the cornea and sclera) and the anterior lens capsule, acc; the angle of the ciliary cleft, cca; the area of the ciliary cleft, asd; the minimum distance between the angle recess and the scleral venous plexus, svpa; the total area of the scleral venous plexus on the ultrasound biomicroscopic image of the iridocorneal angle, st; the scleral thickness at the ccw. nineteen cases received first medical therapy on opuvmc, and the responsiveness to the therapy and the ica structures were evaluated simultaneously. twelve dogs showing no response to the first medical therapy had significant decrease of the corrected width and area of the ciliary cleft, and the total area of the scleral venous plexus, compared with 7 cases responding to the therapy (fig . 5fig . 5.box-plots of each ultrasound biomicrosopic measurement in 7 dogs responding to first medical therapy ( effect) and 12 dogs showing no response to the therapy (no effect): (a) corrected width of the ciliary cleft (ccw); (b) the angle of the ciliary cleft (acc); (c) corrected area of the ciliary cleft (cca); (d) corrected minimum distance / length from the angle recess to the scleral venous plexus (asd); (e) corrected total area of the scleral venous plexus on the ultrasound biomicrosopic image of the iridocorneal angle (svpa); (f) corrected scleral thickness (st). the boxes define the 25th and 75th percentiles, with the median value indicated by a horizontal bar. data of asd in the group of no effect were calculated from 3 cases, because it could not be detected in the other 9 cases due to complete collapse of the ciliary cleft and/or the scleral venous plexus. ). box - plots of each ultrasound biomicrosopic measurement in 7 dogs responding to first medical therapy (effect) and 12 dogs showing no response to the therapy (no effect): (a) corrected width of the ciliary cleft (ccw); (b) the angle of the ciliary cleft (acc); (c) corrected area of the ciliary cleft (cca); (d) corrected minimum distance / length from the angle recess to the scleral venous plexus (asd); (e) corrected total area of the scleral venous plexus on the ultrasound biomicrosopic image of the iridocorneal angle (svpa); (f) corrected scleral thickness (st). the boxes define the 25th and 75th percentiles, with the median value indicated by a horizontal bar. data of asd in the group of no effect were calculated from 3 cases, because it could not be detected in the other 9 cases due to complete collapse of the ciliary cleft and/or the scleral venous plexus. glaucoma is a leading cause of blindness in dogs, and there are no perfect therapies for prevention of vision impairment in glaucomatous dogs. this may be attributable to inadequate assessment of entire structural changes within the ica and lack of a predictive system of canine glaucoma. hence, anatomical structures of the ica were evaluated by ubm for comparison of its cross - sectional microstructures in healthy and glaucomatous live dogs. ubm clearly revealed abnormalities deep within the ica, especially on the ciliary cleft and the scleral venous plexus, in not only glaucomatous dogs but also in healthy canines with a predisposition to pcag, which were anatomical changes similar to those described in previous reports. in predis, ubm values of the width and angle of the ciliary cleft were significantly lower than those of nor, although there were no significant differences in terms of the area of the ciliary cleft, the minimum distance between the angle recess and the scleral venous plexus, the total area of the scleral venous plexus and the scleral thickness between predis and nor, indicating that the pathway of the aqueous humor in the pcag - predisposed dogs would function as well as that in dogs with a normal open angle, despite the presence of structural abnormalities of the angular aperture (the opening of the ciliary cleft). significant differences were found for ubm measurements related to the ciliary cleft between non - glaucomatous healthy dogs and animals with unilateral glaucoma (fig . 4). the width, angle and area of the ciliary cleft of uni were decreased significantly compared with those of nor and predis, and the 75th percentile values of the corrected width (0.041) and angle (6.0) of the ciliary cleft in uni were smaller than the lower limits of their reference ranges in nor. these quantitative suggest unilateral pcag has decreased capacity of aqueous outflow from the anterior chamber in the eyes. however, the aqueous outflow might be able to retain a normal level, because there were no differences in the minimum distance between the angle recess and the scleral venous plexus, the total area of the scleral venous plexus and the scleral thickness between uni and healthy normotensive eye groups (nor and predis). therefore, aqueous humor production should be restricted to decrease the aqueous outflow from the anterior chamber, thereby maintaining the balance of the aqueous inflow and outflow in the ciliary cleft to prevent onset of glaucoma. this proposal has already been accepted as prophylactic therapy in the management of canine glaucoma. prophylactic medical treatment from the time of confirmed or presumed glaucoma in the first eye is helpful for delaying or preventing the onset of the disease in the second normotensive eye, although the median delay was about 22 months, less than 2 years. greater delay of the onset of glaucoma may be provided by early start of prophylactic treatment in dogs with a predisposition to pcag or unilateral type. ubm evaluation of the ica is helpful for providing objective criteria regarding the start of prophylactic treatment in dogs with a risk of glaucoma onset. judging from our presented here, glaucomatous prophylaxis is suggested for cases in which the ubm value of the corrected width, angle or area of the ciliary cleft decreases and is less than the lower limit of the reference range in dogs with normotensive eyes with open angle (nor). in glaucomatous dogs, ubm also revealed ica abnormalities that were available for management of canine glaucoma. 5 ), namely, the opening of the ciliary cleft and the total area of the scleral venous plexus. both the opening of the ciliary cleft (width and area of the ciliary cleft) and the total area of the scleral venous plexus would represent valuable information for prediction of the responsiveness to medical therapies, because the opening of the ciliary cleft and the scleral venous plexus in cases that were responsive to the medical therapies were significantly larger than in dogs that were unresponsive (fig . these findings suggest that the presence of observable ciliary cleft and scleral venous plexus is important for maintaining aqueous outflow from the anterior chamber in the eye . these ubm parameters are useful for prediction of the effects of medical therapies with ocular hypotensive agents . the opening of the ciliary cleft and the scleral venous plexus are key structures contributing to not only the pathophysiology of canine glaucoma but also the responsiveness to medical therapy in glaucomatous eyes . the minimum distance between the angle recess and the scleral venous plexus was only measured in 10 of 19 hypertensive eyes in acg, because it could not be detected in the nine eyes with loss of the angle recess due to complete collapse of the ciliary cleft . no significant differences of the corrected minimum distance between the angle recess and the scleral venous plexus, and the scleral thickness were detected between acg and the other ocular groups, such as nor, predis and uni ( fig . 4), suggesting that the minimum distance between the angle recess and the scleral venous plexus may only contribute to the aqueous outflow resistance in canine eyes, but may not be involved in the causes of increased iop. meanwhile, there were statistically significant differences in the corrected scleral thickness between acg and crg ( fig. these abnormalities might be associated with structural changes, such as scleral stretching or buphthalmos, induced by increased iop in the long term. although gonioscopic examination is an essential diagnostic tool as a conventional procedure for assessing abnormalities of the ica in the field of veterinary ophthalmology, objective and quantitative information of the ica is needed to establish new diagnostic criteria for improving strategies of glaucomatous managements in animals. since ubm can provide objective and quantitative data of entire ica abnormalities which are unable to assess with classical gonioscopic examination, ubm examination should be more widely applied for detecting quantitative abnormalities of the ica in dogs, especially in glaucoma - predisposed dogs or cases suffering from unilateral glaucoma. the potential for ubm examinations to contribute to analysis of glaucomatous mechanisms and provide advanced diagnosis and management of glaucoma warrants further investigation in canine patients.

by using ultrasound biomicroscopy (ubm), the cross - sectional structures of the entire iridocorneal angle (ica) which are unable to assess with gonioscopic examination were evaluated objectively and quantitatively in live healthy and glaucomatous dogs. the icas of normotensive eyes in healthy dogs with normal open angle (nor), a predisposition to primary closed angle glaucoma (pcag) (predis) and suffering from unilateral pcag (uni), as well as the icas of hypertensive eyes with acute and chronic pcag (acg and crg), were assessed. the opening of the ciliary cleft in predis was smaller than that in nor. in uni, the opening and area of the ciliary cleft were significantly decreased compared with those of nor and predis. acg had widespread structural abnormalities including marked decrease in the ciliary cleft and scleral venous plexus, and a thinner sclera than those in normotensive eyes, whereas the ica collapsed in crg with the thinnest sclera. medical therapy - responsive glaucomatous cases had wider ciliary cleft and scleral venous plexus than unresponsive ones. these findings suggest that the ciliary cleft and scleral venous plexus of the ica are key structures contributing to not only the pathophysiology of canine glaucoma but also the responsiveness to medical therapy in glaucomatous eyes, and cross - sectional entire structures of the ica should be evaluated quantitatively with ubm when diagnosing and managing canine glaucoma.

in a series of experiments, rhesus monkeys have been given in their diets 0.3, 1.5, and 25 ppm of a commercial polybrominated biphenyl (pbb) (as firemaster ff-1). the seven adult female monkeys receiving 0.3 ppm pbb have been on the treatment regime for 15 months and have consumed over 22 mg of pbb. during the initial 6 months of exposure, they lost weight and 2 of the animals develop sterile abscesses. at 6 months, 4 of the 7 animals had flattened and lengthened serum progesterone peaks. this change was correlated with an increase in length of their menstrual cycles. after 6 months of pbb exposure, the animals were bred. two of the 7 animals showed excessive and prolonged implantation bleeding. two abortions and 5 live births were recorded. all of the experimental infants were smaller than the controls at birth. the animals receiving a diet containing 1.5 ppm pbb for 36 weeks (total intake 70 mg) have shown a moderate weight loss and decrease in serum cholesterol. similar changes have also been recorded in the group given the 25 ppm pbb diet for 14 weeks (approximately 500 mg total intake). in addition, these animals have also developed a hyperplastic gastritis.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.

schizophrenia is a severe brain disorder that afflicts 0.51% of the world's population and that is typically first diagnosed in late adolescence or early adulthood. the illness is manifest as disturbances in perception, attention, volition, inferential thinking, fluency and production of language, and the recognition and expression of emotion that lead to substantial impairments in social and occupational functioning. many affected individuals suffer from comorbid depression, an increased risk of cardiovascular disease, and excessive nicotine, alcohol, and cannabis use. the first domain is positive or psychotic symptoms that include delusions, false beliefs firmly held in the face of contradictory evidence; perceptual disturbances and hallucinations, which may occur in any sensory modality but are most commonly auditory and experienced as hearing voices distinct from one's own thoughts; abnormalities in form of thought that are usually manifest as loose associations, over - inclusiveness, and/or neologisms; abnormal psychomotor activity that is usually manifest as grossly disorganized behavior, posturing, and/or catatonia. negative symptoms include asociality (withdrawal or isolation from family and friends), avolition (impaired initiative, motivation, and decision - making), alogia (poverty in the amount or content of speech), and anhedonia (reduced capacity to experience pleasure). the third category of symptoms includes a number of cognitive abnormalities such as disturbances in selective attention, working memory, executive control, episodic memory, language comprehension, and social - emotional processing. although positive symptoms are usually the presenting and most striking clinical feature of schizophrenia, disturbances in cognition appear to be the core features of the illness as they are present before the onset of psychosis and are the best predictor of long - term functional outcome for schizophrenia patients. therefore, functional recovery (e.g., recovery of the capacity to maintain employment) is largely dependent on improving cognitive deficits. although schizophrenia was initially characterized over 100 years ago, we still have only a limited understanding of its pathophysiology. moreover, we lack efficient tools for its treatment or prevention. for example, the multicenter, nimh - funded clinical antipsychotic trials in intervention effectiveness project recently found that newer atypical antipsychotics are not significantly more effective for treating psychosis than older typical antipsychotic medications and showed little benefit for improving cognitive symptoms. these findings highlight the need to develop novel therapeutic interventions for schizophrenia. if functional recovery of patients with schizophrenia depends on improving cognitive deficits, then understanding the neural basis of the normal cognitive operations that are impaired in schizophrenia is crucial to develop new therapies. interestingly, a number of findings from postmortem brain studies suggest that schizophrenia is associated with deficits of gaba - mediated synaptic transmission. furthermore, current hypotheses from cellular and systems neurophysiology suggest that a major role of gaba - mediated transmission is to produce synchronized neural network oscillations which by facilitating the processing and flow of information within and between brain regions may be essential for normal cognitive function. here we review convergent findings from schizophrenia research, cellular neurophysiology, and cognitive neuroscience that favor the hypothesis that deficits of cognitive function in schizophrenia from a dysfunction in gaba - mediated synaptic inhibition that disturbs oscillatory neural synchrony. this paper reviews recent evidence further indicating that in subjects with schizophrenia cognitive dysfunction is associated with alterations of oscillations in the gamma frequency band (3080 hz), which are normally induced during tasks that engage cognition. in addition, it reviews the cellular and molecular machinery involved in gaba - mediated synaptic transmission and the mechanisms by which gaba - mediated inhibition may synchronize neural activity in cortical circuits, focusing on the role of parvalbumin- (pv-) positive gaba neurons, whose function has been increasingly linked to the production of synchronized gamma oscillations. furthermore, data on the postnatal development of pv gaba neurons and their synaptic connections and the developmental trajectories of gene products involved in gaba - mediated synaptic inhibition is reviewed. finally, recent studies further suggesting that schizophrenia is associated with alterations in gaba - mediated synaptic transmission, particularly, but not exclusively, from pv neurons are highlighted. alterations of gaba signaling that impair gamma oscillations and therefore cognitive function in schizophrenia suggest potential paths for therapeutic interventions. schizophrenia is associated with deficits in behavioral tasks that assess perceptual and cognitive processes. many such tasks normally increase synchronized oscillatory activity as measured in the electroencephalogram (eeg), and such increase in synchrony is altered in subjects with schizophrenia. for example, gamma - band synchrony during tasks that require visual gestalt perception is attenuated in schizophrenia patients. one of the core cognitive deficits in schizophrenia is a dysfunction of working memory, a system to keep information in mind and to manipulate it while performing complex tasks. gamma oscillatory activity (3080 hz) may play an important role in normal working memory, given that gamma band synchrony increases with increasing working memory load. in patients with schizophrenia, working memory deficits are accompanied by altered patterns of cortical oscillatory activity, since schizophrenia patients actually fail to enhance gamma activity with increasing working memory load and show overall increased gamma band power during working memory. subjects with schizophrenia also have decreased oscillations in various frequency bands during specific phases of the working memory process, including encoding, maintenance, and retrieval. cognitive function, including working memory, implicates an interconnected network of brain regions, many of which show structural and functional abnormalities in schizophrenia. the prefrontal cortex (pfc), which is extensively interconnected with cortical and subcortical regions, is thought to exert top - down control of the flow of neural activity between brain regions to provide cognitive control, coordinating incoming sensory and motor information with representations of internal goals and rules to select a context - appropriate behavioral response. subjects with schizophrenia have significant deficits in cognitive control and attenuated gamma oscillations in pfc during cognitive control tasks. cognitive control - related gamma activity, but not theta activity, is reduced in the frontal cortex of first - episode schizophrenia patients independent of medication status, suggesting a deficit related to the disease process as opposed to medication side effects or the consequences of being chronically ill. interestingly, some studies reported a positive correlation between gamma oscillations and hallucination symptoms score in schizophrenia , indicating that the propensity for auditory hallucinations correlates with an increased tendency to enter states of oscillatory synchrony. it remains to be determined whether the decrease in gamma activity associated with cognitive deficits and the positive correlation between gamma activity and psychotic symptoms are due to different underlying mechanisms, manners of eliciting gamma, or cohorts of subjects. if, as suggested by multiple lines of evidence, altered neural synchrony underlies impairment of cognition in schizophrenia, then understanding the neural mechanisms normally involved in production of synchronized oscillations in neocortical circuits is crucial to develop new therapeutic interventions. whereas several mechanisms have been proposed, production of rhythms via gaba - mediated inhibition is currently a leading candidate mechanism, as reviewed in the following. by definition, gaba neurons have the capacity to synthesize gaba from glutamate via the enzymatic activity of glutamic acid decarboxylase (gad), for which there are two gene products of different molecular weight, gad65 and gad67. whereas the gad isoforms differ in a number of properties, their specific functional roles are not fully understood. interestingly, gad65 and gad67 are differentially expressed in gabaergic terminals in a cell type - specific manner (see below). gad - mediated gaba synthesis occurs in the cytosol, and gaba is transported into synaptic vesicles by the vesicular gaba transporter vgat (figure 1). shortly after an action potential arrives at the nerve terminal, vesicular gaba release is triggered with a certain probability and in a ca - dependent manner. in pv neuron terminals, pv may act as a ca buffer that binds residual ca after activation of the ca sensor that triggers vesicular gaba release. at all synaptic connections from cortical gaba neurons thus far studied, the effects of synaptically released gaba are mediated by gabaa receptors (gabaars), as the postsynaptic response is abolished by gabaar antagonists in hippocampus and neocortex. in contrast, gabab receptors mediate the postsynaptic effects of gaba only at connections from gaba neurons of the neurogliaform cell subtype. postsynaptic gabaars are heteropentamers composed of subunits from 7 different families (16, 13, 13, , , , and 13) typically combined following a 2: 2: stoichiometry to form a gaba - activated chloride channel. importantly, the subunit composition of the gabaar complex determines many of its functional properties. for instance, gaba - activated chloride currents produced by 1 subunit - containing gabaars (1-gabaars) have much faster decay kinetics than currents mediated by gabaars containing other subunits. benzodiazepines bind to gabaars via a binding site localized at the interface between and subunits and may modulate (potentiate or decrease) gabaar function in an subunit - selective manner. for example, zolpidem enhances gaba effects preferentially at 1-gabaars, whereas the 3ia and 5ia compounds are inverse agonists preferentially at 3-gabaars and 5-gabaars, respectively. other drugs, including tpa023 (also named mk0777), tpa023b, tpa123, and tpa003 have comparable binding affinity at 1-, 2-, 3-, and 5-gabaars but may lack pharmacological effects at certain gabaar subtypes. in particular , tpa023 is a partial agonist at 2- and 3-gabaars but has no agonist efficacy at 1- and 5-gabaars. the magnitude and direction of the ionic current flowing through gabaars depends on its driving force or difference between its electrochemical equilibrium potential (egabaa) and the resting membrane potential (vmr) in the plasma membrane compartment where gabaars are located. because gabaar channels are largely permeable to chloride, egabaa is close to the chloride equilibrium potential (ecl) and therefore egabaa depends on the sodium - potassium - chloride cotransporter 1 (nkcc1) and the potassium - chloride co - transporter 2 (kcc2), which mediate chloride uptake and extrusion, respectively. importantly, since the active gabaar conductance tends to clamp the membrane potential at egabaa, if egabaa is negative relative to vmr (vmr > egabaa), the gabaar currents are hyperpolarizing, whereas if egabaa is positive to vmr (egabaa > vmr), the gabaar currents produce depolarization. commonly, egabaa is negative to vmr, however, in certain cell types or subcellular compartments (and in general, early in brain development) egabaa > vmr. whether the gabaar conductance has inhibitory or excitatory effects depends on the relation between egabaa and the voltage threshold for action potential firing (vth), which is always depolarized relative to vmr (vth > vmr). when vth > vmr > egabaa, the chloride current is hyperpolarizing and clearly inhibitory because it shifts the membrane potential away from firing threshold, thus reducing the probability of firing. in contrast, if egabaa > vth > vmr, the chloride current is excitatory because it tends to depolarize the membrane above vth. however, if vth > egabaa > vmr, the gabaar conductance is inhibitory because, even though egabaa > vmr, shunting by the active gabaar conductance keeps the membrane potential below firing threshold. importantly, gabaar - mediated inputs that depolarize the membrane below vth (vth > egabaa > vmr) can have dual, time - dependent inhibitory / excitatory effects. initially, when the gabaar conductance is active, the net effect of the synaptic input is inhibitory because of the shunting effect. however, the depolarizing postsynaptic potential outlasts the duration of the gabaar conductance, thus increasing the firing probability once the gabaar conductance decays. importantly, the depolarizing gabaar - mediated post - synaptic potential may be amplified by voltage - dependent na currents localized perisomatically, possibly in the initial segment of the axon , enhancing its excitatory effect. the inhibitory versus excitatory effect of the gabaar conductance may be dynamic, because vmr and vth are subject to modulation and change over time. in addition, egabaa may vary between cell types and subcellular compartments, depending on the nkcc1/kcc2 activity ratio. it has been commonly suggested that uptake of extracellular gaba by plasma membrane transporters could help terminate the synaptic effect of gaba and thus the duration of the inhibitory postsynaptic current (ipsc). in the cns, gaba uptake is largely mediated by the plasma membrane gaba transporter 1 (gat1) which translocates gaba through the neuronal and glial membrane (figure 1). interestingly, recent experiments indicate that gat1 does not control the time course of gabaar - mediated ipscs, since the duration of ipscs produced at single synapses is not affected by pharmacological inhibition of gat1, nor in gat1 knockout mice. the finding that gat1 does not control ipsc duration may be explained by gat1's predominantly extrasynaptic localization and by the slow gat1-mediated gaba translocation rate compared with the rapid gabaar activation by synaptic gaba. other experiments suggest that gat1's main role is preventing intersynaptic gaba spillover , (e.g., the unintended activation of gabaars at a given synapse by gaba released at adjacent synapses). four different gaba transporters have been cloned: gat1, gat2, gat3, and bgt1; therefore, it is possible that some of the gaba transporters different from gat1 have properties consistent with uptake - mediated control of ipsc duration. however, gat2 is only found during very early brain development and bgt1 is not abundant in brain. gat3 is mostly localized in glia, and the effects of gat3 blockade indicate that, similar to gat1, gat3's main role is reducing the effects of gaba spillover. the mechanisms by which gabaar - mediated inhibition may synchronize postsynaptic cell activity have been reviewed in detail previously. figure 2 illustrates a group of pyramidal neurons firing asynchronously in response to some excitatory inputs that receive common gabaar - mediated hyperpolarizing inhibition. if such gaba - mediated hyperpolarizing input is strong enough, then the postsynaptic neurons will be inhibited together during a certain time window and, as the gabaar inhibitory effect decays, will escape from inhibition to resume firing nearly in synchrony (figure 2). such postinhibition synchronous spiking of pyramidal cells can be elicited by single - gaba neurons and readily generates synchrony throughout large numbers of neurons in computational network models. therefore, post - inhibition synchronous spiking is a strong candidate mechanism for production of neural synchrony. alternative synchronization mechanisms, which are not reviewed here, involve gap junctions connecting pyramidal cell axons or noisy but correlated inputs. importantly, neuronal synchrony is commonly observed during episodes of rhythmic / oscillatory network activity, especially in association with cognitive tasks. therefore, the circuit mechanisms of synchronized oscillations via gabaar - mediated inhibition must involve rhythmic interneuron firing and trains of ipscs in their postsynaptic target cells. as multiple subclasses of gaba neurons exist , a crucial issue is whether specific subtypes are involved in the mechanisms of synchronized oscillations. synchronized oscillations occur at different frequency bands, including theta (410 hz), beta (1530 hz), and gamma (3080 hz). whether oscillations of all frequency bands depend on gabaar - mediated inhibition and, if so, on particular gaba neuron subtypes is still a matter of investigation. here we focus on models for the mechanisms of gamma oscillations, which are commonly induced during cognitive tasks and seem to be impaired in the cortex of patients with schizophrenia. synchronization by the gabaar - mediated mechanism described in figure 2 requires sufficiently strong gaba synapses activating a relatively large gabaa conductance via inhibitory inputs localized near the site of action potential initiation. in pyramidal cells, action potentials are commonly triggered near the axon initial segment (ais), the axonal compartment that is closest to the soma. therefore, inhibitory inputs onto the perisomatic membrane compartment (soma, proximal dendrites, and ais) produce stronger inhibition than inputs onto distal dendrites , suggesting that perisomatic - targeting gaba neurons may be crucially involved in production of synchronized oscillations. three main subtypes of perisomatic - targeting gaba neurons exist in neocortex and hippocampus, namely, the parvalbumin - positive and the cholecystokinin - positive basket cells (pvbcs and cckbcs) and the pv - positive chandelier cells (pvchcs). both pvbcs and cckbcs innervate pyramidal cells at the soma and proximal dendrites (figure 3), whereas pvchcs synapse exclusively onto the ais (figure 3). because a synaptic gabaar conductance has stronger inhibitory effect the closest it is localized to the site of spike initiation, pvchc inputs onto the ais surprisingly, some recent studies suggested that synaptic input from pvchcs is actually excitatory, since stimulation of pvchcs frequently initiates spikes in postsynaptic pyramidal cells via gabaar activation. in addition, electron microscopy studies support the idea that pvchc inputs are excitatory, as they show very low levels of kcc2 in the ais compared to the soma or dendrites. since kcc2 extrudes chloride, lower ais levels of kcc2 should produce a more positive egabaa, ing in a depolarizing gabaar current (figure 3). in fact, in paired recordings using experimental conditions that preserve the physiological intracellular chloride concentration, pvchc inputs depolarize the postsynaptic pyramidal neurons , whereas in identical experimental conditions pvbc inputs are hyperpolarizing , consistent with higher levels of kcc2 transporters in the somatic membrane. experiments with uncaging of gaba onto gabaars in specific membrane compartments similarly showed a more depolarized egabaa at the ais compared with the soma and dendrites. an excitatory depolarizing gabaar - mediated input by pvchcs is inconsistent with their participation in inhibition - based synchronization. however, although egabaa at the ais is 1020 mv depolarized before vmr, it may be negative relative to vth. as highlighted above, when vth > egabaa > vmr, the gabaa conductance has a dual inhibitory / excitatory effect (figure 3), which in the case of pvchc inputs may be amplified by their proximity to the spike initiation site. such inhibitory / excitatory effect may contribute to synchronization of postsynaptic cell activity at gamma band frequency. specifically, since the postsynaptic potential outlasts the duration of the gabaar conductance (figure 3), once the conductance is deactivated, the depolarizing postsynaptic potential accelerates the post - inhibition synchronous spiking, facilitating synchronization at gamma frequency. such a mechanism operates at gabaar - mediated synapses onto gaba neurons and could also apply at pvchc inputs onto pyramidal neurons. the depolarizing effect of pvchcs described in neocortex contrasts with pioneer paired recording experiments showing that either pvbc or pvchc inputs hyperpolarize hippocampal pyramidal neurons. a hyperpolarizing effect of hippocampal pvbcs and pvchcs was also suggested recently by experiments using conditions that preserve the intracellular chloride concentrations, but that are different from the intracellular chloride - preserving conditions employed in studies of neocortical pvchc inputs. interestingly, the ais membrane has low levels of kcc2 in neocortical and hippocampal pyramidal cells. the discrepancies between findings for hippocampal versus neocortical pvchcs highlight the need for further research to clarify the enigmatic role of this class of gaba neurons. whether excitatory or inhibitory, the unique properties of pvchc inputs suggest that the reported alterations of these cells must significantly contribute to cortical circuit dysfunction in schizophrenia. in contrast to pvchcs, both pvbc and cckbc inputs are hyperpolarizing (vmr > egabaa), although egabaa is slightly, but significantly, different for pvbc versus cckbc inputs. such differences in egabaa were attributed to the activity of the voltage- and chloride - dependent chloride channel clc-2, which helps maintaining a low internal chloride concentration (thus a hyperpolarized egabaa) at inputs from pvbcs but not from cckbcs. the clc-2-dependent regulation of internal chloride at pvbc inputs is dependent on the extent of gabaar activation. these findings highlight a degree of functional diversity between bc subtypes, showing that pvbc inputs, but not cckbc inputs, possess a mechanism to prevent internal chloride accumulation during high - frequency neuronal activity such as that observed in gaba neurons participating in gamma oscillations. functional diversity between pvbcs, pvchcs and cckbcs is also suggested by recent data demonstrating that the relative levels of gad65 and gad67 proteins in nerve terminals of these cells vary significantly in a cell - type - specific manner. using immunocytochemical labeling combined with a quantitative fluorescence microscopy methodology, the colocalization of gad65 and gad67 proteins in the same terminals was assessed for pvbcs, pvchcs and cckbcs (figure 4). the latter cells were identified using an antibody that detects the cannabinoid 1 receptors expressed by inhibitory terminals. importantly, in the prefrontal cortex nearly all cannabinoid 1 receptor - expressing cells detected using this antibody are immunoreactive for cck. this assessment showed that the ratio of gad67/gad65 expression varied significantly across each type of terminal, with very high (15.42) and very low (0.18) ratios observed in terminals of pvchcs and cckbcs, respectively, and a ratio of 1.49 for pvbc terminals. these data reinforce the idea that synaptic connections from different perisomatic - targeting gaba neuron subtypes are functionally diverse. the impact of different gad67/gad65 ratio on the properties of the gaba synapses studied remains enigmatic because the differential functional roles of gad67 and gad65 are still poorly understood. in mice, gad67 deficiency produces major alterations, as gad67 animals are born with cleft palate (which is lethal) and with 90% reduction of bulk gaba concentration in brain tissue. in contrast, gad65 mice survive into adulthood, displaying 20% reduction in total brain gaba concentration together with increased susceptibility to seizures , increased anxiety , and altered fear conditioning. in synapses of gad65 mice, gabaar - mediated synaptic transmission appears normal during low - frequency stimulation but is strongly impaired at stimulation frequency of 30 hz or higher. in mice with cerebellum - specific gad67 deficiency , gabaar - mediated transmission is markedly weaker even at low stimulus frequency , suggesting a crucial role of gad67 in baseline synaptic transmission. whether the effects of gad67 deficiency on cerebellar synapses are observed in cortical synapses as well remains to be determined. importantly, gad67 deficiency markedly impairs the maturation of cortical gabaergic synaptic connections. therefore , testing the role of gad67 in synaptic transmission independent of its role in synapse development requires a paradigm in which gad67 is decreased once gaba synapse maturation has been completed. in addition, gad65 and gad67 both comprise 50% of total gad protein in mouse cortex, while gad65 comprises 70% in the rat cortex. the gad65 and gad67 proportions of the total gad protein in human cortex is unknown; however, the differences between mouse and rat strongly stress the need to study gad function in molecularly relevant systems when trying to understand their roles in human disease. gabaar - mediated inputs involved in gamma band synchronization must inhibit their postsynaptic neurons for a time compatible with the gamma oscillation period. for instance, long - lasting postsynaptic currents such as those activated by gabab receptors (which may last hundreds of milliseconds) are inconsistent with gamma synchrony, as postsynaptic neuron inhibition would be longer than the typical gamma cycle period. whereas all gabaar subtypes produce faster currents than gabab receptors, the gabaa current duration depends on the subunit composition. therefore, pvbcs may produce ipscs with faster decay, as 1-gabaars predominate at inputs from pvbcs whereas 2-gabaars predominate at both pvchc and cckbc inputs. interestingly, pvbcs, cckbcs, and pvchcs elicit in pyramidal cells uipscs with nearly identical kinetics. therefore, in addition to the gabaar subunit composition, the ipsc kinetics are determined by additional factors that may not affect recombinant gabaars. such factors include gabaar subunit phosphorylation and other posttranslational modifications , and the time course of the gaba concentration transient to which gabaars are exposed. although pvbcs and cckbcs produce ipscs with similar duration during individual gaba release events, cckbcs distinctively produce multiple asynchronous release events after single - action potentials. multiple gaba release events may prolong the postsynaptic inhibitory effect of each cckbc spike. in contrast, pvbc terminals produce a single synchronous gaba release event per presynaptic action potential. asynchronous release from cckbc terminals has been observed in multiple studies suggesting that it is a fundamental property that prolongs the inhibitory effect of cckbcs on postsynaptic neurons possibly linking their activity with synchronization at frequency bands lower than gamma. the data reviewed above suggest that, relative to cckbcs and pvchcs, pvbcs have unique properties consistent with a crucial role in the mechanisms of gamma band synchrony. however, such data do not directly assess involvement of any of these gaba neuron subtypes in the gamma oscillation mechanisms. interestingly , some electrophysiological studies more directly indicate that among perisomatic - targeting gaba neuron subtypes, pvbcs are most likely to be involved in the production of gamma oscillations. for instance, whereas both bcs and chcs are active during gamma oscillations in vivo and in vitro , the firing of cckbcs and pvchcs is more weakly coupled with the gamma oscillation cycle than pvbc firing, although cckbc and pvchc firing is strongly coupled with theta oscillations. furthermore, gamma oscillations are significantly reduced or abolished by suppressing pv cell activity with optogenetic methods that do not directly affect cckbcs or by stimulation of presynaptic opioid receptors that suppress gaba release from pvbcs but not from cckbcs or chcs. therefore, perisomatic gabaar - mediated currents from pvbcs appear to be the main source of gabaar - mediated synchronization in the gamma frequency band. essential for modeling the circuit mechanisms of gamma synchronization is to understand how pvbcs are normally recruited to fire rhythmically at gamma frequency. recruitment of pvbc firing depends on activation of not only excitatory but also inhibitory synaptic inputs onto them since pvbcs target other gaba neurons, including nearby pvbcs , and are also inhibited by inputs from different classes of gaba neurons, including the cckbcs. gamma oscillations are successfully generated in computational model networks that rely on reciprocal inhibition between gaba neurons and are thus called ing, for interneuron network gamma. in ing models, gaba neurons are recruited by a strong tonic excitation that drives them to fire at a frequency above gamma, and the reciprocal inhibition synchronizes gaba neuron firing at a frequency inversely related to the ipsc duration, falling within gamma range for durations typical of 1-gabaar - mediated ipscs. whereas gamma rhythms possibly induced by ing - like mechanisms have been observed experimentally, the actual source of the strong tonic excitation onto gaba neurons required by ing models is unclear. metabotropic glutamate receptors or kainate receptors could provide such a tonic drive, although this possibility is supported, only indirectly, by findings obtained with ampa- and nmda - mediated synaptic transmission blocked. a recent study employing genetically engineered mice with a deletion of gabaar expression in pvbcs directly tested whether inhibition onto pvbcs is necessary to generate gamma oscillations, as predicted by the ing models. in such mice, gabaar - mediated ipscs were abolished exclusively in pvbcs and theta oscillations and their coupling with gamma oscillations were severely disrupted. however, in such mice hippocampal gamma oscillations in vivo were intact as compared with wild - type mice. these data argue against the ing model for gamma band synchrony and suggest that inhibition onto pvbcs, potentially from cckbcs , is crucial for coupling theta and gamma oscillations. such theta - gamma coupling is thought to be important for cognitive function. as the role of ing mechanisms in gamma oscillation production continues to be tested , some studies favor an alternative model, known as pyramidal interneuron network gamma (ping), which depends on recurrent excitatory - inhibitory synaptic interactions. in ping, pvbcs are recruited by phasic glutamate - mediated inputs from the pyramidal cells, and the pvbcs provide strong feedback inhibition that synchronizes pyramidal cell firing. the ping model predicts that during the gamma oscillation cycle pvbcs fire after the pyramidal neurons, with timing consistent with monosynaptic recruitment by pyramidal cells. the spike timing of pyramidal cells and putative bcs during gamma oscillations in awake behaving animals is actually consistent with the ping model, as bcs fire 2 - 3 ms later than pyramidal neurons. similar findings were obtained for pyramidal and pvbc spikes during gamma oscillations in hippocampal and neocortical brain slices. ping models also predict the presence of trains of gamma frequency ipscs in pyramidal neurons and trains of gamma frequency epscs in pvbcs. evidence consistent with ipsc trains in pyramidal cells was obtained for gamma oscillations in vivo and in vitro. in addition, during gamma oscillations in vitro, pvbcs display rhythmic epscs highly synchronized with the gamma rhythm. interestingly, optogenetics experiments show that driving pv neurons by nonrhythmic excitatory inputs is sufficient to generate gamma synchrony via feedback inhibition, a finding also consistent with the ping model of gamma. ping mechanisms rely on recruitment of pvbcs by phasic excitatory input; therefore, the properties of glutamate synapses onto pv neurons are extremely relevant for models of gamma oscillations. interestingly, schizophrenia has been hypothesized to be associated with a deficit of glutamate transmission, more specifically with hypofunction of nmda receptors, particularly in gaba neurons. moreover, some studies have suggested that nmda hypofunction could especially affect pv cells. therefore, an important question is the following: what are the subtypes of glutamate receptors that mediate synaptic activation of pv gaba neurons? the answer to this question is relevant in the context of alterations of gamma synchrony in schizophrenia, because if nmda receptors are important to recruit pv neurons in a ping mechanism of gamma, then nmda hypofunction could be linked to deficits of gamma synchrony in schizophrenia. data from recent studies showed that systemic administration of nmda receptor antagonists increase the firing rate of putative pyramidal neurons and decrease the firing of putative inhibitory cells in the pfc in vivo, suggesting that nmda receptors may be crucial to recruit inhibition. an important question not directly addressed by such studies is whether the inhibitory neurons dependent on nmda receptors belong to the pv - positive class of gaba neurons. whereas several studies demonstrated that synaptic excitation of pv neurons is relatively nmda receptor independent, until recently no studies directly compared the importance of nmda receptors in synaptically evoked recruitment of pvbcs versus pyramidal neurons in neocortical circuits. recent data from recordings in mouse pfc show that, compared with pyramidal cells, glutamate synapses onto pvbcs have epscs with faster decay and weaker nmda receptor contribution, supporting the idea that the rapid activation of pvbcs is largely dependent on fast ampa receptor - mediated excitation. moreover, in a computational model producing gamma oscillations via ping mechanisms, fast ampa - mediated excitation of pvbcs was critical for gamma band synchronization because the slower decay time course of nmda - mediated epscs disrupted gamma band synchrony. some studies indeed showed that gamma oscillations are not affected or are enhanced by nmda receptor antagonists, whereas ampa receptor antagonism completely abolished them. similarly, in mice with ampa receptor deletion genetically engineered to occur exclusively in pv - positive neurons, gamma oscillations are strongly reduced; however, nmda receptor deletion selectively in pv - positive cells does not decrease and in fact increases gamma oscillation power. the of recent studies therefore suggest that, in mature cortical circuits, nmda receptors only play a minor role in synaptically evoked excitation of pv - positive neurons and therefore on gamma oscillations produced via ping mechanisms. if indeed pv neuron excitation is normally weakly dependent on nmda receptors, then such data suggest that excitatory synapses onto pv cells are an unlikely target of nmda receptor hypofunction mechanisms in schizophrenia. importantly, although in most cases gamma synchrony is unaffected by nmda receptor blockade, the effects of nmda receptor antagonists on gamma oscillations may vary with cortical region or layer, in some cases ketamine producing a decrease, in others producing an increase in gamma power. in addition, some data show that whereas mature pvbcs display a relatively small nmda receptor mediated component in synaptic responses , such nmda component is substantially more prominent in immature pvbcs. such findings suggest the interesting possibility that alterations of nmda receptor - signaling during early brain development could alter pvbc function in ways that persist into adulthood, thus changing the role of pvbcs in mature local circuits. interestingly, a recent study showed that a genetically engineered deletion of nmda receptors from pv - positive cells does not have significant effects if the deletion is produced in the brain of adult mice. however, a similar deletion produced during early brain development caused behavioral alterations in adult mice, some of which resemble behavioral dysfunction in patients with schizophrenia. schizophrenia is hypothesized to be a neurodevelopmental disorder based on data linking the disease with adverse events during pre- and perinatal periods and the presence of cognitive and behavioral deficits in childhood many years prior to the onset of psychosis during late adolescence and early adulthood. adolescence, the developmental transition from parent - dependent childhood to independent adulthood, is associated with significant changes in behavior and with marked improvements in cognitive control. moreover, gamma band synchrony emerges during childhood and continues to mature until early adulthood. the postnatal developmental trajectory of gaba - synaptic function may therefore suggest critical periods of vulnerability during which the mechanisms producing neural synchrony could become dysfunctional in schizophrenia. in what follows we review developmental studies of the functional properties of pvbcs and their synaptic connections in rodents and of gaba - related gene products studied in the human and nonhuman primate brain. mature pv neurons have a unique fast - spiking (fs) firing pattern (figure 5), which includes very narrow action potentials and high frequency firing without the spike - frequency adaptation typically observed in pyramidal cells and other gaba neuron subtypes. although the nonadapting properties of fs cells are revealed with artificial stimuli (rectangular currents steps lasting several hundred ms), they correlate strongly with the particular ability of fs cells to respond to oscillatory inputs at gamma frequency (figure 5), which is likely due to the gamma frequency resonance of the fs cell membrane. immature fs neurons, in contrast, have significantly slower action potentials and stronger spike - frequency adaptation, fail to respond efficiently to gamma frequency oscillatory inputs, and show much slower conduction velocity of action potentials along their axon. in rodent hippocampus, as well as in auditory, somatosensory, and prefrontal cortices, maturation of fs neuron electrical properties is complete by postnatal day 25 (p25) after which fs neurons display adult - like electrical properties. functional properties of the inhibitory connections onto excitatory neurons also differ markedly between developing and mature pvbcs: unitary ipscs (uipscs), from immature pvbcs, are weaker and have slower decay time than uipscs produced by mature pvbcs. such acceleration of uipsc decay is explained by an increase in the contribution of 1-gabaars with maturation. a gabaar subunit switch may also contribute to the developmental acceleration of the uipsp decay although developmental changes in the pyramidal cell membrane time constant contribute as well. mature fsbcs produce highly synchronous gaba release, in contrast to asynchronous release from mature cckbcs. however, gaba release is less synchronous and less reliable in synapses from immature fsbcs. postnatal maturation of the fsbc connections takes place relatively rapidly, as uipscs acquire mature properties by p28. inhibition onto fs cells also undergoes significant developmental changes. for example, uipscs at fsbc - to - fsbc connections mature within 3 - 4 weeks postnatally , whereas miniature ipscs (mipscs, which represent gaba release at single synapses) recorded from fsbcs acquire adult - like properties by p25. unitary epsps (uepsps) at immature pyramidal cell - to - fsbc connections have slow decay time, which accelerates markedly during development, reaching adult - like values by p22. similarly, miniature epscs (mepscs) recorded from immature fsbcs are slow and show a developmental acceleration of their decay to reach very fast mepsc decay values in mature fs cells. fast - decaying epscs and epsps in mature fsbcs are largely mediated by ampa receptors , suggesting that the rapid epsc decay in fsbcs is due, at least in part, to a weak contribution from the slow - decaying nmda currents. actually, the developmental acceleration of epsc decay is accompanied by a marked decrease in the contribution of nmda currents which is still ongoing at p40 to p96 , which in rodents corresponds to the transition from adolescence to adulthood. in contrast to pfc, in hippocampus, auditory, and somatosensory cortices, nmda receptor contribution in fsbcs decreases prior to adolescence. although the mechanisms controlling postnatal development of pvbc firing properties and their synaptic inputs and outputs are important candidates as the substrate of alterations in schizophrenia, they are still poorly understood. neuregulin-1 is a trophic factor crucial for brain development that is encoded by a schizophrenia susceptibility gene and is highly expressed during late developmental periods and in adulthood. among various neuregulin-1 receptors, the erbb4 receptor, whose gene also confers schizophrenia susceptibility , is enriched in gaba neurons, particularly in pv - positive cells where it facilitates gaba release, possibly mediating neuregulin-1 enhancement of gamma oscillations. neuregulin-1 signaling appears to regulate early development of gaba synapses and, via erbb4 receptors, may control development of pv neuron synapses. interestingly, erbb4-mediated neuregulin-1 effects are crucial for development of excitatory synapses onto pvbcs. in parallel to the maturation of their firing pattern and synaptic connections, pvbcs undergo significant developmental morphological changes. for instance, the total length of the dendritic and axonal trees of pvbcs increases significantly from p6 to p25 , the number of axonal branch points increasing five times during this developmental period. the number of postsynaptic neurons innervated by individual pvbcs also increases markedly with postnatal development , ing in a higher functional connectivity between mature pvbcs and excitatory neurons. a crucial factor regulating development of innervation patterns by pvbcs for instance, gad67 knockdown in single pvbcs dramatically decreases formation of axon branches and synapses, as well as the number of postsynaptic neurons innervated by each pvbc. such effects of gad67 knock - down are observed in organotypic cell cultures and in the primary visual cortex in vivo with gad67 knock - down induced at p13 and the patterns of innervation examined at p20 or p32. the role of gad67-mediated gaba synthesis in formation and/or stability of pvbc synapses may involve neuroligin - neurexin interactions and modulation of gaba receptor trafficking. because detailed molecular and biophysical analysis of developmental changes in gaba neuron and gaba synapse function is feasible only using animal models, especially rodents, an important question is how developmental time scales translate from animal to human brain. proper translation would require understanding if similar developmental stages are found in rodent and human brains, whether such developmental stages involve similar processes and underlying mechanisms, and whether developmental periods cover similar fractions of the total lifespan. some developmental changes, for instance, excitatory synapse pruning, have similar proportional duration, with only the extent of synaptic pruning differing across mammalian species. also, functional maturation of glutamate synapses onto pyramidal cells occurs prior to adolescence in nonhuman primates, and in rodents. whereas some unique studies have assessed functional properties of pvbcs and pvchcs in the cortex of adult humans and nonhuman primates , we lack information on the developmental trajectory of synaptic inhibition from primate pv neurons. interestingly, functional properties of yet unidentified gaba synapses onto primate pyramidal neurons change during development through adolescence in a manner consistent with changes in the expression of gene products involved in gaba - mediated transmission. specifically, the decay time of gabaar - mediated synaptic potentials accelerates during adolescence in parallel to changes in the protein and/or mrna levels for 1 and 2 gabaar subunits that would predict such acceleration. the synaptic connections from pvchcs onto the pyramidal cell ais form vertical arrays of multiple synaptic boutons that are usually easy to distinguish and typically called cartridges. developmental properties of inputs from pvchcs onto the ais can be studied using immunocytochemistry to detect biochemical markers that are concentrated at the cartridges in the ais. in monkey pfc, the density of chandelier neuron axon cartridges immunoreactive for either pv or gat1 changes markedly during postnatal development. although the precise time course differs for the two markers, the density of labeled cartridges is low in the newborn, increases to reach a peak prior to the onset of puberty, and then declines markedly to adult levels. because cartridges are readily visualized with the golgi technique over this same time period, the changes in pv- and gat1-immunoreactive cartridges may reflect developmental shifts in the concentration of these proteins, rather than in the number of axon terminals, but this remains to be experimentally assessed. the detectability of gabaa 2 subunits in ais is very high in the early postnatal period and then steadily declines through adolescence into adulthood. immunoreactivity for ankyrin - g, iv spectrin, and gephyrin (a scaffolding protein that regulates the clustering of gabaars containing 2 subunits at ais) also exhibit substantial changes during postnatal development. the densities of ankyrin - g and iv spectrin immunoreactive aiss are greatest at birth and then sharply decline to reach relatively stable values by one year of age. in contrast, the relative density of gephyrin - immunoreactive ais did not appear to change through the two postnatal years but then sharply decline through adolescence and into adulthood. the high density of ais with detectable levels of ankyrin - g immunoreactivity in the first three postnatal months may reflect the recruitment to this location of a portion of the large number of gaba synapses that are formed in the monkey dlpfc during this developmental epoch. binding to ankyrin - g is also essential for the localization of many other membrane proteins to the ais, including the voltage - gated na channels that are required for action potential generation. thus, the high levels of ankyrin - g immunoreactivity may also indicate an increased capacity of pyramidal neurons for repetitive firing that parallels their increase in excitatory inputs during early postnatal development. the parallel relative densities of ankyrin - g - ir and iv spectrin - sd - ir ais likely reflect that ankyrin - g is required for the recruitment of iv spectrin to ais. although iv spectrin is not essential for the formation of the ais, it does appear necessary for maintenance of membrane structure and molecular organization , and thus the stability , of the ais. given the general role of spectrins in maintaining membrane integrity and elasticity, high levels of iv spectrin during early postnatal development might insure the stability of ais structure while pfc thickness is increasing. interestingly, in human pfc the levels of gad67 mrna increase progressively during prenatal and postnatal development through childhood until around the peripubertal period, followed by a plateau or mild decline during aging. a similar pattern was reported for gad67 mrna expression during mouse and monkey cortical development, suggesting a highly conserved developmental trajectory of gad67 expression across mammals. studying protein expression by immunoblotting, a recent study found that gad67 protein levels did not change across the lifespan in human visual cortex. in contrast, gad65 showed a progressive 60% increase until teenage years and young adulthood, followed by slight decline in older adults. two other presynaptic proteins involved in gaba transmission, the cannabinoid receptor 1 and vgat, showed higher levels in infants and young children, which declined to adult - like levels in preteenage years. the levels of pv mrna increase markedly in postnatal human pfc, from very low perinatal levels until adult - like levels are reached by 25 years of age, an early developmental trajectory which is similar to that reported for pv mrna and protein in rodent neocortex. interestingly, a comparative analysis using immunolocalization of the chloride transporters nkcc1 and kcc2 revealed a very similar developmental trajectory in rat and human cortex. nkcc1 levels peaking during perinatal development and decaying rapidly thereafter reach adult - like levels during childhood; conversely, kcc2 is undetectable perinatally and increases until reaching adult levels during childhood. since the nkcc1/kcc2 activity ratio determines whether gabaar - mediated ipscs depolarize or hyperpolarize the postsynaptic membrane, these data suggest that the very early developmental switch from excitatory to inhibitory effects at most gaba synapses is highly conserved between rodent and human neocortex. analysis of gabaar subunit proteins during postnatal development in human visual cortex showed that 1 gabaar subunit levels increase from < 1 years until reaching adult levels at 13.5 years of age, whereas 2 gabaar subunits decreased significantly with age to reach adult levels by ~10 years and 3 gabaar subunit levels do not change significantly with age. consequently, the 1/2 subunit protein ratio increased markedly with development attaining adult - like ratios at 4.5 years of age. remarkably, very similar developmental trajectories were found for the levels of gabaar subunit mrnas in postmortem samples of human pfc. for example, 1 gabaar subunit mrna levels are very low perinatally and increase markedly until toddler ages, thereafter remaining consistently high through to adulthood. in contrast, 2 subunit mrna increased during the first postnatal months, decreasing subsequently until reaching mature levels at teenage years or young adulthood. the mrnas for 4 and 5 gabaa subunits in human pfc showed a developmental pattern similar to that of 2 mrna, whereas 3 subunit mrna did not change significantly with age. postnatal expression of mrna for and gabaar subunits similarly shows significant age - dependent changes, with 1 subunits showing a very early developmental decrease between neonate and infant ages, remaining constant thereafter, and 2 increasing somewhat later, between toddler and teenage years. on the other hand, 1 and 3 subunit mrna levels decrease with age during childhood and teenage the developmental trajectories reviewed above suggest that similar processes underlie developmental changes in gabaar - mediated synapses across various areas of human and rodent cortex, although further studies are necessary to properly compare developmental trajectories across species. a major difference between species is that the maturation of gaba - related markers in humans involves progressive change over one to two decades, whereas in rodents gaba synapse maturation appears to be complete within 3 - 4 postnatal weeks. such difference suggests that the absolute time window during which activity and experience may influence gaba synapse development is markedly expanded in primate versus rodent brains. the prolonged period that may be necessary for the normal developmental tuning of the more complex circuitry of the primate cortex probably also prolongs the time window during which environmental factors can produce subtle developmental alterations that may contribute to the pathophysiology of schizophrenia. the hypothesis that a deficit in gabaar - mediated transmission underlies cortical circuit dysfunction in schizophrenia is supported by convergent lines of evidence from post - mortem studies of the brain of subjects with schizophrenia. furthermore, such hypothesis is strengthened by the fact that one of the most reliably replicated findings in schizophrenia research is the decrease in gad67 mrna (for review, see). interestingly, equivalent measurements of gad65 levels thus far failed to reveal alterations, suggesting that the role of gad65 in gaba - mediated transmission maybe intact in schizophrenia. a remarkable recent study found that in schizophrenia several gaba - related transcripts, including those for gad67, pv, gat1, somatostatin, and the gabaar subunits 1 and , show decreased levels in dorsolateral pfc as well as in the anterior cingulate, primary motor, and primary visual cortices. such conserved regional pattern of gaba alterations suggests that dysfunctional gaba neurotransmission contributes to multiple clinical features of schizophrenia including perceptual and motor deficits that could contribute to impaired cognitive function. the disruption of pv expression across cortical areas confirms the previous findings and, moreover, suggests that alterations of pv - positive cells are central to the schizophrenia disease process, although the consequences of such decrease in pv are not completely understood. pv is a ca buffer that is present in nerve terminals of pv - positive neurons (figure 1). due to its slow kinetics of ca binding, pv is unable to bind intracellular ca before ca influx activates the ca sensor that triggers gaba release, because in pv - positive terminals ca influx is tightly coupled with gaba release. interestingly, gaba release by single stimuli does not differ between pv - deficient and wild - type mice, but pv deficiency facilitates repetitive gaba release. in synapses from pv - deficient mice, the amplitude of intracellular ca transients in nerve terminals is not affected, but their decay is slowed, indicating that pv normally accelerates such decay. therefore, one possibility is that the decrease of pv in schizophrenia, instead of contributing to deficits, is a compensatory response to enhance gaba release in the face of decreased gaba synthesis. alternatively, reduced pv levels may produce synaptic dysfunction via loss of some asynchronous release normally produced when caunbinds from pv, well after the presynaptic action potential ended. a pathological loss of asynchronous gaba release by decreased pv levels, however, requires the existence of asynchronous release when pv is intact, a feature that is not observed in cortical pv - positive cells , although it is found in pv - positive cerebellar interneurons. whereas schizophrenia may be associated with an increased density of 2-gabaars at the ais synapses from pvchcs, one study failed to detect significant changes in total tissue levels of 2-gabaar mrna. one possibility is that changes in 2-gabaars are synapse- or layer - specific and perhaps found exclusively at ais synapses in superficial cortical layers, as initially reported. consistent with this interpretation, laminar analysis of mrna levels for gabaar subunits in the cortex of subjects with schizophrenia revealed significantly increased levels of mrna for 2 gabaar subunits exclusively in layer 2. moreover, the same study revealed lower levels of 1 gabaar subunit mrna in layers 3 and 4, which is consistent with a decrease in total tissue levels of 1 gabaar subunit mrna observed using quantitative pcr. given that 1 subunit - containing gabaars constitute about 60% of the total gabaars in adult brain, it is possible that 1 subunit mrna is significantly more abundant than that for 2 subunits, thus increasing the chance of detecting changes of total tissue 1 mrna levels in schizophrenia. importantly, a decrease in 1 subunits in schizophrenia is consistent with weaker synaptic transmission from pvbcs, since 1 subunit - containing gabaars are predominant at synapses from mature pvbcs. in addition to gabaar levels, the strength of the postsynaptic response to gaba depends on the driving force for the gabaar current which is determined by its reversal potential egabaa. as egabaa depends on chloride extrusion by kcc2 and chloride uptake by nkcc1, a recent study examined mrna expression for both chloride transporters in the cortex of subjects with schizophrenia. interestingly, kcc2 and nkcc1 transcript levels were not altered in subjects with schizophrenia; however, transcripts for two kinases (oxsr1 and wnk3) that strongly regulate kcc2 and nkcc1 activity in opposite directions, are overexpressed in schizophrenia. if increased levels of oxsr1 and wnk3 mrna actually represent increased kinase activity, then the chloride gradient across the postsynaptic membrane may be decreased, ing in an egabaa significantly more depolarized than normal. since normally egabaa varies with cell type and subcellular compartment, understanding the consequences of alterations in chloride transport requires a detailed quantitative analysis of protein localization and activity, a challenging task in this case, given that postmortem interval effects alter the integrity of some of these proteins. direct demonstration that gaba - mediated synaptic inhibition is decreased in the cortex of subjects with schizophrenia is challenging. interestingly, magnetic resonance spectroscopy (mrs) was recently applied to noninvasively measured gaba concentration in human neocortex and determined whether a decrease of gaba is observed in schizophrenia. mrs does not distinguish extracellular gaba from transmitter stored in particular cellular compartments or cell types and also lacks adequate temporal resolution but nevertheless reveals activity - dependent changes in gaba levels. for example, acute psychological stress which elevates subjective anxiety produces a short - term decrease in gaba concentration in human dorsolateral pfc that can be detected with mrs. combining mrs and eeg in the same subjects, the relations between brain gaba content and oscillatory neural activity in schizophrenia may be explored. interestingly, in normal human subjects, gaba concentration measured in visual cortex with mrs was positively correlated with the strength of gamma oscillations induced by visual stimulation. moreover, interindividual variation in gaba concentration determined by mrs in visual cortex was correlated with variability of performance in a visual stimulus orientation discrimination task that induces gamma oscillations. measurements of tissue gaba concentration with mrs may help in clarifying the relations between gad67 levels, gamma oscillations, and cognitive performance in schizophrenia. one such study did not detect differences in gaba concentration in the anterior cingulate cortex of schizophrenia versus control subjects, whereas gaba concentration was apparently decreased by antipsychotic medications. another mrs study reported reduced gaba concentration in basal ganglia but not frontal cortex of schizophrenia patients. in patients with relatively low antipsychotic exposure , mrs revealed a significant reduction of gaba concentration in visual cortex that did not covary with medication dosage but was correlated with behavioral abnormalities in a visual surround - suppression task thought to depend on gaba - mediated inhibition. moreover, a longitudinal study of early - stage first - episode schizophrenia patients showed that 6 months of treatment with atypical antipsychotics did not change gaba concentrations measured with mrs in frontal and parietal lobe cortices nor in basal ganglia. in contrast, mrs revealed elevated gaba concentration in anterior cingulate and parietal cortex of subjects with chronically treated schizophrenia compared to control subjects. the mrs findings suggesting that antipsychotics may change brain gaba concentration highlight the importance of addressing the effects of confounding factors such as medications. significantly, both postmortem studies in humans and experimental studies in animals have failed to show an effect of antipsychotic medications on gad67 mrna levels. the data from mrs studies therefore underscore the importance of combining neurochemical, electrophysiological, and behavioral assessment, given the large interindividual variability in bulk gaba concentration, gamma activity levels, and behavioral performance. instead of or in addition to medication effects, the large variability in cortical gaba content measured with mrs in human cortex may be explained by the effects of genetic variants in the gad1 gene that may differentially confer risk of schizophrenia. the findings reviewed here suggest that alterations of gaba transmission produce cognitive deficits in schizophrenia by altering the circuit mechanisms of gamma oscillations. these observations suggest a molecular and cellular basis for the development of new therapeutic interventions. importantly, the proposal that gaba alterations are linked to altered gamma oscillations and cognition is supported, at least in part, by animal model studies showing that producing a functional loss of gaba - mediated inhibition diminishes gamma oscillations and impairs cognitive function. whereas work in animal models is essential , the difficulty of capturing in animals the complexity of behavioral alterations in a uniquely human disorder may explain the relative lack of success in developing drugs to treat schizophrenia compared with other disease areas. interpretation of studies in human subjects based on comparisons between healthy controls and patients is complicated as well, given that schizophrenia versus control differences may actually reflect pathogenesis but also could represent compensatory changes or effects of confounding factors. for example, the effects of producing a transient deficit in gabaar - mediated signaling were tested recently in human subjects using iomazenil, a compound that binds to the benzodiazepine site of gabaars and negatively modulates the effects of gaba (i.e., an inverse agonist). consistent with dysfunctional gabaar signaling in schizophrenia, iomazenil produced perceptual deficits and psychotic symptoms in schizophrenia patients at doses that did not affect healthy control subjects. however, the schizophrenia patients in such study chronically received antipsychotics and anxiolytics, raising the question of whether an interaction between acute iomazenil and chronic medications influenced such findings. preliminary tests of the idea that enhancing gabaar signaling improves behavioral and electrophysiological measures in subjects with schizophrenia were conducted in two recent studies evaluating the effects of mk-0777, an 2/3 gabaar - preferring positive allosteric modulator. in one study, randomized administration of mk-0777 or placebo in double - blind fashion improved performance of schizophrenia patients in a cognitive control task, simultaneously increasing gamma oscillation power in frontal cortex. in contrast, the second study did not find significant effects of mk-0777 compared with placebo in the performance of patients in a battery of tests designed to assess cognitive function in schizophrenia. the inconsistent beneficial effects of mk-0777 administration might be explained by the fact that mk-0777 is a partial agonist at 2/3 subunit - containing gabaars with only about 1020% potency compared to a full agonist. thus, one possibility is that more potent 2/3 benzodiazepine site agonists need to be employed. such drugs should also be more selective because, compared with placebo, mk-0777 had a tendency to produce sedation and somnolence , which could mask improvements in cognitive performance. importantly, the 2/3 gabaar modulator mk-0777 was selected based on the compelling rationale that inputs from pvchcs onto the ais show important alterations in schizophrenia and that, depending on cortical layer, such inputs involve 2-gabaars or 3-gabaars. however, whether or not pvchcs play a role in production of gamma band synchrony remains unclear, and so it is possible that pvchc alterations in schizophrenia produce cognitive deficits unrelated to dysfunctional gamma band synchrony. therefore, further information from both basic and clinical research studies is necessary to further assess the effectiveness of 2/3 gabaar modulators for treatment of gamma synchrony - related cognitive deficits in schizophrenia. indeed, basic research studies continue to provide insight into the role of specific subtypes of gaba neurons on inhibition - mediated cortical network oscillations, and molecular pharmacology studies are identifying novel compounds acting at different sites within the gabaar receptor complex. importantly, a potential role of gaba - mediated neural synchrony in cortical circuits is to enable spike - timing - dependent plasticity, indirectly modifying the strength and stability of excitatory synaptic connections. whether spike - timing - dependent plasticity at glutamate synapses is impaired in schizophrenia is not yet clear, but it is possible that the decrease of dendritic spine density in pyramidal neurons in schizophrenia is due to glutamate synapse loss produced by altered plasticity mechanisms. if neural synchrony - dependent glutamate synaptic plasticity is dysfunctional in schizophrenia, then cognitive enhancement behavioral therapies that involve learning paradigms may help in preventing or reversing the consequences of altered circuitry on the induction of synaptic plasticity. interestingly, cognitive enhancement behavioral therapy was recently shown to improve cognition and prevent gray matter loss in schizophrenia. potentially, combining cognitive therapies with pharmacological treatment that boosts otherwise weakened neural synchrony may constitute an effective treatment intervention in schizophrenia, as for other psychiatric disorders.

schizophrenia is a brain disorder associated with cognitive deficits that severely affect the patients' capacity for daily functioning. whereas our understanding of its pathophysiology is limited, postmortem studies suggest that schizophrenia is associated with deficits of gaba - mediated synaptic transmission. a major role of gaba - mediated transmission may be producing synchronized network oscillations which are currently hypothesized to be essential for normal cognitive function. therefore, cognitive deficits in schizophrenia may from a gaba synapse dysfunction that disturbs neural synchrony. here, we highlight recent studies further suggesting alterations of gaba transmission and network oscillations in schizophrenia. we also review current models for the mechanisms of gaba - mediated synchronization of neural activity, focusing on parvalbumin - positive gaba neurons, which are altered in schizophrenia and whose function has been strongly linked to the production of neural synchrony. alterations of gaba signaling that impair gamma oscillations and, as a , cognitive function suggest paths for novel therapeutic interventions.

records were examined from all hospitals that treated patients with influenza (h5n1) virus in northwest frontier province during 2007. data were rendered anonymous and entered into a secure database with predetermined clinical and epidemiologic fields. cases matching predefined criteria (table 1) were classified as laboratory confirmed, likely, or possible. we slightly modified who criteria to resemble criteria that clinicians might adopt during an actual outbreak, especially in a resource - poor setting. we identified 20 cases4 laboratory confirmed, 7 likely, and 9 possible ing in a ratio of 4 likely / possible cases for each laboratory - confirmed case. median age was 29 years (range 760 years) for all patients and 30 years (range 2335 years) for confirmed case - patients; 16 (80%) patients were male. the infecting exposure could not be established for all patients because multiple exposures, human and avian, were recorded for some. of the 4 patients with laboratory - confirmed cases, 3 were treated with oseltamivir (2 of whom survived), and 1 had asymptomatic disease and received no antiviral treatment. signs and symptoms were mainly those of a febrile influenza - like illness (table 2), although 1 patient with a laboratory - confirmed case was asymptomatic (microneutralization titer 320, western blot positive, throat swab positive for h5 by reverse transcription pcr); this case - patient was also described in a previous epidemiologic investigation. the index case - patient (patient 1) had culled influenza (h5n1) virus infected poultry. after becoming febrile (38c) while in abbottabad, he traveled by public transportation to his family home in peshawar. his illness progressed and on november 5, 2007, he was admitted to khyber teaching hospital, where the diagnosis of influenza (h5n1) infection was made. infection appeared to spread initially from household family contacts (patients 26) to medical staff (patient 7, who had positive pcr but negative microneutralization test ) and to a frequent visitor to the intensive care unit (patient 8). path of infection of influenza (h5n1), pakistan, 2007. during october 2230, patient 1 worked culling infected chickens; on november 2, he moved home and had contact with 4 brothers (patients 25) and possibly a cousin (patient 6). he was hospitalized on november 5 and transferred to an intensive care unit the next day. his cousin cared for him and became patient 6; his attending doctor became patient 7. on november 23, patient 3 was hospitalized and on november 28 was transferred to an intensive care unit; during this time, patient 8 frequently visited his wife in the same intensive care unit. as previously noted, the extended period from the time persons were exposed to the index case - patient, during which family members became ill, points to human - to - human - to - human transmission; patient 2 probably accounted for intermediary or second - generation infection. the chain of infection illustrated in figure 2 suggests that further human - to - human - to - human transmission might have occurred and suggests nosocomial transmission. of note, patient 6 (a cousin of the index case - patient) had a microneutralization titer of 80 but a negative western blot . although 4 contacts of patient 6 exhibited no signs or symptoms of influenza, they did have positive h5 microneutralization titers ranging from 80 to 160. no evidence epidemiologically links the remaining 12 patients to the 8 patients in the cluster; each of the 12 either had direct contact with influenza (h5n1) virus infected poultry or was near healthy or diseased poultry before symptom onset. three patients worked on poultry farms: 1 had taken a sample from an influenza (h5n1) virus infected chicken, 1 was directly involved in culling, and 1 was indirectly exposed to live poultry. eight patients had negative test for influenza (h5n1) virus, and 3 had positive from the national institute of health islamabad but negative confirmatory - testing from who; 1 patient died before samples could be taken. different laboratories reported conflicting with respect to confirmation of infection, possibly because of the difficulties of complying with specimen - handling requirements in resource - poor settings. the preponderance of male patients is probably explained by sociocultural factors; the index case - patient was a poultry culler, a male - dominated task, and shared accommodation with male family members. the human - to - human transmission from the index case - patient to at least some household contacts seems clear, and the extended period over which these contacts became ill supports subsequent human - to - human transmission. figure 2 supports the that patient 2 initiated a chain of infection in which further human - to - human transmission to patients 7 and 8 occurred. possible nosocomial transmission is of concern because full implementation of isolation procedures in resource - poor settings may be problematic. although virologically supported probable human - to - human transmission of influenza (h5n1) virus has been documented, it has been thought to occur only with prolonged and close contact. household clustering and the difficulty of establishing exact virus exposures have encumbered efforts to investigate possible human - to - human transmission. modeling has suggested human - to - human transmission in indonesia, but the utility of statistical modeling unsupported by field data has been questioned. although the index case - patient traveled by public transportation from abbottabad, where he acquired his infection, no infections were reported for anyone other than household contacts, who were all related and exposed at his family home at peshawar. in contrast, patients 2 and 6 might have spread infection through less intimate contact, which raises 2 questions. might some persons shed virus more efficiently than others, possibly in greater quantity? and what role might host factors play in susceptibility to influenza (h5n1) virus infection and disease? a degree of virus adaptation to humans might also have occurred, although absence of sustained community transmission argues against this possibility. of concern is the 4:1 ratio of likely / possible to laboratory - confirmed cases, suggesting that official tallies understate true incidence of infection. factors that may contribute to undercounting are the difficulty of obtaining virologic confirmation or of storing and transporting samples in resource - poor settings and reluctance by relatives to consent to autopsy. another reason to believe that less fulminant cases may go unreported is the occurrence in pakistan, and elsewhere, of clinically mild and asymptomatic cases, indicating that influenza (h5n1) virus may cause a spectrum of illness. the demonstration during the 1997 hong kong outbreak of influenza (h5n1) with seroconversion in apparently asymptomatic health care workers and social contacts suggests human - to - human transmission, although in hanoi, no transmission to health care workers was detected. also contributing to underreporting are the predominant clinical signs of undifferentiated influenza - like illness observed in pakistan and elsewhere, which, unless clinical deterioration occurred, would be unremarkable in many tropical settings. although the survival rate was greater for patients who received oseltamivir, the small number of patients and the inclusion of those with mild and asymptomatic illness prevent meaningful statistical comparison. several features of the outbreak are unusual or give cause for concern: human - to - human - to - human transmission, possible nosocomial transmission, occurrence of mild and asymptomatic cases, and difficulties of establishing laboratory confirmation of likely and possible cases (which also prevented genotypic matching of specimens from primary and putative secondary cases). taken together, these features suggest that current surveillance might undercount the extent of human infection with influenza (h5n1) virus and that human - to - human transmission might possibly be associated with less severe disease.

human infection with avian influenza (h5n1) virus raises concern for the possibility of a pandemic. we report 20 cases, which ranged from asymptomatic to fatal, in pakistan in 2007. these cases indicate human - to - human - to - human transmission of this virus, and the number of cases may be higher than realized.

wheelchairs with reclining back supports are often used for individuals with leg and trunk disorders, including those with post - stroke hemiplegia and spinal cord injuries. individuals who have difficulty sitting in the hospital can sometimes be more easily transported sometime in wheelchairs with reclining back support. in a previous study of reclining wheelchairs, individuals with flaccid hemiplegia were often found to often slide forward when returning to a seated position from a reclined position1. many wheelchair users who need reclining back support can not correct this slouching posture unassisted. this leads to a sacral sitting posture which in increased shear force on the sacrum, predisposing the individual to a sacral decubitus ulcer2. to address these problems, reclining wheelchairs with tilting seats jan et al.3 suggested that wheelchairs that can tilt and recline can enhance skin perfusion over ischial tuberosities without reducing sacral skin perfusion when moving the person from an upright to a tilted and reclined position. however, wheelchairs with tilting seats remain uncommon. therefore, it is important to evaluate the reduction in the shear force acting on the buttocks when such a wheelchair s back support is reclined. in our previous research4,5,6, we investigated the functions that reclining wheelchairs should possess to reduce the shear force acting on the buttocks when reclining the back support. the obtained suggested that reclining wheelchairs should be adjustable with the rotational axis position of the back support closer to the hip joint. carlson and payette7 described other techniques for minimizing friction and shear forces in wheelchair seating through orientation of the seat surface, positioning of the foot supports, and the use of low - friction seat cover materials. when patients are transported in wheelchairs with a reclining back support, their lower legs are elevated by leg supports. elevated leg support has been described as being important for easy patient transfer and wheelchair stability8,9,10. elevated leg supports are generally used to augment venous circulation and reduce dependent edema, or to fix the knee in extension because of orthopedic deformity, surgical immobilization, or severe hypertonicity in the extensors11. however, the influence of any of these factors on the shear force acting on the buttocks has not yet investigated. the rationale behind footplate adjustment usually appears to be empirically based and differs from researcher to researcher. hobson12 reported that the foot plates should be adjusted to support approximately 10% of the body weight. gilsdorf et al.13 found that the lowest ischial interface pressures on any cushion occurred with the legs dangling. the of these studies suggested that the position of the lower extremities affects the forces acting on the buttocks. thus, we hypothesized that the forces acting on the buttocks, which in the formation of decubitus ulcers, are influenced by the elevation of the leg supports. aissaoui et al.14 investigated the effects of elevated leg support on posture and pressure distribution in healthy subjects sitting in a wheelchair. their showed that the elevating the leg support induces an increase in pressure under ischial tuberosities. no studies exist, however, on the relationship between the elevation of leg support and the shear force acting on the buttocks when a wheelchair s back support is reclined. moreover, the timing of leg support elevation, i.e., whether leg support elevation before or after reclining of the back support is better, has not been investigated. therefore, the purpose of this study was to investigate the influence of elevating wheelchair leg supports and the timing of the leg support elevation on the shear forces acting on the buttocks in wheelchairs with reclining back support. it was presumed that the size and shape of the pelvis would affect the of this study. the participants were 17 healthy adult men without leg or trunk disease: mean age, 22.6 6.6 years; mean height, 170.1 4.4 cm; mean body weight, 62.4 8.9 kg. exclusion criteria were: the participants that had back pain, a history of surgery, rheumatism, or neurologic disorders. this study was conducted with the approval of the research ethics committee of kawasaki university of medical welfare (no . 415), and informed consent was obtained from all the participants prior to their participation. we used an experimental chair with electric controls for reclining the back support (hashimoto artificial limb manufacturer, okayama, japan). the dimensions of the experimental chair were as follows: back support height, 104 cm; seat depth, 40 cm; backward angle of the seat, 0; reclining angle of the back support, 1040; angular velocity at which the back support reclined, 3/s. the rotational axis of the back support, the joint between the seat and back support, were located at the same height as the seat. the elevation angle of the leg supports could be adjusted between 10 and 80 backward from the vertical line. the rotational axes of the leg supports, which the joint between the seat and the top edge of the leg support frame, were located at the same height as the seat (fig . 1fig . the dimensions of the experimental chair were as follows : back support height, 104 cm ; seat depth, 40 cm ; backward angle of seat, 0 ; reclining angle of back support, 1040 ; angular velocity at which the back support reclined, 3/s . the elevation angle of the leg supports could be adjusted between 10 and 80 backward from the vertical . a : level goniometer, b : leg - support, c : rotational axis of back support, c : rotational axis of leg support). the dimensions of the experimental chair were as follows: back support height, 104 cm; seat depth, 40 cm; backward angle of seat, 0; reclining angle of back support, 1040; angular velocity at which the back support reclined, 3/s. the elevation angle of the leg supports could be adjusted between 10 and 80 backward from the vertical. a: level goniometer, b: leg - support, c: rotational axis of back support, c: rotational axis of leg support measurements were obtained which each participant sat comfortably with bilateral symmetry and rested on the back support and force plate located on the chair seat. hirose15 reported that inclinations of the sternum and abdominal line are correlated with inclinations of the thoracic and lumbar spine in both the frontal and sagittal planes. therefore, the posture of each participant was checked, by visually and manually inspecting the sternum and abdominal line, to ensure that the thoracic and lumbar spine in the frontal plane did not lean laterally. in addition, to maintain constant friction between the clothing of each participant and the seat surface, all participants wore 100% cotton clothing during the experiment. because the smooth metal surface of the force plate was conducive to the participant sliding forward in the chair, a rubber net was laid over the plate to minimize sliding and the risk of postural collapse. the coefficients of friction were calculated on the basis of the maximum static friction force, measured using a pull - tension gauge and weight. the measured coefficients of friction between the clothing and the rubber net, between the rubber net and the surface of the force plate, and between the surface of the back support and the clothing were 0.9, 0.8, and 0.4, respectively. the participants were instructed to fold their arms in front of their chests in a relaxed state and to not intentionally change their body position during the experiment. kemmoku et al.16 reported that the vertical and horizontal forces acting on the sacrococcygeal and ischial tuberosity areas increase in a seated posture as the angle of pelvic tilt increases. thus, to ensure consistency in the pelvic tilt angle, each participant s buttocks were positioned so that the back support and dorsal surface were in contact. , the positions of the lower legs were adjusted to be perpendicular to the feet on the floor, and the horizontal thigh angle was then adjusted by stacking wooden boards under the experimental chair. furthermore, to reduce the resistance of the lower extremities, a roller board was placed under the participants feet. in contrast, under the leg - up condition, the positions of the lower legs were elevated upward to an 80 incline backward from the vertical line by elevating the leg supports. aissaoui et al.14 reported that the angls of lower leg supports can affect the posture of a sitter s hip if the angular modification changes the distance from the seat front to the foot plates. this change from the fact that the leg support s axis of motion is not aligned with the knee joint axis. this interdependency between leg support angles and the distance between the footplate and the seat requires the linear placement of the lower leg and foot plate to be adjusted in synchrony with the leg support s angular modification. in addition, the soles of the feet did not counteract the horizontal force applied to the buttocks by applying pressure to the foot plates during the experiment. thus, under the leg - up condition, the foot plates were not used in this study (fig . measurements were made while each participant sat comfortably with bilateral symmetry and resting on the back support and force plate located on the chair seat .). measurements were made while each participant sat comfortably with bilateral symmetry and resting on the back support and force plate located on the chair seat. to investigate the cause of the increased shear force acting on the buttocks, we measured the horizontal and vertical forces when the back support was reclined. in addition, we also measured the center of pressure (cop) on the force plate and the trunk sliding distance along the back support to examine how the shear force increased. shear as an action or stress ing from applied forces which causes or tends to cause two contiguous internal parts of the body to deform in the transverse plane. as measuring shear force is difficult, we measured the horizontal and vertical forces to determine the shear force as described by kemmoku et al16. the horizontal and vertical forces acting on the buttocks were measured using a force plate (400 400 mm ; sampling frequency, 100 hz ; kyowa electronic instruments, tokyo, japan) that measured the reaction force in the posterior direction, which is equivalent to the horizontal force in the anterior direction when the back support was inclined. in addition, the anterior - posterior position of the cop on the force plate was measured. the point of origin of the measured cop was the center of the plate. thus, a positive value indicated that the cop was located farther forward than the center of the plate. furthermore, we measured the trunk sliding distance along the back support (bs) was using a video camcorder. videos of the trunk and back support were taken from the left side using a digital video camera (panasonic, osaka, japan) for the duration of the back support movement. dartfish teampro data 6.0 video analysis software (dartfish, fribourg, switzerland) was used to measure the trunk sliding distance along the back support. the distance was defined as follows: bs = va vs where va and vs correspond to the distances between the acromion and the reference base point (b), projected on the back support plane, from a position of back support after reclining (a) and at each start position (s), respectively18. a positive value indicates that the trunk was slid farther downward from the start position. to correct for the influence of each participant s postural collapse during measurement, measurements were performed 10 seconds after the posture was set. with respect to the angle of back support inclination, park et al.19 reported that decubitus ulcers may be prevented or diminished in tetraplegia patients when the back support angle of the wheelchair is more than 120, which is similar to 30 from the vertical line. accordingly, the experimental back support was reclined at increasing angles, beginning at 10 from the vertical (the initial upright position, iup), proceeding to a fully reclined position (frp) of 40 from the vertical, and then returning to the upright position (rup). in this study, the reclining cycle of back support was divided into two phases (i.e., the posterior inclination phase and the returning inclination phase). the posterior inclination phase was from iup to frp, and the returning inclination phase was from frp to rup. the position of the lower legs was set according to the experimental condition before the back support was reclined. between each phase, the participants were asked to stand up and relax for one minute to release the residual forces on the back support and the seat. in both inclination phases, the time required to measure the forces in each position of back support was 5 seconds. for each experimental condition , we used the average of the forces acting on the buttocks and the position of the cop after measuring 201 stable samples for each participant. the two conditions were measured in a random order, with three trials for each experimental condition. if the participant could not continue sitting because of intolerance of the position or danger of sliding out of the wheelchair, the experiment was stopped for safety reasons. between each trial, the participants were asked to reset and relax for one minute. to correct for the effects of body weight, the measured horizontal and vertical forces acting on the buttocks were normalized by body weight (percent body weight ; % bw), on using of the raw data measured by the force plate. preliminary analysis of the forces acting on the buttocks and the distance sliding along the back support was performed using the shapiro - wilk normality test. to investigate the influence of elevating the leg supports, the forces acting on the buttocks, the position of the cop, and the sliding distance were compared between the two experimental conditions in each of the reclining positions in the inclination phase. in addition, to investigate the influence of the reclining the back support, the forces and the position of cop were compared between the two positions of the back support in each inclination phase. the statistical analyses were performed using the statistical package for the social sciences (spss) version 16.0 j for windows (spss, chicago, il, usa), and a significance level of p < 0.05. tables 1 and 2table 1.horizontal force acting on the buttocks in the various positions of back support (n=17)phasepositionthe leg - down conditionthe leg - up conditionthe posterior inclinationiup**10.2 1.015.5 2.8frp**10.3 1.014.9 1.5 phasepositionthe leg - down conditionthe leg - up conditionthe returning inclinationfrp**13.2 0.916.4 1.7rup**17.4 2.022.8 3.4mean sd (% bw). * * p < 0.01 (significant differnce between two experimental conditions). p < 0.01 (significant differnce between the two positions of back support)table 2.vertical force acting on the buttocks in the various positions of back support (n=17)phasepositionthe leg - down condition the leg - up condition the posterior inclination iup**71.8 2.974.8 4.1frp**60.3 5.068.0 5.6the returning inclination frp**60.4 3.367.3 3.0rup**79.4 4.687.5 3.6mean sd (% bw). * * p < 0.01 (significant differnce between the two experimental conditions). p < 0.01 (significant difference between the two positions of back support) show the horizontal and vertical forces acting on the buttocks, table 3table 3.position of the cop on the force plate in the various positions of back support (n=17)phasepositionthe leg - down condition the leg - up condition the posterior inclination iup**87.3 14.346.1 11.8frp**114.7 14.569.8 12.4the returning inclination frp**111.8 13.767.1 10.5rup**82.7 16.649.5 12.3mean sd (mm). * * p < 0.01 (significant differnce between the two experimental conditions). p < 0.01 (significant differnce between the two positions of back support). a negative value indicates that the cop was located farther backward than the center of the plate. shows the position of the cop on the force plate, and table 4table 4.trunk sliding distances of the back support in the various inclination phases (n=17)phasethe leg - down conditionthe leg - up conditionthe posterior inclination78.8 10.481.2 12.9the returning inclination66.8 13.268.8 13.1mean sd (mm). a positive value indicates that the trunk was slid farther downward than the start position. mean sd (% bw). * * p < 0.01 (significant differnce between two experimental conditions). < 0.01 (significant differnce between the two positions of back support) mean sd (% bw). * * p < 0.01 (significant differnce between the two experimental conditions). < 0.01 (significant difference between the two positions of back support) mean sd (mm). * * p < 0.01 (significant differnce between the two experimental conditions). p < 0.01 (significant differnce between the two positions of back support). a negative value indicates that the cop was located farther backward than the center of the plate. a positive value indicates that the trunk was slid farther downward than the start position. regarding the horizontal force acting on the buttocks, there were significant differences in the horizontal forces measured under the two experimental conditions in all the reclining positions of the back support. in addition, there were not significant differences between the two positions of back support in the posterior inclination phase, but there were significant differences for that in the return - to - the - upright phase (table 1). regarding the vertical force acting on the buttocks, there were significant differences in the vertical forces between the two leg elevation conditions in all reclining positions of the back support. in addition, there were significant differences between the two leg elevation positions in each inclination phase (table 2). regarding the anterior - posterior position of cop on the force plate , there were significant differences in the anterior - posterior positions of the cop between the two leg elevation conditions in all reclining positions of the back support. in addition, there were significant differences between the two leg elevation positions in each inclination phase (table 3). regarding the trunk sliding distance along the back support, the differences in the sliding distances of the back support under the two conditions were not significant (table 4). this study examined the influence of elevating leg support elevation on the shear force acting on the buttocks during reclining of the back support to aid in the prevention of decubitus ulcers in supposed individuals who use wheelchairs with reclining back supports. in the rup, under both experimental conditions, the forward horizontal force acting on the buttocks increased significantly in comparison with that in the frp as the back support reclined. in previous research4,5,6, we investigated the mechanism of the increase in the horizontal force acting on buttocks from the frp to rup. the suggested that the increase in horizontal force was produced by the friction force on the back support and the difference in the position of the rotational axes of the back supports and the trunk - pelvis. with respect to the influence of elevating leg support elevation, in the iup as the start position of the posterior inclination phase and the rup as the end position of the returning inclination phase, the horizontal forces acting on the buttocks under the leg - up condition were significantly greater than under the leg - down condition. kemmoku et al.16 reported that the horizontal forces acting on the sacrococcygeal and ischial tuberosity areas increased in a seated posture as the angle of pelvic tilt increased. in the present study, the angle of backward pelvic tilt was increased by extending the hamstrings as a of elevating the leg support. moreover, aissaoui et al.14 reported that the pressure on the back support during sitting on a chair was increased by elevating leg support. in addition, there is a strong relationship between the reaction force on the back support and the horizontal force acting on the buttocks20. the pressure, i.e., the reaction force, on the back support is increased by increasing the angle of backward pelvic tilt. with respect to the present study, therefore, it might be suggests that the horizontal force acting on the buttocks under the leg - up condition was also increased significantly in the iup and rup. incidentally, with the frp as the start and end positions of each inclination phase, the hamstrings should not be extended by elevating the leg support so that the pelvis is inclined backward by reclining the back support. however, the horizontal and vertical forces acting on the buttocks under the leg - up condition were significantly higher than under the leg - down condition. gilsdorf et al.13 reported that the anterior - posterior position of the cop is located forward on the seat, so that the mass of the thighs produces a load on the front of the seat that can be adjusted by adjusting the thigh angle downward from the horizontal plane when sitting on a chair. under the leg - up condition in the present study, the cop was located significantly more forward on the seat than under the leg - down condition, even although the differences in the sliding distances of the back support of the two conditions were not significant. the lower legs were elevated by the leg supports under the leg - up condition. the angle of the lower legs was 10 less than the horizontal plane at the maximum elevating on angle of the leg supports. the inclination of the leg support and the mass of the lower legs ed in rotated the thighs forward. under the leg - up condition, the vertical force on the seat was significantly increased, and the position of the cop was shifted forward as a of the thighs pressing the seat due to the rotation. these findings suggest that the leg support used in this study did not sufficiently support the mass of the lower legs. the vector of the lower leg weight was separated into parallel and vertical components for analysis of the leg support inclination. the resistance force in the parallel direction was the static friction force between the leg support surface and the lower legs. the maximum static friction force just before sliding of the lower legs occurs is defined as the product of the friction coefficient and the vertical force on the surface of the leg support. this implies that in regions where the vertical force is relatively high, the static friction can become high as well, suggesting that the static friction value was low, and thus that the vertical force on the leg support was low because the vector of the lower leg mass was separated into two components for the leg - up condition in this study. therefore, in the frp, the horizontal force acting on the buttocks under the leg - up condition, which did not sufficiently support the lower legs, was significantly higher than that under the leg - down condition, in which sufficient support was perpendicularly provided to the lower legs through the soles of the feet. the main limitation of this study was that it included only healthy adult males. in addition, because the measurement times were short, the effect of delayed postural collapse was not evaluated. however, we did not measure body alignment in this study. furthermore, the form (i.e., the rotational axis position of the leg support), material, and coefficient of friction of the experimental wheelchair s seat differed from those used to measure the horizontal forces. for example, the foot plates were not used in this study so that the soles of the feet did not resist the horizontal force acting on the buttocks by pressing against the foot plates. if the foot plates had been used in this study, it might be presumed that the fluctuation of the horizontal force applied to the buttocks under the leg - up condition was decreased by resistance to the parallel force forward and downward on the leg support. moreover, we did not consider factors that interact with the friction force, such as urinary incontinence and sweat, which affect many wheelchair users. therefore, the present should be extrapolated to actual wheelchair users with great caution. the of this study suggest that leg supports should be positioned downward before reclining the back support of a wheelchair to prevent decubitus ulcer formation. we plan to investigate the influences of the seat material and the friction force of the back support on the horizontal force acting on the buttocks while the wheelchair is reclining. these will aid in the development of reclining wheelchairs and ultimately reduce the occurrence of decubitus ulcers.

the purpose of this study was to investigate the effect of the timing of leg support elevation on the horizontal force acting on the buttocks in a reclining wheelchair. the participants were 17 healthy men. two experimental conditions were tested: the leg - down and leg - up conditions. the back support was reclined at increasing angles, from the initial upright position (iup), proceeding to the fully reclined position (frp), and returned to the upright position (rup). the posterior inclination phase was from iup to frp, and the returning inclination phase was from frp to rup. the horizontal force under the leg - up condition was significantly higher than that under the leg - down condition in all positions of back support. the leg supports should be positioned downward before reclining the back support of a wheelchair.

the kinetic visual field has been classically interpreted by measuring one or more isopter field areas. areas are expressed in degrees squared, centimeters squared, average diameter, or steradians. however, these units are usually limited to one isopter field area, and no information about the sensitivity to target luminance is included. this study presents a method that quantifies the kinetic visual field as a three - dimensional form by expressing both isopter area and sensitivity to target luminances of a particular target size. this quantification of a visual field can be used clinically to follow changes in the visual fields of patients over time.1 kinetic perimetry was performed with a goldmann perimeter (haag - streit inc, bern, switzerland) using target sizes of i4e, i3e, i2e, and i1e. twenty - five normal subjects, comprising eight males and 17 females of mean age 33.9 10.1 (range 1764) years were selected. one subject was under 20 years of age, 10 were aged 2029 years, nine were 3039 years, and five were 40 years or older (table 1). the study was approved by the national eye institute institutional review board, and was performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki. best corrected visual acuity using the early treatment of diabetic retinopathy study lighthouse chart was measured and a complete dilated ophthalmologic examination was performed on each volunteer. all patients had best corrected visual acuity of 20/20 or better in the tested eye, and there were no media opacities or other abnormal findings on dilated slit lamp and fundus examination that would affect perimetric testing. in addition, visual fields of four patients with representative visual defects obtained during the course of their normal clinical visits were chosen to illustrate this method. these were concentric contraction (gyrate atrophy), temporal hemianopia (craniopharyngioma), arcuate scotoma (glaucoma), and central scotoma (macular dystrophy). one steradian is the area on the surface of a sphere that is subtended by a solid angle emanating from the center of a sphere.2 the two - dimensional kinetic visual field is an equidistant polar projection of the interior of the perimetry bowl onto a flat surface (azimuthal). such projections contain equally spaced circles that are equidistant only along meridians.3 units such as degrees squared, centimeters squared, or average diameter, that are measured on a planar surface, distort the true size of peripheral field regions by making them appear larger than equivalent central field regions. tangential azimuthal deformation increases greatly with eccentricities beyond 50 from the center (20% at 60 and 57% at 90).4 small distortions using the solid angle to measure visual fields still exist with regard to retinal eccentricity, and require certain assumptions and approximations in order to be corrected mathematically.2,68 despite these, the steradian is a more appropriate unit to quantify visual field area than other units. one steradian is one radian squared and since one radian subtends 57.3, one steradian equals (57.3) or 3283.3 squared. the visual fields obtained were scanned and each of the four isopters was then digitized. the area of each isopter was calculated in steradians using a method described by weleber and tobler that corrects for cartographic distortion due to polar projection.2 the series of formulae is listed in table 2. the digitized x and y coordinates for each isopter are listed in columns a and b. the formulae in each of the columns can be copied and pasted into a spreadsheet application, such as microsoft excel. the total area of an isopter in steradians is the sum of all of the values in column p. it should be noted that an x coordinate of 0.000 will be read as infinity by the formula in column c (= atan b1/a1), therefore an x coordinate of 0.000 is converted to 0.001. thus, a dim target luminance, such as 1e, will require greater sensitivity to be perceived and will have a greater vertical height. we utilized cd / m, ie, si units, rather than apostilbs, to describe luminance per area. apostilbs can be converted to si units (cd / m) by dividing apostilbs by. the inverse of the target luminance values is used to describe target luminance sensitivity. the log of the sensitivity compresses the axis scale and the units are multiplied by 1000 to keep the log units positive. the volume of each visual field isopter is the product of area and sensitivity to target luminance. the ing units are steradians log 10(cd / m), referred to as goldmanns. the volume of the visual field of the four luminances for the size i target is calculated by summing the products of area and height for each isopter. in this study, the same target size (i) for each isopter measurement is used, so the only variable change is target luminance. in order to avoid duplication of visual field volume between isopters, the height of each larger target sizes can be used by multiplying the area by the appropriate target luminance factor. this would increase the height of the i target because of its decreased luminance relative to larger target sizes (table 3). if there is a visual field deficit present, the volume of the scotoma is similarly calculated for each isopter and the total is subtracted from the total visual field volume. the area of the blind spot is 0.012 steradians or 0.048 goldmanns and may or may not be subtracted from the total volume measurement as long as the measurements between studies are consistent. this method of calculating the volume of the visual field can also be applied to automated static perimetry. in threshold static perimetry, differential luminance sensitivity units are expressed in decibels of target luminance attenuation. each threshold value already represents the height of a specific area of the visual field. the humphrey field analyzer (carl zeiss, meditec, dublin, ca) 30 - 2 program contains 76 threshold measurements, each one representing 36 squared degrees (or 0.011 steradians). thus, there are 76 columns of visual space in the humphrey field analyzer 30 - 2 program. the volume of each column is the product of the area of the base and its height, and the total volume (db) is the sum of all the column volumes. the foveal threshold is not included in this calculation because it overlaps with the four surrounding threshold columns. the total volume can be converted to goldmann units by multiplying the decibels of luminance attenuation with a conversion factor. the humphrey field analyzer 30 - 2 represents a total area of 76/(3283.3)/steradians or 0.8333 steradians. the height of the visual field is determined by converting the mean target luminance attenuation in decibels (dbmean or log ( cd / m) ) to apostilbs using a conversion chart. in static perimetry, thus, to keep the log units positive, (cd / m) is multiplied by a factor of 10,000 instead of the 1000 used in kinetic perimetry. the relationship between decibels of target luminance attenuation and log 10(cd / m) is linear, and is described by the following equation: log 10(cd / m) = 0.1 (dbmean) + 0.5. for the humphrey field analyzer 30 - 2, the sensitivity to luminance can be converted to goldmanns by multiplying by 0.8333 steradians. thus: gn = 0.8333 + 0.5 ). because of inherent differences, units obtained by static perimetry can not be interchanged with the volumetric measurements obtained from kinetic perimetry. the assignment of a vertical dimension for luminance sensitivity reinterprets the island of vision for a particular target size from the classical planar figure that is used in kinetic perimetry to a three - dimensional form (figures 1a and 1b). isopter areas and volumes, as well as total volume obtained with goldmann perimetry for each subject and their age, are listed in table 1. the mean total volume of the 25 normal subjects for the i isopter was 3.467 0.37 goldmanns, but these decreased in the older age groups. the mean for the 10 subjects aged 2029 years was 3.615 0.36 goldmanns, that of the nine subjects aged 3039 years was 3.459 0.407 goldmanns, and for the five subjects older than 40 years, was 3.176 0.200 goldmanns. table 4 depicts the isopter volumes and total volume of the four representative visual field defects illustrated in figures 2a d. the field with concentric contraction (figure 2a) demonstrates a large decrease in the total isopter i volume (0.212 goldmanns). in addition, the differential sensitivity to target luminances dimmer than i4e decreases abruptly in this particular patient, ing in a volume that is mostly comprised of the i4e isopter (62.3% of total i isopter volume versus 44.7% for age - matched controls). the patient with temporal hemianopia (figure 2b) demonstrates a proportional decrease in volume between isopters. although the i-2e isopter area of the central scotoma is 0.2605 steradians, or 5.9 times larger than that of the arcuate scotoma (0.044 steradians), the central scotoma contains greater depth because it extends across three isopters and is 11.5 times the volume of the arcuate scotoma (0.254 goldmanns versus 0.022 goldmanns). figure 3 illustrates three - dimensional examples of 30 - 2 humphrey visual field measurements in a normal subject. the average differential luminance sensitivity over 76 points represented in the 30 field was 30.54 db or 2.96 goldmanns. this report presents a quantitative method to interpret kinetic visual fields by using a single volumetric measurement that incorporates all isopter areas of one target size as well as differential luminance sensitivity. this can be used as a global index comparable with mean sensitivity in static perimetry to describe quantitatively the hill of vision as assessed by kinetic perimetry. this measurement can be used to follow the visual field of a patient over time or to compare between patients. although it can be cumbersome to scan and digitize each isopter using a manual kinetic perimeter, the process can be greatly ameliorated with the use of more recent semiautomated perimeters, such as the octopus 101 (haag - streit, kniz, switzerland), that can digitally store the coordinates at each measured isopter point and calculate the isopter areas.913 the area of each isopter and total field volume can then be calculated with appropriate software. in static perimetry, conversion to a volume unit simply involves multiplying the sum of the decibels of target luminance attenuation with a conversion factor. because current static perimeters are automated, they could be programmed to perform such a function. three - dimensional modeling of the hill of vision using both semiautomated kinetic and automated static perimetry has been previously described.11,1416 these offer the possibility of modeling visual field volumes using normal age - dependent local reference values, thereby generating age - normalized mean defect values.17 in goldmann perimetry, the volume of the island of vision that is measured is an underestimate of the true volume. first, target differential luminance sensitivity for an isopter target size is often well below threshold levels. second, a finite number of points for each isopter is collected by the perimetrist. third, a small number of target luminances (usually four) are used in clinical kinetic perimetry. thus, the illustration in figure 1b representing a normal visual field depicts a four - stepped pyramid, when a cone - like structure would be a closer approximation of the true island of vision for a particular isopter target. based on the stepped structure from the measured isopter volumes, a smoother hill of vision could be modeled with interpolation software. nevertheless, standard goldmann perimetry provides a method of kinetic visual field testing that is consistent, practical, and requires few assumptions for measuring the size of a visual field. the use of a volumetric parameter in kinetic perimetry can be used to evaluate more accurately the amount of field contraction or scotoma enlargement over time than the area of one isopter alone. in patients with dense scotomas or, in conditions such as retinitis pigmentosa or gyrate atrophy with reduced light sensitivity, kinetic visual fields contain smaller i4e isopter areas when compared with normal fields, but field volumes that are often proportionally much smaller than the isopter area. in the patient with gyrate atrophy, the i4e isopter volume was 8.5% compared with the mean of our age - matched controls, while the volume was 6.1% because of decreased differential luminance sensitivity to dimmer targets. by contrast, in a patient with a proportionate decrease in visual field across all isopter luminances for a particular target size, such as in hemianopia, the visual field demonstrates a more consistent decrease between area and volume (27.6% and 26.2%, respectively, in the patient with craniopharyngioma). the use of semiautomated kinetic perimetry can be used to measure scotoma size and to relate scotoma depth with age - related normal values as mean defect, a function not available with standard goldmann perimetry.17 we chose one target size (i) to represent the island of the visual field because this limits the number of assumptions in constructing the volume. it avoids the added variable of spatial summation that occurs when using different target sizes with similar luminances, in goldmann perimetry, dimmer luminances (3e, 2e, and 1e) are most frequently used with i target rather than iii or v target size. however, use of the i target alone truncates the true island of vision considerably, in essence measuring the top portion of the island of vision. nevertheless, the use of one target size requires few assumptions about the luminance levels of other target sizes and avoids variance seen with far peripheral field measurements using larger target sizes. larger target sizes, such as v4e and iii4e, can be added by multiplying the isopter by the appropriate luminance attenuation factor. although standard luminance levels for larger target sizes can be found in table 1,18 it would be useful to calibrate the luminance of each target to obtain a more accurate conversion factor for luminance sensitivity. however, considerable differences in its size between individuals has been recently demonstrated with semiautomated kinetic perimetry.16 whether the physiologic blind spot is included in isopter size calculations or whether one or more target sizes are used, it is consistency in visual field volume formulation that allows for more accurate evaluation of visual field changes over time. in our study, 20 of 25 subjects were under the age of 40 years, so our do not represent normative data from a widely ranged age group. the decrease in differential luminance sensitivity in the peripheral portions of the visual field and decreased sensitivity to smaller and dimmer targets has been eloquently demonstrated to correlate with increasing age, particularly after the age of 40 years.12,1316,19 in addition, semiautomated kinetic perimeters, such as the octopus 101, allow for measurement of reaction time or the interval between stimulus onset and patient response. isopter and scotoma sizes derived by semiautomated kinetic perimetry can be corrected for variable response times reducing variance of isopter area.12,17,20 although not available with standard goldmann perimetry in this study, reaction time corrected isopters could be used with newer automated perimeters to derive visual field volume. the 30 - 2 humphrey visual field analyzer measures the central 30 core of the island of vision. there is a small amount of cartographic distortion inherent with two - dimensional projection from a curved surface. for the central 30 surface, the volumetric increase is small, approximately 2% of the entire central 30 surface, and was not accounted for in this study.4 it should be noted that volumetric measurements between static and kinetic perimetry can not be compared because of inherent differences between the two methods. physiologic dissociation between kinetic and static stimuli in perimetry is well known, particularly with achromatic target perception.21 moreover, the sensitivity for kinetic stimuli has been shown to be greater than for static stimuli independent of age, stimulus size, and eccentricity.22 static perimetry may leave areas within a measured visual field untested, especially with the use of small target sizes and scattered grids with limited coverage. thus, direct comparison of static and kinetic visual field volumes is inaccurate, and extrapolation between the two methods by a simple conversion factor is inaccurate. finally, unlike manual perimetry, differences in individual local differential luminance sensitivity values in static perimetry are related to age - corrected normal differential luminance sensitivity values, and the ing deviations are used to determine global indices, such as mean defect. nevertheless, description of a visual field as a volume represents a useful index of quantifying the visual field for each of these perimetric techniques. this study presents a method to quantify the volume of the island of vision using a method to correct for cartographic distortion due to polar projection, with a single value containing information about both visual field area and sensitivity to luminance. this technique can be applied to assess the progression or stability of visual field defects quantitatively over time in both kinetic and static perimetry. newer semiautomated kinetic perimeters may be able to incorporate this type of volumetric measurement in the assessment of the visual field.

: the purpose of this study was to quantify the volume of the kinetic visual field with a single unit that accounts for visual field area and differential luminance sensitivity.methods:kinetic visual field perimetry was performed with a goldmann perimeter using i4e, i3e, i2e, and i1e targets. the visual fields of 25 normal volunteers (17 women, eight men) of mean age 33.9 10.1 (range 1764) years were obtained and digitized. isopter areas were measured with a method devised to correct cartographic distortion due to polar projection inherent in perimetry and are expressed in steradians. the third dimension of each isopter represents sensitivity to target luminance and was calculated as log (target luminance1). if luminance is expressed in cd / m2, the values for the third dimension are 0.5 for i4e, 1.0 for i3e, 1.5 for i2e, and 2.0 for i1e. the ing unit is a steradian (log 103 ( cd / m2)1 which is referred to as a goldmann. in addition, the visual fields of four patients with representative visual defect patterns were examined and compared with normal subjects.:mean isopter areas for normal subjects were 3.092 0.242 steradians for i4e, 2.349 0.280 steradians for i3e, 1.242 0.263 steradians for i2e, and 0.251 0.114 steradians for the i1e target. isopter volumes were 1.546 0.121 goldmanns for the i4e target, 1.174 0.140 goldmanns for i3e, 0.621 0.131 goldmanns for i2e, and 0.126 0.057 goldmanns for i1e. the total mean visual field volume in our study for the i target was 3.467 0.371 goldmanns.:the volume of the island of vision may be used to quantify a visual field with a single value which contains information about both visual field extension and differential luminance sensitivity. this technique may be used to assess the progression or stability of visual field defects over time. a similar method may be applied to static perimetry.

the online version of this article (doi:10.1007/s00894 - 014 - 2272-y) contains supplementary material, which is available to authorized users. one of the most important goals of theoretical chemistry is to understand the origin of conformational changes in molecules. in order to achieve this goal many methods can be applied to the description of electronic structures: molecular orbitals (mos) , localized molecular orbitals (lmos) , bond orders , atoms in molecules (aim), fermi hole, kinetic energy and information theory based quantities , and various charge and energy decomposition schemes. a useful and elegant approach suitable for description of energy profiles of chemical reactions was proposed by torro - labbe and coworkers based on the reaction force concept. bickelhaupt and zeist proposed the activation strain model , which also appears to be very useful in the analysis of chemical reactions. carbon bond is one of the most important conformational transitions in organic chemistry. a typical example is ethane, which exhibits staggered and eclipsed conformations; the former minimum energy structure is more stable than the transition state eclipsed structure by 3.0 kcal mol. the classical and intuitive explanation of the barrier suggested in organic textbooks is based on the steric repulsion between c an alternative explanation is based on hyperconjugation stabilization, which is stronger in the staggered conformation. however, as pointed out by mulliken, hyperconjugation effect should have only a minor influence on the barrier.. stated that the barrier to rotation in ethane can be related to the polarization of charge density along the carbon carbon bond. goodman and coworkers have shown, based on the natural bond orbitals (nbo) method, that ethane s staggered conformation is the of hyperconjugation. goodman s based on the nbo method have been challenged by the work of bickelhaupt and baerends based on the model of a chemical bond originating from fragmented molecular orbitals; according to these the internal rotational barrier in ethane is due to pauli repulsion acting between the ch bonds of opposite ch3 units. subsequent calculations by mo and coworkers and then by pendas et al. , confirmed the classical, steric - based interpretation of the barrier; in addition, the former authors have shown that hyperconjugation stabilizes the staggered conformer only by about 4 kj mol relative to the eclipsed form. a very elegant recent paper by mo and gao provided a compact overview of the most important studies on this subject; the main is that the internal rotational barrier in ethane is due predominantly to steric effects acting between c we have recently developed the ets - nocv scheme by combining the extended transition state (ets) energy decomposition approach with the natural orbitals for chemical valence (nocv) method. ets - nocv has proved suitable for qualitative and quantitative description of the crucial components (, , , etc .) in addition, the energy profiles of some chemical reactions can be also characterized. more importantly for this study , it was shown that nocv representation allows for qualitative and quantitative description of hyperconjugation effects. furthermore, the ets energy decomposition scheme provides quantitative information on the pauli repulsion effects. therefore, the main goal of this article was to apply for the first time the ets - nocv charge and energy decomposition scheme to analysis of the internal rotation in ammonia borane. hyperconjugation and steric factors will be discussed in a detailed way. it should be noted that ammonia borane is considered nowadays as one of the most promising hydrogen storage materials. in addition, it was already proven that ammonia borane exhibits dissimilar features as compared to isoelectronic ethane. furthermore, the present study sheds additional qualitative and quantitative light on the steric repulsion in ammonia borane by decomposition of total pauli repulsion into specific contributions stemming from different symmetry (and). in order to achieve this goal, we defined for the first time the eigenvectors for pauli repulsion; in this representation, one can thus discuss the pauli repulsion components originating from different symmetries. all dft calculations presented here were based on the amsterdam density functional (adf 2009.01) program in which the ets - nocv scheme was implemented. the becke - perdew exchange - correlation functional was applied (bp86). a standard triple - zeta sto basis containing two sets of polarization functions (tz2p) our analysis is based on the ets - nocv approach, which is a combination of the extended transition state (ets) method with the natural orbitals for chemical valence (nocv) scheme. the basic concept of the ets scheme involves partitioning of the total bonding energy etotal between interacting fragments into four components:1\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {e}_{\mathrm{total}}=\vardelta {e}_{\mathrm{dist}}+\vardelta {e}_{\mathrm{elstat}}+\vardelta {e}_{\mathrm{pauli}}+\vardelta {e}_{\mathrm{orb} } $ $ \end{document}etotal=edist+eelstat+epauli+eorb the first component, edist, referred to as the distortion term, represents the amount of energy required to promote the separated fragments from their equilibrium geometry to the structure they will take up in the combined molecule; it can also be seen as strain energy. the second term, eelstat, corresponds to the classical electrostatic interaction between the promoted fragments as they are brought to their positions in the final complex. the third term, epauli, accounts for the repulsive pauli interaction between occupied orbitals on the two fragments in the combined molecule. it is calculated as the difference between the energies of orthogonalized and non - orthogonalized fragments. finally, the last stabilizing term, eorb, represents the interactions between the occupied molecular orbitals of one fragment with the unoccupied molecular orbitals of the other fragment as well as the mixing of occupied and virtual orbitals within the same fragment (inner - fragment polarization). this energy term, eorb, may be linked to the electronic bonding effect coming from the formation of a chemical bond (eq . 2). the nocv are eigenvectors that diagonalize deformation density matrix p = pmolecule p0, where p0 corresponds to the sum of density matrices for orthogonalized fragments; it has been shown that the natural orbitals for chemical valence pairs (-k,k) decompose the deformation density orb into nocv - contributions, orbk:1\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {\rho}_{orb}(r)={\displaystyle \sum_{k=1}^{m/2}{v}_k\big={\displaystyle \sum_{k=1}^{m/2}\vardelta {\rho}_{orb}^k } $ $ \end{document}orbr=k=1m/2vk=k=1m/2orbkwhere k and m are the nocv eigenvalues and the number of basis functions, respectively. visual inspection of deformation density plots (orbk) helps to attribute symmetry and the direction of the charge flow. in addition, information gained from the analysis of deformation density plots can be enriched by providing the energetic estimations, eorbk, for each orbk within ets - nocv scheme:2\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {e}_{orb}={\displaystyle \sum_k\vardelta { e}_{orb}^k=}{\displaystyle \sum_{k=1}^{m/2}{v}_k\big $ $ \end{document}eorb=keorbk=k=1m/2vkwhere fi, its are diagonal kohn - sham matrix elements defined over nocv with respect to the transition state density (at the midpoint between density of the molecule and the sum of fragment densities). the above components eorbk provide the energetic estimation of orbk that may be related to the importance of a particular electron flow channel for the bonding between the considered molecular fragments. in the present study, in analogy to nocvs, we defined for the first time the natural orbitals (eigenvectors) for pauli repulsion, k, that diagonalize the pauli deformation density matrix, p = p0pisolated, where pisolated is the sum of density matrices for non - orthogonalized fragments, whereas p0 correspond to the sum of density matrices for orthogonalized fragments. such eigenvectors decompose the total pauli deformation density, =0 (orthogonalized - fragments) (non - orthogonalized - fragments), into the nocv - like contributions (kpauli) (in analogy to eq . 1):3\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {\rho}^{pauli}(r)={\displaystyle \sum_{k=1}^{n/2}{v}_k^{pauli}\big={\displaystyle \sum_{k=1}^{n/2}\vardelta {\rho}_k^{pauli}(r) } $ $ \end{document}paulir=k=1n/2vkpauli=k=1n/2kpaulir the total charge transferred in this channel can be considered as:4\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {q}_k^{pauli}={\nu}_k^{pauli} $ $ \end{document}qkpauli=kpauli the present study characterized not only the total values of pauli repulsion (epauli) in ammonia borane based on the original ets scheme (eq . 1) but, in addition, provided a more detailed picture by analyses of both the pauli repulsion contributions kpauli (eq . 3) and the corresponding quantitative charge estimations qkpauli (eq . 4). this approach (eqs . 3, 4) was implemented by one of us in the home version of adf2009.01. at present, the energetic pauli repulsion contributions (epaulik) from kpauli (calculated in an analogous way to eq . 2) hence, we focused our attention on the quantitative measures of kpauli based on eq. 4. red areas of deformation density channels correspond to charge depletion, whereas blue indicates charge accumulation upon bond formation. due to the fact that the steric interaction, which is a non - observable quantity, is very often attributed in the literature to pauli repulsion quantum effect , we use both terms interchangeably throughout the text. finally, we should note that pauli repulsion is one of the bonding components in various energy decomposition schemes; hence, we believe that a more detailed description of this term based on eqs. 3, 4, could be of wide interest. it is very important to point out that the main source of the pauli repulsion is related to an increase in the kinetic energy contribution; so we could also refer to the pauli repulsion term as kinetic repulsion due to the pauli exclusion principle. such a concept, which relates the steric repulsion to the kinetic energy pressure has already been put forward by various authors. in addition, the pauli repulsion contribution appears to qualitatively correlate very well with the experimental taft s steric parameters. all dft calculations presented here were based on the amsterdam density functional (adf 2009.01) program in which the ets - nocv scheme was implemented. the becke - perdew exchange - correlation functional was applied (bp86). a standard triple - zeta sto basis containing two sets of polarization functions (tz2p) our analysis is based on the ets - nocv approach, which is a combination of the extended transition state (ets) method with the natural orbitals for chemical valence (nocv) scheme. the basic concept of the ets scheme involves partitioning of the total bonding energy etotal between interacting fragments into four components:1\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {e}_{\mathrm{total}}=\vardelta {e}_{\mathrm{dist}}+\vardelta {e}_{\mathrm{elstat}}+\vardelta {e}_{\mathrm{pauli}}+\vardelta {e}_{\mathrm{orb} } $ $ \end{document}etotal=edist+eelstat+epauli+eorb the first component, edist, referred to as the distortion term, represents the amount of energy required to promote the separated fragments from their equilibrium geometry to the structure they will take up in the combined molecule; it can also be seen as strain energy. the second term, eelstat, corresponds to the classical electrostatic interaction between the promoted fragments as they are brought to their positions in the final complex. the third term, epauli, accounts for the repulsive pauli interaction between occupied orbitals on the two fragments in the combined molecule. it is calculated as the difference between the energies of orthogonalized and non - orthogonalized fragments. finally, the last stabilizing term, eorb, represents the interactions between the occupied molecular orbitals of one fragment with the unoccupied molecular orbitals of the other fragment as well as the mixing of occupied and virtual orbitals within the same fragment (inner - fragment polarization). this energy term, eorb, may be linked to the electronic bonding effect coming from the formation of a chemical bond (eq . 2). the nocv are eigenvectors that diagonalize deformation density matrix p = pmolecule p0, where p0 corresponds to the sum of density matrices for orthogonalized fragments; it has been shown that the natural orbitals for chemical valence pairs (-k,k) decompose the deformation density orb into nocv - contributions, orbk:1\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {\rho}_{orb}(r)={\displaystyle \sum_{k=1}^{m/2}{v}_k\big={\displaystyle \sum_{k=1}^{m/2}\vardelta {\rho}_{orb}^k } $ $ \end{document}orbr=k=1m/2vk=k=1m/2orbkwhere k and m are the nocv eigenvalues and the number of basis functions, respectively. visual inspection of deformation density plots (orbk) helps to attribute symmetry and the direction of the charge flow. in addition, information gained from the analysis of deformation density plots can be enriched by providing the energetic estimations, eorbk, for each orbk within ets - nocv scheme:2\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {e}_{orb}={\displaystyle \sum_k\vardelta { e}_{orb}^k=}{\displaystyle \sum_{k=1}^{m/2}{v}_k\big $ $ \end{document}eorb=keorbk=k=1m/2vkwhere fi, its are diagonal kohn - sham matrix elements defined over nocv with respect to the transition state density (at the midpoint between density of the molecule and the sum of fragment densities). the above components eorbk provide the energetic estimation of orbk that may be related to the importance of a particular electron flow channel for the bonding between the considered molecular fragments. in the present study, in analogy to nocvs, we defined for the first time the natural orbitals (eigenvectors) for pauli repulsion, k, that diagonalize the pauli deformation density matrix, p = p0pisolated, where pisolated is the sum of density matrices for non - orthogonalized fragments, whereas p0 correspond to the sum of density matrices for orthogonalized fragments. such eigenvectors decompose the total pauli deformation density, =0 (orthogonalized - fragments) (non - orthogonalized - fragments), into the nocv - like contributions (kpauli) (in analogy to eq . 1):3\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {\rho}^{pauli}(r)={\displaystyle \sum_{k=1}^{n/2}{v}_k^{pauli}\big={\displaystyle \sum_{k=1}^{n/2}\vardelta {\rho}_k^{pauli}(r) } $ $ \end{document}paulir=k=1n/2vkpauli=k=1n/2kpaulir the total charge transferred in this channel can be considered as:4\documentclass{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \vardelta {q}_k^{pauli}={\nu}_k^{pauli} $ $ \end{document}qkpauli=kpauli the present study characterized not only the total values of pauli repulsion (epauli) in ammonia borane based on the original ets scheme (eq . 1) but, in addition, provided a more detailed picture by analyses of both the pauli repulsion contributions kpauli (eq . 3) and the corresponding quantitative charge estimations qkpauli (eq . 4). was implemented by one of us in the home version of adf2009.01. at present, the energetic pauli repulsion contributions (epaulik) from kpauli (calculated in an analogous way to eq . hence, we focused our attention on the quantitative measures of kpauli based on eq . 4 . red areas of deformation density channels correspond to charge depletion, whereas blue indicates charge accumulation upon bond formation . due to the fact that the steric interaction, which is a non - observable quantity, is very often attributed in the literature to pauli repulsion quantum effect , we use both terms interchangeably throughout the text . finally, we should note that pauli repulsion is one of the bonding components in various energy decomposition schemes ; hence, we believe that a more detailed description of this term based on eqs . 3, 4, could be of wide interest . it is very important to point out that the main source of the pauli repulsion is related to an increase in the kinetic energy contribution ; so we could also refer to the pauli repulsion term as kinetic repulsion due to the pauli exclusion principle . such a concept, which relates the steric repulsion to the kinetic energy pressure has already been put forward by various authors . in addition , the pauli repulsion contribution appears to qualitatively correlate very well with the experimental taft s steric parameters . we will start with a brief description of the bonding situation in the most stable staggered conformation ( s) of ammonia borane (fig . 1). it can be seen from table 1 that the bond dissociation energy (etotal) amounts to 31.94 kcal mol (bp86/tz2p). this value fits well to the experimental enthalpy estimated by haaland (31.1 1 kcal mol) as well as to other theoretical estimations. in line with previous studies , we found a slight dominance (by 0.7 kcal mol) of the electrostatic stabilization over the orbital interaction term (table 1). decomposition of the latter stabilizing term into nocv - based deformation density channels leads to the that donation (orb) from the lone electron pair of ammonia to the lowest unoccupied orbital of bh3 is by far most dominant (eorb = 66.32 kcal mol) as compared to the two hyperconjugation contributions, orbhyp1, orbhyp2; the corresponding orbital interaction stabilizations are eorbhyp1 = eorbhyp2 = 2.30 kcal mol (fig . 2). the latter two degenerated contributions stem from charge transfer from the occupied (b h) orbitals into the empty *(n h) (fig . 2). it is noteworthy that, in the isoelectronic ethane, the sum of stabilization arising from the two orthogonal hyperconjugation components was found to be significantly stronger (10 kcal mol).fig. b n bond lengths (in) are indicatedtable 1extended transition state (ets) energy decomposition describing the h3n bh3 bond in various isomers of ammonia borane. charge estimates for pauli repulsion contributions are indicated s e es e s e total 31.9430.011.9329.87e dist 12.6513.020.3712.65e elstat 77.3273.184.1477.8e pauli 109.39102.127.27111.5e orb 76.6671.974.6976.22 q globalpauli(q 1pauli + q 2pauli + q 3pauli)1.18331.16930.0141.2135 q 1pauli 0.72610.70490.02120.7267 q 2pauli 0.22860.23250.00390.2434 q 3pauli 0.22860.23190.00330.2434 e total = e orb + e pauli + e elstat + e dist labels assigned in fig. 1; e s corresponds to the eclipsed structure in the staggered geometry see eqs. 3, 4 in computational methods and fig. 2dominant natural orbitals for chemical valence (nocv)-based deformation density channels, orb, orbhyp1, orbhyp1, with the corresponding orbital interaction energies for the alternative isomers of ammonia borane. s fully optimized staggered isomer, e s eclipsed structure in the staggered geometry, e fully optimized eclipsed isomer energy profile for internal rotation in ammonia borane. n bond lengths (in) are indicated extended transition state (ets) energy decomposition describing the h3n bh3 bond in various isomers of ammonia borane. charge estimates for pauli repulsion contributions are indicated e total = e orb + e pauli + e elstat + e dist labels assigned in fig. 1; e s corresponds to the eclipsed structure in the staggered geometry see eqs. 3, 4 in computational methods and fig. 5 dominant natural orbitals for chemical valence (nocv)-based deformation density channels, orb, orbhyp1, orbhyp1, with the corresponding orbital interaction energies for the alternative isomers of ammonia borane. the contour value is || = 0.005 a.u. for orb, whereas for remaining hyperconjugation contributions 0.001 a.u. was applied. s fully optimized staggered isomer, e s eclipsed structure in the staggered geometry, e fully optimized eclipsed isomer it is clear from fig. 1 that rotation from the staggered to the eclipsed form leads to a change in energy, by 1.93 kcal mol. this barrier agrees quite well with the experimental value of 2.07 kcal mol determined based on microwave spectra, and with other high level computations. it is very important to point out that when going from the staggered (s) to the eclipsed isomer (e), one observes a notable stretch of the b such elongation leads expectedly to a significant decrease in pauli repulsion, by 7.27 kcal mol; at the same time the electrostatic (eelstat) and orbital interaction (eorb) contributions become less stabilizing, by 4.14 kcal mol and 4.69 kcal mol, respectively (see table 1 and the blue line in fig . 3). from the examples of ethane or biphenyl, it is known that this type of elongation when going from one isomer to the other is due to the steric (pauli) repulsion. as indicated in a series of recent works , in order to estimate and characterize the forces leading to such elongation , one must first consider rigid rotation from the staggered to the eclipsed conformation; we have labeled such eclipsed conformation (in the staggered geometry) as es. we can clearly see now from table 1 and fig. 3 (the orange curve), that an increase in the pauli repulsion contribution, by 2.11 kcal mol, is noted when going from s to es a similar trend, i.e., the maximum pauli repulsion in ammonia borane with the dihedral angle (h b n h) = 0.0, is noted when considering the rigid rotation from the geometry of the eclipsed structure to the staggered one (se) (gray curve in fig . thus, the pauli ( steric) repulsion contribution is responsible for stretching of the b n bond and, accordingly, for the rotational barrier in ammonia borane; the analogous situation holds true for the ethane molecule, as demonstrated first by bickelhaupt et al. and then by others. an increased kinetic repulsion (the main source of the pauli term) in the es geometry is related through the virial theorem to the existence of repulsive forces acting predominantly on nitrogen and boron nuclei. it must be added that hypercongutation stabilizations stemming from the charge transfer from the occupied (b h) orbitals into the empty *(n h) (orbhyp1, orbhyp2), favors the staggered conformation (fig . 2), although the effect is minor (0.4 kcal mol) compared to changes in the remaining bonding contributions (table 1). a quantitatively similar effect is observed for the change in the energy distortion contribution (edist) (table 1).fig. s fully optimized staggered isomer, e s eclipsed structure in the staggered geometry, s e staggered structure in the eclipsed geometry, e fully optimized eclipsed isomer pauli repulsion energies in the alternative isomers of ammonia borane. s fully optimized staggered isomer, e s eclipsed structure in the staggered geometry, s e staggered structure in the eclipsed geometry, e fully optimized eclipsed isomer let us now focus our attention on detailed changes in the pauli repulsion contributions in the three ammonia borane isomers, s, es and e. figure 4 presents the total pauli deformation density contours together with the corresponding energy values (epauli).fig. in addition, charge - based estimations are presented based on eq. 4. the blue / red contours correspond to accumulation / depletion of electron density due to the pauli exclusion principle contours of the total pauli deformation density together with the corresponding energies. the blue / red contours correspond to accumulation / depletion of electron density due to the pauli exclusion principle as already stated, the s es transition leads to a jump in the pauli term that it is then the important question that arises at this point is how the total pauli repulsion is an analysis of function leads to the observation that electrons are removed from the n b binding region (in fact, it is a manifestation of the pauli exclusion principle); although one can see that the red lobes extend also to the areas of nhhb interaction. however, such contours do not allow us to extract information on whether the total changes in pauli repulsion are determined by the repulsive interaction between the lone electron pair of ammonia with the occupied (b h) orbitals or directly by n - hh - b repulsion. in order to obtain such separated information, we have decomposed total pauli repulsion into the contributions (kpauli) according to eq. the three leading pauli deformation density channels, 1pauli, 2pauli, 3pauli, together with the corresponding quantitative charge estimations (eq . 4) are presented in fig. it should be noted that the total charge, qglobalpauli = q1pauli + q2pauli + q3pauli, that is removed from the h3n bh3 binding region correlates well with the trend based on the pauli repulsion energy (table 1, fig . qualitative inspection of the contours ipauli leads to the important observation that the first channel ( 1pauli) corresponds solely to the interaction between the lone electron pair of ammonia with the b h bonds, whereas the two latter orthogonal contributions (2pauli, 3pauli) show nhhb repulsion (fig . more importantly, quantitative analysis of the charge depletion, based on the eigenvalues ( eq . 3), leads to the that, when going from s es, the major changes (by 0.0148 a.u .) are within the second and third values of q2pauli, q3pauli. these show that an increase in the total repulsion in the eclipsed conformation compared to staggered (s es) is determined solely by the nhhb repulsion (of the kinetic origin) due to the pauli exclusion principle (an interaction between the electrons with the same spin as within the b h and n the repulsive contribution from the interaction between the lone electron pair of ammonia with the electrons of b h bonds ( 1pauli) is dominating in absolute terms; however, it does not influence the barrier. classical view that the internal rotational barrier in ammonia borane can be understood solely in terms of nhhb steric (pauli) effects, with minor participation stemming from the hyperconjugation (fig . 2) and geometry distortion term. it must be further noted that we performed a detailed study of the changes in qipauli values (based on various sets of molecules) and have found that differences in the second decimal place are quantitatively meaningful.fig. 5dominant pauli repulsion deformation density channels, 1pauli, 2pauli, 3pauli, together with the corresponding charge estimations, q 1pauli, q 2pauli, q 3pauli in the selected ammonia borane conformations. the blue / red contours correspond to accumulation / depletion of electron density. s fully optimized staggered isomer, e s eclipsed structure in the staggered geometry, e fully optimized eclipsed isomer dominant pauli repulsion deformation density channels, 1pauli, 2pauli, 3pauli, together with the corresponding charge estimations, q 1pauli, q 2pauli, q 3pauli in the selected ammonia borane conformations. s fully optimized staggered isomer, e s eclipsed structure in the staggered geometry, e fully optimized eclipsed isomer finally, we performed similar ets - nocv and pauli repulsion analyses for ethane and found that chhc pauli (kinetic) repulsion channels are responsible for the rotation of methyl groups (see supplementary electronic material); this is in line with the reached first by bickelhaupt and subsequently by other researchers. it is important at this point to cite another important work in the field explaining the origin of rotation in ethane. it is possible to decompose the barrier into changes in the kinetic (t) and potential energy contributions: nuclei-nuclei (vnn), nuclei-electrons (vne) and electrons - electrons (vee), as done by bader and others for ethane. such an approach, while very physical and compelling, does not allow, for example, separate discussion of the role of the hyperconjugation effect, which is well rooted in chemistry. it was shown that rigid rotation s es leads to a rise in all of the destabilizing terms (t + 9 kcal mol, vnn+vee + 16 kcal mol), whereas the electron - nuclei stabilization is vne 22 kcal mol. the rise in kinetic energy is related, through the virial theorem, to the repulsion force acting on the nuclei. accordingly, in the next step, ese, the cc bond elongates, which leads to weakening of the attraction (vne + 207 kcal mol) and decrease in the repulsion (vnn+vee 201 kcal mol ; t 3 kcal mol, the values are provided with respect to ethane in staggered geometry). one should also note that various authors have combined the above contributions in different ways. finally, liu and govind, defined in an elegant way at dft level, the steric contribution (equal to the kinetic weizscker term) from a difference between the total electronic energy and the sum of electrostatic (vne+vee+vnn) and quantum energy terms eq (comprising the sum exc+epauli); the change in the kinetic term due to the pauli exclusion principle is incorporated in epauli. it was shown that rigid rotation s es in the appearance of destabilizing forces originating from the fermionic quantum contribution eq; closer inspection of the author s data shows that this change is due entirely to a rise in the kinetic energy term. finally, one should cite the separate work of nagy, who discussed the fisher information based on the kinetic term; the role of kinetic energy and the information origin of the chemical bonding have been studied by nalewajski. the present work studied for the first time the internal rotation in ammonia borane based on our recently developed charge and energy decomposition scheme, ets - nocv, as well as the eigenvectors for pauli repulsion. detailed analyses of the electronic and the steric factors were performed in order to understand the origin of the barrier to rotation in ammonia borane. we found that the barrier to rotation, staggered eclipsed, is only 2 kcal mol. it was demonstrated using the ets - nocv scheme that the hyperconjugation, originating from the charge transfer from the occupied (b h) orbitals into the empty *(n h), favors the staggered isomer, although, quantitatively it leads to only a slight stabilization (5 kcal mol). for ethane, we have found, based on our newly proposed scheme, the natural orbitals for pauli repulsion, that rigid rotation from the staggered to the eclipsed conformation causes predominantly the enhancement of steric (pauli) repulsion acting solely between n h and b h bonds; this is subsequently relieved, leading to elongation of the b accordingly, the barrier to rotation in ammonia borane can be understood in a classical way; namely, as originating from the steric (pauli) repulsion contributions that act solely between n h and b h bonds. h bonds is dominant in absolute terms; however, it does not influence the barrier.

the internal rotation in ammonia borane (ab) was studied on the basis of natural orbitals for chemical valence (nocv) and eigenvectors for pauli repulsion (nopr). we found that the total hyperconjugation stabilization (ca . 5 kcal mol1), based on the charge transfer from the occupied (b h) orbitals into the empty *(n h), slightly favors the staggered conformation over the eclipsed one; however, the barrier to internal rotation in ammonia borane can be understood predominantly in a classical way, as originating from the steric (pauli) repulsion contributions (of the kinetic origin) that act solely between n h and b h bonds. repulsion between the lone pair of ammonia and the adjacent b h bonds was found to be dominant in absolute terms; however, it does not determine the rotational barrier. similar on the role of chhc repulsion appeared to be valid for isoelectronic ethane.figurepauli (kinetic) repulsion acting between the n - h and b - h bonds of ammonia boraneelectronic supplementary materialthe online version of this article (doi:10.1007/s00894 - 014 - 2272-y) contains supplementary material, which is available to authorized users.

the value of positron emission tomography (pet) and pet combined with computed tomography (ct) imaging in pediatric lymphomas has been fairly well described. however, because pediatric solid tumors are rare, the role of pet in their management is less well established. there is a growing body of literature about the use of pet - ct imaging in the diagnosis and management of pediatric sarcomas, the most common of which are the bone tumors, osteosarcoma (os) and ewing sarcoma (ews), and the soft - tissue malignancy, rhabdomyosarcoma (rms). therefore, this review focuses on the value of pet - ct imaging in the assessment of these malignancies. when pet - ct is performed in children, challenges unique to pediatric patients these include the need for sedation, pregnancy screening of patients and caregivers, and potential artifact caused by metabolically active brown fat. several pitfalls in interpretation of pet - ct in children with sarcomas are also presented. accurate anatomic coregistration of pet and ct images requires that the patient remain completely still throughout the procedure. because pet - ct examinations can be lengthy, sedation or general anesthesia may be required if children are unable to cooperate. when a sedation team is used, its members should rotate through the pet - ct area to minimize their radiation exposure. the need for sedation or anesthesia will influence patient scheduling and the timing of oral contrast administration when a diagnostic ct is also being performed. before sedation or anesthesia, (npo) period including 2 h of clear liquids only immediately before the procedure. therefore, when pet - ct patients must ingest oral contrast material for diagnostic ct, appointments are scheduled to allow adequate time between examinations so that sedation or anesthesia, if needed, can be safely administered. pregnant guardians of young patients must not be allowed in the room where radionuclide tracer (usually the glucose analogue fluorine-18 fluorodeoxyglucose) administration and uptake occur. further, before administration of any radioisotope, young female patients must be questioned about the possibility of pregnancy. this delicate subject is best handled by a technologist who is experienced in working with young girls. before fdg is administered at our institution, a nuclear medicine technologist questions female patients who are10 years of age or older, and all female guardians, about the possibility of pregnancy. when a patient or guardian is unsure of her pregnancy status, a pregnancy test is performed and must be negative before fdg is administered. brown adipose tissue, or brown fat, is metabolically active in pediatric patients, women, and persons with a low body mass index. the primary function of brown fat is the production of heat through an anaerobic, glycolytic pathway that in the uptake of glucose and hence of fdg. intense fdg activity is often observed within brown fat in the supraclavicular regions, axillae, paraspinal regions of the posterior mediastinum, and adjacent to the adrenal glands. intense fdg avidity in brown fat can be difficult to distinguish from cervical, supraclavicular, or axillary pathology (which are commonly seen in lymphoma). in such cases, we administer an anxiolytic agent, such as diazepam, the night before and the morning of pet - ct to reduce fdg activity in brown fat and improve the prominence of lesions (fig . ( a) this maximum intensity projection (mip) pet image shows intense fdg activity in metabolically active brown fat in the neck, supraclavicular areas and axilla. (b) the examination was repeated after administration of diazepam the evening before and on the morning of the pet - ct scan. brown fat fdg activity resolved and the site of pathologic uptake in the right neck (arrow) became more apparent. an 8-year - old boy with hodgkin (a) this maximum intensity projection (mip) pet image shows intense fdg activity in metabolically active brown fat in the neck, supraclavicular areas and axilla. (b) the examination was repeated after administration of diazepam the evening before and on the morning of the pet - ct scan. brown fat fdg activity resolved and the site of pathologic uptake in the right neck (arrow) became more apparent. because the value of pet imaging in lymphoma has been well described, this review focuses on the emerging role of pet - ct in the assessment of children with sarcomas. the conventional staging evaluation of these children includes magnetic resonance imaging (mri) or ct of the primary tumor and regional nodal beds, chest ct for detection of pulmonary metastases, and technetium-99 m methyldiphosphonate (mdp) bone scan for detection of bone metastases. the response evaluation criteria in solid tumors (recist), introduced in 2000 as an alternative to the world health organization (who) criteria, provide a standard, reproducible, and objective method of assessing the efficacy of solid tumor therapies. these criteria incorporate important advances in imaging technology and simplify the who scheme, but they still rely on changes in unidimensional tumor measurements to define tumor response or progression. unfortunately, solid malignancies, such as bone sarcomas and cystic soft - tissue sarcomas, may respond well to chemotherapy without substantially changing in size. further, sarcomas do not shrink or grow in a uniform manner; therefore, unidimensional measurements may not accurately reflect response or progression. tumors that respond poorly to therapy may be followed for months before a unidimensional measurement increases significantly. meanwhile, patients are exposed to toxic but ineffective chemotherapy and are likely to have a diminished probability of survival. fdg - pet has the advantage of revealing viable tumor tissue, which is highly metabolically active and therefore accumulates fdg. a decrease in tumor glucose uptake after treatment is correlated with a reduction in the percentage of viable tumor cells. therefore, fdg - pet may offer a more sensitive evaluation of the response or progression of some pediatric solid malignancies than do conventional, anatomic imaging modalities. in children with bone and soft - tissue sarcomas, the lung is the first site of distant spread and is involved in 2025% of patients at diagnosis. the prognosis is poor for children with os and pulmonary metastases unless all metastases are surgically resected. the survival of patients with ews and pulmonary metastases may be improved by whole - lung irradiation. therefore, chest ct is the gold standard for detection of pulmonary metastases; however, in a retrospective study of 41 children with solid malignancies who underwent biopsy of pulmonary nodules, we found that it has limited accuracy in distinguishing benign from malignant nodule histology. we also found only slight to moderate interreviewer agreement among 3 experienced pediatric radiologists who classified nodules as benign or malignant on the basis of ct features. there is clearly a need to improve the imaging distinction of benign from malignant pulmonary nodules to reduce the frequency of unnecessary thoracotomy. to assess the ability of pet alone to detect pulmonary metastases of os or ews, franzius and colleagues compared pet to ct, clinical follow - up, and histologic interpretation of resected nodules. among 30 pet scans performed at diagnosis or recurrence (before initiation of salvage therapy) , pet had a sensitivity of 14%, specificity of 91%, and accuracy of 73%. the ability of pet to detect malignant nodules increased with nodule size: 12 of 16 malignant nodules 10 mm were identified, whereas none measuring < 5 mm were detected (fig . their were supported by the recent work of vlker et al ., who investigated the value of pet alone for staging 46 cases of childhood ews ( n = 23), os (n = 11), and rms (n = 12). they found that 21 of 28 lung metastases detected by ct were missed by pet (sensitivity 25%) and that pet - positive nodules were 8 mm in diameter whereas pet - negative nodules were < 7 mm. gertha and colleagues showed that the fusion modality pet - ct detected pulmonary ews metastases better than pet alone in 53 patients. however, because pet - ct is generally performed with a low milliampere - second technique for ct scanning, image quality may be insufficient to detect very small pulmonary nodules. increasingly smaller nodules can now be detected by using current - generation, multislice helical scanners coupled with picture archiving and communication systems (pacs), whose window and level adjustment features and magnification tools improve image interpretation. we have shown that small pulmonary nodules (< 5 mm) are as likely to be malignant as larger nodules in children with solid malignancies. given the need to aggressively treat pulmonary metastatic disease in children with sarcomas, pet - ct is preferred over pet alone as a potential adjunct to distinguish benign from malignant nodules, but the inherent limitation of spatial resolution of the pet and ct components must be taken into account. we have also found that benign processes, such as active infection, can in substantial fdg uptake within pulmonary nodules, mimicking that seen in metastases (fig . 3). figure 2(a) these 0.8-cm nodules (arrows), metastatic from ewing sarcoma, seen on ct, show (b) no fdg activity on correlative pet (arrows indicate approximate location of nodules). in contrast, (c) this 1.1-cm nodule (arrow), metastatic from rhabdoid tumor, shows (d) intense fdg activity on correlative pet imaging (arrow). in children, nodule size affects the detection of fdg activity but not the likelihood of malignant histology. figure 3this 14-year - old boy was suspected of having lymphoma and underwent pet - ct. (a) ct shows an ill - defined, 1.8-cm pulmonary nodule (arrow) that on (b) pet showed intense fdg activity (arrow). (a) these 0.8-cm nodules (arrows), metastatic from ewing sarcoma, seen on ct, show (b) no fdg activity on correlative pet (arrows indicate approximate location of nodules). in contrast, (c) this 1.1-cm nodule (arrow), metastatic from rhabdoid tumor, shows (d) intense fdg activity on correlative pet imaging (arrow). in children, nodule this 14-year - old boy was suspected of having lymphoma and underwent pet - ct. (a) ct shows an ill - defined, 1.8-cm pulmonary nodule (arrow) that on (b) pet showed intense fdg activity (arrow). about 10% of os patients develop bone metastases, with or without concurrent pulmonary metastases. as many as 25% of patients with ews or rms experience bone or bone marrow involvement. because ews is radiosensitive, targeted radiation can be beneficial if these metastases are few and well defined. however, when more than 50% of the marrow is involved, irradiation can cause significant myelosuppression and compound the myelosuppressive toxicity of chemotherapy. unfortunately, the prognosis of patients with rms and bone or bone marrow metastases is not improved by surgical metastatectomy or targeted radiotherapy. because os metastases often contain calcification or ossification, they can typically be detected by the bone - seeking radioisotope tc mdp. franzius and colleagues directly compared pet to tc mdp bone scintigraphy for the detection of bone metastases in 32 patients with os. they found 5 bone metastases in 2 patients; all foci were evident on tc mdp bone scan, whereas none were demonstrated by pet. found 4 of 12 patients to have 31 bone metastases at the time of diagnosis of os; the sensitivity of detection was 90% for pet, compared with 81% for tc mdp scintigraphy. pet is more convincingly superior to tc mdp scintigraphy for the detection of osseous metastases. found that on 66 paired pet and tc mdp bone scans performed before or after initiation of therapy for ews, osseous metastases were detected by pet with a sensitivity of 100% (19/19), specificity of 96% (45/47), and accuracy of 97% (64/66), compared with 68% (13/19), 87% (41/47) and 82% (54/66), respectively, for tc mdp scintigraphy. 6 of whom had 49 osseous metastases, they found that the sensitivity of pet was 88%, compared with 37% sensitivity for tc bone scan (p < 0.01). in our practice , we have found pet - ct to be more sensitive than tc bone scan for the detection of osseous metastases in several children with rms or ews (fig . the greater sensitivity of pet to tc mdp scintigraphy in detecting osseous metastases of ews and rms is probably multifactorial . fdg - pet is postulated to detect the increased glucose metabolism of bone marrow metastases before an osteoblastic reaction is appreciable . in contrast, detection of bone metastases by tc bone scintigraphy depends on ossification within the metastatic deposit, the degree of associated cortical destruction, and the intensity of osteoblastic activity, all of which are greater in os bone metastases than in those of ews or rms . it is also possible that cellular glucose uptake and metabolism differ in these sarcomas . ( b) this mip pet anterior image, obtained 1 day later, shows widely metastatic disease throughout the axial and appendicular skeleton. the primary tumor is the focus of intense soft - tissue activity in the lower left calf (arrow). (b) this mip pet anterior image, obtained 1 day later, shows widely metastatic disease throughout the axial and appendicular skeleton. the primary tumor is the focus of intense soft - tissue activity in the lower left calf (arrow). (reprinted with permission from ajr 2005 ; 184 : 1293304 .) in assessing children with malignancies that metastasize to bone, it is important to consider benign processes that can mimic metastatic disease on pet imaging. akoi and colleagues assessed the value of pet in distinguishing benign (n = 33) from malignant (n = 19) bone tumors in 52 children and adults. they measured the mean standardized uptake value (suv) within a region of interest (roi) in the area of tumor with maximum fdg accumulation and found no significant difference between os and giant cell tumors (p = 0.171), os and fibrous dysplasia (p = 0.127), or fibrous dysplasia and chondrosarcomas (p = 0.667). further, they found no suv threshold value that reliably distinguished benign from malignant bone lesions. we reviewed pet - ct imaging performed to evaluate underlying cancers (n = 13) or aggressive fibromatosis (n = 1) in 14 children who also had benign fibrocortical defects, nonossifying fibromas, or cortical desmoids that were discovered as foci of fdg avidity. in some cases, the intense fdg avidity of these benign lesions mimicked the appearance of bony metastatic disease (fig . these common, benign lesions are often found incidentally on radiographs of children and young adults . they undergo spontaneous regression over time and are rarely seen after the second decade of life . because they have characteristic radiographic features that are well described in the literature, the information gained from correlative ct ( when pet - ct is performed) or plain radiographs is invaluable in determining whether further imaging or biopsy is necessary. (a) mip pet image shows a focus of intense activity above the left knee (arrow) that on (b) correlative ct and (c) axial pet images localized to a benign fibrocortical defect (arrows). (a) mip pet image shows a focus of intense activity above the left knee (arrow) that on (b) correlative ct and (c) axial pet images localized to a benign fibrocortical defect (arrows). local - regional lymph node metastasis is uncommon in os and ews but is present at diagnosis in as many as 20% of children with rms. nodal spread is present at diagnosis in approximately half of children with extremity rms and occurs more frequently in older boys (10 years) with paratesticular rms and in association with the alveolar rms subtype. when lymph node metastasis is present, the likelihood of survival can be improved by intensification of chemotherapy and irradiation of the affected region. therefore, identification of sites of lymph node involvement is crucial to the management and outcome of childhood rms. klem et al. investigated the value of pet in detecting metastases during the baseline staging of 24 patients with rms by comparing it to ct, mri, clinical assessment, and histology of resected lesions. the measured suv of suspicious foci was considered abnormal if it exceeded that of tissue and did not reflect a normal physiologic process. although the nodes were seen on conventional imaging, klem et al. did not report node size or the presence or absence of pathologic enlargement. an additional patient in the study had a lower extremity tumor and a pet interpretation a ct of the inguinal area was uninformative, and therefore chemotherapy was initiated after resection of the primary tumor, without inguinal node biopsy. tumor progression in the involved inguinal node subsequently became apparent on physical examination, pet, and mri and was confirmed by biopsy. the authors concluded that had the nodal involvement been confirmed at diagnosis, the patient might have benefited from more aggressive therapy. described 20 presumed lymph node metastases identified in 8 of 46 children with bone and soft - tissue sarcomas (1 with os, 1 with ews, and 6 with rms). a lesion - based analysis showed a sensitivity of 95% (19/20) for pet but only 25% (5/20) for ct and mri. this study was limited by the absence of size criteria defining abnormal lymph nodes on conventional imaging; further, 16 of the 20 presumed nodal metastases were not confirmed by pathology studies. in a small case series by ben arush and colleagues, 3 children with alveolar rms underwent pet - ct and conventional imaging for baseline staging; 2 had moderately fdg - avid regional lymph nodes from which biopsies were taken. the third patient had mild fdg avidity within a regional node but no biopsy was taken. the patient was started on therapy for high - risk disease; at the time of scheduled follow - up, adenopathy was palpable in the area of the previously mildly fdg - avid node. we have found that benign processes, such as reactive hyperplasia, can cause regional node enlargement and fdg avidity on pet imaging in children with sarcomas (fig . the available evidence suggests that although pet may identify sites of nodal disease not appreciated on conventional imaging, care should be used in interpreting pet findings . metastatic lymph nodes may demonstrate only mild fdg avidity, whereas nonmetastatic nodes may show intense avidity . therefore, biopsy of suspicious lymph nodes should be considered when there is discordance between the findings of different imaging modalities or between imaging and clinical findings and when identification of nodal disease will affect patient management and outcome . figure 6a 20-year - old woman with malignant peripheral nerve sheath tumor in the left thigh, underwent baseline pet - ct . ( a) ct shows enlarged iliac nodes (arrows) ipsilateral to thigh tumor that on (b) pet showed intense fdg activity. biopsy of these nodes revealed reactive hyperplasia. a 20-year - old woman with malignant peripheral nerve sheath tumor in the left thigh, underwent baseline pet - ct. (a) ct shows enlarged iliac nodes (arrows) ipsilateral to thigh tumor that on (b) pet showed intense fdg activity. patients with nonmetastatic os and ews are treated with neoadjuvant therapy before surgical resection of the primary tumor and subsequent adjuvant therapy. chances of survival are improved when 90% or more of the resected tumor is necrotic and resection margins are void of tumor. factors that predict the histologic tumor response to neoadjuvant therapy might also predict patient outcome and help to identify candidates for limb - sparing surgery versus amputation or early resection. a reliable, noninvasive method for assessing tumor metabolism and viability, such as pet, could allow the oncologist and surgeon to individualize management of tumors that are aggressive, respond poorly, or arise in surgically challenging sites. osteogenic sarcoma and ews are metabolically heterogeneous tumors. because tumor histologic grade and predicted patient outcome are determined by the percentage of viable tumor in the resected specimen, the maximum tumor suv is believed to provide the most accurate noninvasive assessment of tumor grade. several investigators have compared the maximum tumor suv before and after neoadjuvant therapy with tumor grade and patient outcome. in 2002, hawkins and colleagues reported a comparison of the histologic response of resected tumors (18 os and 13 ews) to tumor suv at diagnosis (suv1) and at the end of neoadjuvant therapy (suv2). in these 31 patients, histologic response was significantly associated with the suv2 (p = 0.01) and the suv2/suv1 ratio (p = 0.01). an suv2 < 2 had a positive predictive value (ppv) of 93% for identifying tumors that were 90% or more necrotic and a 75% negative predictive value (npv) for identifying tumors that were < 90% necrotic (unfavorable response). when an suv2/suv1 cutoff point of 0.5 was used, the ppv and npv were 78% and 63%, respectively. in 2005, hawkins and coworkers reported similar findings in a follow - up study of 36 patients with ews who had pet imaging before and after neoadjuvant therapy. among the 32 patients who underwent resection of the primary tumor, an suv2 < 2.5 had a ppv of 79% for a favorable response and an npv of 40% for an unfavorable response. the ppv and npv for an suv2/suv1 ratio 0.5 were 77% and 33%, respectively. an suv2 < 2.5 was also associated with greater 4-year progression - free survival (pfs) in all 36 patients (p = 0.006) and in the 24 patients who had no metastatic disease at diagnosis (p = 0.036). these findings and those of others suggest that the maximum suv of primary os and ews after neoadjuvant therapy can predict tumor response and patient outcome. further studies are needed to determine whether the suv can be used to identify unresponsive or poorly responsive tumors earlier in the course of neoadjuvant therapy, so that therapy can be appropriately modified. pet imaging of children requires an expert team of professionals to manage the unique technical challenges encountered in this age group. further, the interpreting physician must be aware of the aspects of pediatric anatomy and physiology that differ from those in adults. whereas the role of pet in the assessment of lymphomas is fairly well established, the growing body of evidence supports the continued investigation of pet imaging in the management of pediatric sarcomas. pet may provide valuable information about pulmonary nodules in children, but accurate interpretation requires awareness of its limitations. small malignant pulmonary nodules may not be appreciable on pet imaging, whereas large benign nodules may show intense fdg avidity. pet may detect osseous metastases of childhood ews and rms earlier than mdp bone scintigraphy, because bone marrow infiltration precedes cortical destruction and an osteoblastic reaction. on the other hand, the bone - seeking radiotracer mdp may offer superior detection of lytic and ossifying bone metastases often associated with os. when pet is used to detect bone metastasis in children, an awareness of the common benign bone lesions in this age group is essential, as they can mimic metastatic disease. in such cases the correlative ct imaging obtained during pet - ct, or plain - film radiography, can preclude the need for further imaging or biopsy. pet imaging that shows sites of local - regional nodal fdg uptake in children with bone and soft - tissue sarcomas should be interpreted with caution. biopsy of suspicious lymph nodes is probably indicated when the findings of different imaging modalities or of imaging and physical examination are inconsistent. because the size of bone tumors does not change substantially in response to neoadjuvant therapy, ct and mri have limited value for assessment of tumor response. because fdg - pet reflects the metabolic activity of tumors, which can be semi - quantitatively measured as the suv, pet holds promise as an alternative method of assessing response in these tumors. additional studies are needed to better define the optimal timing of pet during neoadjuvant therapy to detect poorly responsive tumors and allow early intervention.

abstractpositron emission tomography (pet)-computed tomography (ct) is emerging as a valuable tool for assessing a wide variety of pediatric malignancies, including lymphomas, soft - tissue tumors, and bone sarcomas. pet - ct may provide information that is not apparent on conventional imaging performed to stage these diseases and monitor their response to treatment. the use of pet - ct in children requires an awareness of the technical and logistical issues unique to this patient population. in addition, interpretation of pediatric pet - ct imaging requires familiarity with aspects of pediatric anatomy and physiology that differ from those of adults. in this article, the technical considerations in performing pediatric pet - ct, pitfalls in the diagnostic use of pet - ct in children, and current and emerging applications of pet - ct in pediatric oncology are reviewed.

although hundreds of thousands of papers on the topic can easily be found in computerized bibliographies, as well as in works from earlier times and obstetrical manuals published over the last two centuries, there is still no universally accepted way to manage breech delivery. even metaanalyses do not give a clear answer to the question as to which mode of delivery is better. a recent study by hannah (toronto) has proven that cesarean section (c - section) seems to be safer for the infant. when weighing the pros and cons of c - section, however , one should be aware that these may be due to the safety of low segment surgery and the safer anesthesia now available. on the other hand, the problem of the uterine scar has to be considered, especially in families that want to have more children. there have also been some suggestions that babies delivered by c - section more frequently suffer from respiratory problems. two radically different approaches to ceserean section become apparent among mothers. while some actually this kind of delivery, others regard it as a last resort, not only in view of possible complications for mother and child, but also because of the delayed return to professional activity as a of surgical complications. the last follow - up performed in this study was done at two years of age. the question may fairly be asked as to whether or not it suffices to do the follow - up at this age, since most babies are doing well at this time. some of them may be suffering from various kinds of damages that present only at a later age, and we do not know how they cope during the school years or in later life. this was the main reason we decide to analyze our by extending the follow - up study to late childhood. the aim of our study was to assess the presence of signs of organic disorders, using psychological examination tools, in children delivered vaginally versus c - section, separately for primiparas and multiparas. in the department of obstetrics at the medical university of gdansk, 917 breech deliveries took place between 1981 and 1990. all the mothers in question received a questionnaire, sent to the address we found in the hospital records; they were asked to answer some questions and to indicate whether they might be interested in a follow - up psychological and neurological examination of their breech delivered child. we received positive responses from 232 mothers, who provided us with considerable information about the children s further development and problems that had arisen during their school years. all the respondents were contacted by telephone, and 83 of them agreed to visit our department with their children to undergo a psychological examination. the ages of the children included in this study ranged from 9 to 18 years. the whole study was carried out with the consent of our university bioethical committee. in this paper we analyze the of psychological tests intended to assess the possibility of organic brain disorders that could have been due to the breech delivery. the following tests were performed: the bender - kopitz test (bkt); the benton visual retention test (bvrt). the bkt is a modified version of the lauretta bender test, a familiar method for detecting organic brain damage in adult patients. the version used in our study was developed by elisabeth kopitz to examine children, in order to detect organic brain disorders. it is also used to investigate minimal brain dysfunction, the level of development of visuo - motor integration, and emotional disorders. in order to classify the as normal, almost normal, or pathological they were assessed on the basis of polish norms, where a score between 090 is treated as relatively normal, 91100 is borderline normal, and a score of 101 or higher indicates organic lesions of the central nervous system. it allows us to assess memory disorders, concentration disorders, and orientation and motor disorders in the case of many patients suffering from psychiatric and neurological disorders. the subject draws an image from memory immediately after having looked at it for 10 seconds, so that the test detects the abnormal decay of visual information. there are 10 images, each of which contains one or more figures (usually three). the of the bvrt were assessed both on the basis of the number of mistakes made by the patent, and the number of correct representations. in both cases, depending on the level of the patient s intelligence and his age, both the expected number of mistakes and the expected number of correct representations were calculated on the basis of the instruction. if the score obtained in our study in terms of the number of correct answers was 2 points lower than expected, this was treated as a somewhere between normal and pathological. when the was 3 points lower, it was treated as pathological. as for the number of mistakes made by the patient, the was treated as a borderline when it was higher than expected by 3 points, while a 4 points higher than expected was considered to be pathological. the were analyzed statistically using the test, with the level of significance established at p<0.05. since delivering for the first time is thought to have an impact on the prognosis in breech delivery, we analyzed the separately for primiparas and multiparas. we analyzed each group as a whole and with respect to the presenting part, dividing each group into complete breech, frank breech and footling breech cases. nearly 90% of children born by primiparas presented with no organic brain damage assessed by the bvrt. half of these (3 cases) were in the vaginal delivery group, and the other half in the c - section group. only one abnormal from the bender - kopitz test was noted in the vaginal delivery group. statistical analysis of from both tests revealed no statistically significant differences between groups. in only 1 child with complete breech at delivery abnormal bvrt were found in 4 cases, 2 cases each in the vaginal delivery and c - section groups, with frank breech presentation at delivery. only 1 abnormal bender - kopitz test was found in the vaginal delivery group. statistical analysis revealed no influence of the mode of delivery on the incidence of abnormal test . in all deliveries from the footling presentation at delivery in primiparas a c - section was performed, thus no statistical analysis could be carried out. although no statistical analysis was possible for the footling presentation in primiparas, we mention our here to stress that abnormal were also found in the c - section group. we may therefore safely assume that the protective value of the c - section is disputable. 81.3% of babies born by multiparas from the breech presentation presented no signs of organic brain disorders as assessed by the benton test. statistical analysis showed no influence of the mode of delivery on the incidence of brain damage in these children (table 8). abnormal from the bender - kopitz test were found in only one child born from the breech presentation. statistical analysis did not reveal any difference between the mode of delivery groups. in the complete breech delivery group in multiparas, thus no comparison of the mode of delivery was possible, and our have only a purely informative function. one 8-year - old child in this group had abnormal on both tests (12.5%). although comparative analysis was not possible, we can assume that most children born vaginally from complete breech presentation by multiparas presented no signs of organic brain disorders. 78.6% of the children born vaginally from frank breech by multiparas and 85.7% of those born by c - section presented no signs of brain damage in the benton visual retention test. all children delivered from frank breech presentation by multiparas had normal on the bender - kopitz test regardless of the mode of delivery. the presented above show no influence of the mode of delivery on the incidence of organic brain dysfunction in multiparas with frank breech presentation at delivery. we were unable to perform a comparison of the mode of delivery in this group. even though we had no deliveries by c - section in this group, all were found normal in the bender - kopitz test, and only 1 was abnormal in the benton visual retention test, so we can consider the vaginal mode for breech delivery to be a safe one. a normal on the bender - kopitz test is an indirect indicator of a correct, or at least satisfactory level of development of visual perception and visual coordination. since many authors, e.g. losiowski et al. , connect the score in the apgar scale in the fifth minute of life with, among other things, the level of psychomotor development and neurological status, this relation was also investigated in our research. no correlation was found between the number of points scored by a child on the apgar scale and the of the benton test (r=0.001, f=0.000025, p<0.996) or the bender test (r=0.206, f=2.084, p<0.156). nearly 90% of children born by primiparas presented with no organic brain damage assessed by the bvrt. half of these (3 cases) were in the vaginal delivery group, and the other half in the c - section group. only one abnormal from the bender - kopitz test was noted in the vaginal delivery group. statistical analysis of from both tests revealed no statistically significant differences between groups. in only 1 child with complete breech at delivery abnormal bvrt were found in 4 cases, 2 cases each in the vaginal delivery and c - section groups, with frank breech presentation at delivery. only 1 abnormal bender - kopitz test was found in the vaginal delivery group. statistical analysis revealed no influence of the mode of delivery on the incidence of abnormal test . in all deliveries from the footling presentation at delivery in primiparas a c - section was performed, thus no statistical analysis could be carried out. although no statistical analysis was possible for the footling presentation in primiparas, we mention our here to stress that abnormal were also found in the c - section group. we may therefore safely assume that the protective value of the c - section is disputable. nearly 90% of children born by primiparas presented with no organic brain damage assessed by the bvrt. half of these (3 cases) were in the vaginal delivery group, and the other half in the c - section group. only one abnormal from the bender - kopitz test was noted in the vaginal delivery group. statistical analysis of from both tests revealed no statistically significant differences between groups. in only 1 child with complete breech at delivery did we find abnormal bvrt and bender - kopitz test . abnormal bvrt were found in 4 cases, 2 cases each in the vaginal delivery and c - section groups, with frank breech presentation at delivery. only 1 abnormal bender - kopitz test was found in the vaginal delivery group. statistical analysis revealed no influence of the mode of delivery on the incidence of abnormal test . in all deliveries from the footling presentation at delivery in primiparas a c - section was performed, thus no statistical analysis could be carried out. although no statistical analysis was possible for the footling presentation in primiparas, we mention our here to stress that abnormal were also found in the c - section group. we may therefore safely assume that the protective value of the c - section is disputable. 81.3% of babies born by multiparas from the breech presentation presented no signs of organic brain disorders as assessed by the benton test. statistical analysis showed no influence of the mode of delivery on the incidence of brain damage in these children (table 8). abnormal from the bender - kopitz test were found in only one child born from the breech presentation. statistical analysis did not reveal any difference between the mode of delivery groups. in the complete breech delivery group in multiparas, thus no comparison of the mode of delivery was possible, and our have only a purely informative function. one 8-year - old child in this group had abnormal on both tests (12.5%). although comparative analysis was not possible, we can assume that most children born vaginally from complete breech presentation by multiparas presented no signs of organic brain disorders. 78.6% of the children born vaginally from frank breech by multiparas and 85.7% of those born by c - section presented no signs of brain damage in the benton visual retention test. all children delivered from frank breech presentation by multiparas had normal on the bender - kopitz test regardless of the mode of delivery. the presented above show no influence of the mode of delivery on the incidence of organic brain dysfunction in multiparas with frank breech presentation at delivery. we were unable to perform a comparison of the mode of delivery in this group. even though we had no deliveries by c - section in this group, all were found normal in the bender - kopitz test, and only 1 was abnormal in the benton visual retention test, so we can consider the vaginal mode for breech delivery to be a safe one. a normal on the bender - kopitz test is an indirect indicator of a correct, or at least satisfactory level of development of visual perception and visual coordination. since many authors, e.g. losiowski et al. , connect the score in the apgar scale in the fifth minute of life with, among other things, the level of psychomotor development and neurological status, this relation was also investigated in our research. no correlation was found between the number of points scored by a child on the apgar scale and the of the benton test (r=0.001, f=0.000025, p<0.996) or the bender test (r=0.206, f=2.084, p<0.156). 81.3% of babies born by multiparas from the breech presentation presented no signs of organic brain disorders as assessed by the benton test. statistical analysis showed no influence of the mode of delivery on the incidence of brain damage in these children (table 8). abnormal from the bender - kopitz test were found in only one child born from the breech presentation. in the complete breech delivery group in multiparas, all the children were delivered vaginally. thus no comparison of the mode of delivery was possible, and our have only a purely informative function. one 8-year - old child in this group had abnormal on both tests (12.5%). although comparative analysis was not possible, we can assume that most children born vaginally from complete breech presentation by multiparas presented no signs of organic brain disorders. 78.6% of the children born vaginally from frank breech by multiparas and 85.7% of those born by c - section presented no signs of brain damage in the benton visual retention test. all children delivered from frank breech presentation by multiparas had normal on the bender - kopitz test regardless of the mode of delivery. the presented above show no influence of the mode of delivery on the incidence of organic brain dysfunction in multiparas with frank breech presentation at delivery. we were unable to perform a comparison of the mode of delivery in this group. even though we had no deliveries by c - section in this group, all were found normal in the bender - kopitz test, and only 1 was abnormal in the benton visual retention test, so we can consider the vaginal mode for breech delivery to be a safe one. a normal on the bender - kopitz test is an indirect indicator of a correct, or at least satisfactory level of development of visual perception and visual coordination. since many authors, e.g. losiowski et al. , connect the score in the apgar scale in the fifth minute of life with, among other things, the level of psychomotor development and neurological status, this relation was also investigated in our research. no correlation was found between the number of points scored by a child on the apgar scale and the of the benton test (r=0.001, f=0.000025, p<0.996) or the bender test (r=0.206, f=2.084, p<0.156). controversies surrounding breech deliveries have engaged many researchers and considerable financial resources. a new approach to this subject was achieved by mary hannah in a multicentre study coordinated by the university of toronto. the achieved in this study have indicated that the c - section is safer for breech delivered babies, but there has been much discussion on its reliability. many obstetricians remained unconvinced generally, not due to other, contradictory published afterwards, or doubts about the methodology, but usually on the basis of their own experience in breech deliveries. the idea of terminating each breech delivery by c - section has led to the loss of obstetrical skills among older obstetricians and the lack thereof in the younger practicioners. after many years of such a policy in obstetrics in the developed countries, nowadays, the lack of the requisite skill on the part of the physician has become yet another indication for doing a c - section routinely. we decided to analyze our own data and to present them in the present study. despite the general division into the group of primiparas and multiparas, each group of deliveries was analyzed in total and in subgroups of complete breech, frank breech and footling breech presentation. we were motivated to perform this study after the appearance of papers suggesting that babies delivered by c - section more often present respiratory disorders in comparison to those delivered vaginally. in all groups and subgroups we found no influence of the mode of delivery on the incidence of organic brain disorders in later childhood, as assessed by the benton visual retention test and by the bender - kopitz test. it should be emphasized, however, that not every brain damage in a worse mastery of the visual memory tasks measured by the benton visual retention test and by the bender - kopitz test. many factors come into play, including the size, kind and location of the damage and its persistence. it is also worth pointing out that mistakes in the drawing tests are connected first of all with the dysfunction of the posterior (occipital, parietal and temporal) lobes. this in difficulties in so called organic tests, which could be a genuine indicator confirming organic brain dysfunction; nevertheless, the lack of such difficulties can not be the basis for excluding organic brain damage. these children also underwent examination for neurological and other psychological disorders for other studies, and no differences between groups were found. some researchers have investigated the relation between the number of points on the apgar scale and intellectual - cognitive functioning in early childhood. it should nevertheless be stressed that methods of assessing infants are not definite prognosticators of the eventual intellectual level, if they are considered in separation from other risk factors [ 20,21. the vaginal route is a safe method of delivery in breech presentations in both primiparas and multiparas.

summarythe authors performed a long term outcome analysis of minimal brain damage in children delivered in breech presentation, and related the to the mode of delivery (vaginal or by cesarean section).material / methodsin the department of obstetrics at the medical university of gdansk (poland), 917 breech deliveries took place between 1981 and 1990. excluding stillbirths and multiple pregnancies, 874 deliveries were analyzed. we received positive responses from 232 mothers, who provided us with considerable information about the children s further development and problems that had arisen during their school years. all the respondents were contacted by telephone, and 83 of them agreed to visit our department with their children to undergo a psychological examination the following tests were performed: 1 ) the bender - kopitz test (bkt), and 2 ) the benton visual retention test (bvrt).the mode of delivery for all groups and subgroups had no influence on the incidence of organic brain disorders in later childhood, assessed by the benton visual retention test and by the bender - kopitz test.vaginal breech deliveries are safe in both primiparous and multiparous mothers.

the german research network on schizophrenia (grns) is one of 17 existing medical research networks funded by the german federal ministry of education and research (bmbf) since 1999 in order to improve care of patients with illnesses characterized by high morbidity and/or mortality. each of these networks is funded for a maximum of 8 years, with up to 2.5 million euro per year during the first 5 years and up to 0.5 million euro per year for the last 3 years. additional sponsoring (about 5% of the budget) by the industry is provided for research projects or public relations activities of the network. one of the main reasons that the bmbf established such networks originates from the fact, that new knowledge from research is only insufficiently transferred to practice, and problems in everyday care worth being scientifically investigated are only insufficiently recognized by researchers. thus, the structural aim of the grns - in a way to be taken as a precondition - consists of the establishment of long - term communication structures between research, services, consumers, and the public. a means for attaining these aims is the creation or expansion, intensified utilization, and routine application of collaboration and knowledge exchange within (horizontal networking) and between (vertical networking) the two levels of research and care. with regard to the funding by the federal ministry of research , the framework for attaining these objectives is that, of research projects, which constitute the means for intensified collaboration between institutions and optimized care for patients with schizophrenia. the network comprises about 25 interrelated research studies and projects on health care education with high practice relevance. a current total of 16 psychiatric university departments and 14 state and district hospitals, as well as six local networks of psychiatric practices and general practitioners throughout germany, participate in these studies. as regards content, the major objective of the grns is to create the scientific preconditions for the implementation of strategics for early detection and early intervention in the prodromal stage of the first episode (project network i), for the optimization of acute and long - term treatment in first - episode patients and for the rehabilitation in patients with residual symptoms (project network ii). quality of care in hospitals and practices is evaluated and improved by quality assurance programs implementing the existing guidelines for outand inpatient treatment. basic research on structural and functional brain imaging and genetic markers investigates underlying determinants of the manifestation and re - manifestation of the illness as well as the individual response to drug treatment (special network on molecular and pharmacogenetics). a number of more general projects on fighting stigma and discrimination, health care economy, postgraduate training, quality assurance, and methodology complete the spectrum of network projects (see ref 1 and www.kompetenznetz-schizophrenie.de for more information). generally, the studies are multicenter studies designed in such a manner that vertical and horizontal networking is forced, essential, or at least, supported. in order to create synergy and added value as important criteria for successful networking, most, of the projects are strongly interrelated regarding conceptual , method, and organization. the superordinate aim of these studies is to allow for an improvement of the course and the outcome of schizophrenia along with considering cost - benefit aspects. since some of the studies are long - term studies which finished recruitment only recently, reliable will only be available later in 2006. thus, the description will mainly focus on the concept of the studies with regard to the improvement of the management of schizophrenia. it is known that the first, treatment, contact of people suffering from schizophrenia is preceded by a period of manifest, psychotic symptoms, on average lasting for 1 year, and a prepsychotic prodromal period of about 5 years with increasing negative and unspecific symptoms and functional impairment. at, the same time , it has been shown that a delayed treatment, is associated with significant disadvantages for schizophrenia patients, often ing in functional and social decline. thus, in order to optimize outcome it seems essential to recognize and treat at - risk persons and schizophrenia patients as early as possible. for this purpose awareness programs are being carried out as a first step in several german cities within the grns, in order to improve utilization of newly founded early - recognition centers by at - risk persons. as a second step, two early recognition and intervention studies are carried out; these are also used to prospectively validate a two - step early - recognition inventory (eri) and a set, of cognitive tests developed for early detection of at - risk persons. for the two early intervention studies, two groups of atrisk persons are selected from the larger group of persons referred to the early - recognition centers, according to their presumed prodromal stage. early initial prodromal stage is assumed in case subjects report predictive basic symptoms in the eri or in case they have a first - degree relative with schizophrenia and show a marked decline in global functioning. late initial prodromal stages are defined by the occurrence of brief limited intermittent psychotic symptoms (blips) or by attenuated positive symptoms. persons at risk for psychosis in the early prodromal state are included into an early intervention study examining the effects of a newly developed cognitivebehavioral therapy (cbt) strategy for prodromal persons, which is compared with clinical management, within a randomized control design over a 24-month period. persons in the late prodromal state of psychosis are included into a second early intervention study, which compares the effects of atypical antipsychotic medication with amisulpride in combination with clinical management (supported by crisis intervention or family counseling in case of need, but no regular psychotherapy) to such clinical management alone. this is a phase - ill study with an open - label, randomized parallel design, with a treatment period of 2 years. effects of both studies will be evaluated with regard to improvement of prodromal symptoms, prevention of social decline, and suppression, or at least, delay, of progression to psychosis. preliminary of both studies are encouraging, indicating a benefit for at - risk persons treated with cbt or amisulpride, respectively, compared with the control treatments with regard to these outcome variables. should these trends be validated in the final analyses, the use of early recognition and early intervention strategies as developed within the grns would be an important, step in the management, of developing psychosis. though a number of studies have shown advantages of atypical second - generation antipsychotics compared with conventional antipsychotics in acute treatment (for review see ref 12) as well as in long - term treatment, of schizophrenia (for review see ref 13) it is still under debate whether these may be biased by the high dosages of conventional antipsychotics usually used in these studies. low - potency neuroleptics even might not induce more extrapyramidal side effects under a lowdose strategy than second - generation drugs and the potential advantage of atypical antipsychotics may be compromised by their side effects, such as weight, gain and metabolic effects. nevertheless, atypical antipsychotics are recommended as first - choice treatment for both first- and multiple - episode schizophrenia or for first - episode schizophrenia preferentially. however, independent, long - term studies in first - episode patients substantiating these recommendations are lacking or are still under way, such as the clinical antipsychotic trials of intervention effectiveness (catie) trial in the us and the european first episode schizophrenia trial (eufest) study in europe; beyond this uncertainty regarding the best kind of antipsychotic treatment for the special group of first - episode patients, it is furthermore unclear how long treatment should be continued after cessation of the first, acute phase. published guidelines recommend treatment durations of minimum 1 year; the appropriate duration of further treatment in case of symptom remission, however, has not been adequately specified. in order to contribute to these open questions, a comprehensive acute and long - term treatment, study in patients with first - episode schizophrenia is currently been conducted in up to 13 german university hospitals within the grns. the study comprises a prospective doubleblind, randomized, parallel - group comparison of risperidone as a new - generation antipsychotic with halopcridol as a conventional antipsychotic. both drugs are administered in rather low daily dosages of 2 to 8 mg per day during the 8 weeks of acute treatment, and thereafter in a reduced dosage - where possiblc - of 2 to 4 mg per day during a 2-year long - term treatment period. to investigate the necessary duration of long - term treatment in first - episode patients, patients completing the first treatment year without, relapse are randomly allocated to either maintenance treatment, or stepwise drug discontinuation in the second treatment year. in case of impending re - exacerbations, prodrome - based early intervention, either by means of resumption or augmentation of neuroleptic treatment (depending on the basic treatment strategy of discontinuation or maintenance treatment) or by means of treatment / additional treatment, with the benzodiazepine lorazepam is applied in the second treatment year to prevent relapses. this randomized, double - blind comparison shall contribute to the open question of whether prodromes are unspecific consequences of stress experience, treatable with benzodiazepines, or have to be regarded as more specific, prepsychotic symptoms requiring neuroleptic treatment. preliminary findings so far suggest that the treatment, with low dosages of antipsychotics is feasible and effective, and leads to a significant improvement, in positive, negative, and prodromal symptoms in first - episode schizophrenia patients. none of the patients has fulfilled the criteria for relapse within the first year of treatment. because the medication is only about, to be unblinded, comparison of differential treatment or side effects between both drugs is not yet available. schizophrenia patients often exhibit, impairments in facial affect recognition (see ref 31 for review), which is already present in first - episode patients, and even in unaffected siblings of schizophrenia patients. such impairments are strongly associated with more global social dysfunctions characteristic of schizophrenia and may have adverse effects on psychosocial functioning independent of the presence and severity of positive and negative symptoms and cognitive deficits. thus, these impairments represent, a core feature of the disorder and are of high relevance for the psychosocial functioning of the patients. the traditional drug and psychological treatment usually administered to schizophrenia patients seem to be ineffective in this regard, as indicated by the stability of the impairment, across different stages of the disorder despite treatment. against, this , a new training program for the remediation of such impairments has been developed within the grns. (tar) have been compared with a cognitive remediation program (crt) primarily aiming at improving attention, memory, and executive functioning, and with treatment, as usual (tau) without participation in a specific remediation program within a randomized three - group pre - post design. indicated that, patients on tar significantly improved in facial affect recognition, with recognition performance after training approaching the level of healthy controls from former studies. patients on crt and those without special training (tau) did not improve in affect recognition, though patients on crt improved in verbal memory functions. according to these , remediation of disturbed facial affect recognition in schizophrenia patients is possible, but not achievable with a traditional cognitive rehabilitation program such as the crt. instead, functional specialized remediation programs such as the newly developed tar arc a more suitable option. whether these promising training effects of the tar endure across time and pervade into everyday social functioning has to be investigated by future studies. if these effects can be validated, such training programs could become an important, module of psychosocial rehabilitation programs in future. optimizing treatment, of schizophrenia through implementation of guidelines is essential for early and acute as well as long - term and chronic phases of schizophrenia. such measures of quality assurance shall guarantee optimal care in accordance with the state - of - the - art knowledge under consideration of available resources. at present , it is estimated that only 40% to 50% of schizophrenia patients arc treated according to scientific standards and treatment guidelines. although several treatment, guidelines for schizophrenia have been published in recent, years, it has been supposed that only a case - focused implementation of these guidelines will lead to an improvement in outcome quality measures. therefore, two projects targeting quality assurance, either in inpatient, care or in outpatient care, have been performed within the grns. the first of these projects targeted the systematic development, implementation, and evaluation of specific measures of quality management in inpatient treatment of 597 schizophrenia patients at seven psychiatric hospitals, mostly district hospitals. using an experimental control group design with preand post - assessments, quality - orientated interventions according to the concept of total quality management, (tqm) and with reference to the german treatment guidelines were compared in four experimental hospitals with documentation of structural parameters (hospital and patient characteristics), of treatment, and of outcome in three control hospitals. experimental hospitals received feedback by means of comparative benchmarking, and were guided in implementing quality circles for specific problem areas identified from the benchmarking process. indicated that, poorer average clinical outcome was associated with lower guideline conformity in a variety of treatment domains. after case - mix adjustment, benchmarking proved to be an opportunity to improve quality of treatment and promote guideline conformity. the main focus was to implement guidelines, but also other elements of internal (documentation system, monitoring) and external (benchmarking) quality management, in four hospital - associated networks of private psychiatric practices in three different, german cities (dsseldorf, freiburg, and munich). one of the three experimental groups used a computer - based documentation system with implemented treatment, guidelines and decisionsupport, and received comparative benchmarking. this computerized documentation system draws the attention of the physician to the treatment guidelines by means of a pop - up window showing the relevant guideline algorithm whenever the entered data indicate critical changes in the patient's clinical status. two further experimental groups used either the computer - based documentation system without, implemented guidelines and benchmarking, or papcr - and - pencil documentation with additional organization in quality circles. a control group used paper - and - pencil documentation without additional organization in quality circles. in 583 patients with schizophrenia treated by 55 psychiatrists for at least 16 months demonstrated a significantly better outcome in patients in the experimental practices, either using the decision support, system or working with quality circles, as compared with those practices merely documenting their treatment, either computer - based or with paper and pencil, but without, further measures of quality assurance. the stigma associated with mental illness and psychiatric treatment, and the discrimination toward people with mental illnesses that frequently from this, are the main obstacles preventing early and successful treatment. to reduce such stigma and discrimination, especially towards people with schizophrenia, the world psychiatric association's (wpa) global anti - stigma program fighting stigma and discrimination because of schizophrenia - open the doors is currently being implemented in 27 countries. since august 1999, the campaign has also been carried out in seven cities in germany, partly within, and with funding of, the grns. a survey of attitudes towards people with mental illness was conducted at the beginning of this campaign in 7246 persons in six german cities by telephone using a standardized questionnaire. the respondents were asked about, their knowledge with regard to schizophrenia, their social distance from people with schizophrenia, and estimations of the social stigmatization of mental patients in general. thereafter public information programs and educative measures aimed at selected target groups were performed, and the opportunity for personal contact with mentally ill people was promoted in two of the cities in order to improve the public's knowledge regarding symptomatology, causes, and treatment options for schizophrenia. the first of a recently executed second survey of the same persons indicate that such improvement, could indeed be partly obtained in these two cities, whereas no comparable changes occurred in the cities not participating in the antistigma campaign. the next step to be performed is to investigate whether improved knowledge in turn also contributes to abolishing prejudice and negative perceptions and facilitates the social reintegration of those suffering from mental illness. significant structural improvements regarding intensified collaboration between and within the research and care levels have already been achieved. moreover, significant contributions to improved management of schizophrenia have already been obtained, for instance in the area of quality assurance in inpatient, and outpatient treatment. several studies regarding early detection and early intervention, as well as treatment, of first - episode schizophrenia, were initially designed as long - term studies lasting up to 5 years, which only recently reached the phase of analysis. due to the comprehensive design of these carefully coordinated studies targeting a number of important and open questions in schizophrenia the next essential task will be to transfer these into health care. based on a successful midterm evaluation, further funding has recently been granted by the bmbf until mid2008 in order to promote this transfer process. thus, development, and implementation of measures for early recognition and intervention, for treatment of firstepisode schizophrenia, for quality management, and for destigmatization will be in the focus of this last funding period before the grns has to finance itself by other resources. these measures will comprise development, of manuals and brochures, and continued medical education measures, as well as the setting up of special competence centers for each of these topics. nevertheless, an ongoing aim of the grns will still be to offer a research platform, particularly for clinical studies, in order to continue successful horizontal networking between the institutes of research. maintenance and extension of the existing dna and clinical data banks will be an important part, of this effort. the complexity of psychiatric disorders on the one hand, and the progressive specialization in research, especially when using complex biological methods, on the other hand, in an increasing necessity for inter- and intradisciplinary collaboration organized into larger networks like the grns. primarily, such a strategy seems promising to find answers to the urgent and complex questions regarding schizophrenia that are still unresolved.

the german research network on schizophrenia (grns) is a nationwide network currently comprising 16 psychiatric university departments and 14 state and district hospitals, as well as six local networks of psychiatric practices and general practitioners collaborating on about 25 interrelated, multicenter projects on schizophrenia research. the grns aims to intensify collaboration and knowledge exchange betvi / een leading research institutions and qualified routine care facilities, both within (horizontal network) and between (vertical network) the two levels of research and care, in order to create the scientific preconditions for optimization of the management of schizophrenia. the concept and the first of studies aiming at the investigation of(i) strategies for early detection and early intervention in the prodromal stage of psychosis; (ii) treatment in first - episode schizophrenia; (iii) quality management; and (iv) destigmatization, are described as examples of this effort.

three - dimensional (3d) image - based planning approaches to high dose rate (hdr) brachytherapy of cervical cancer have only been introduced in most departmental brachytherapy programs in the past decade, with magnetic resonance (mr) imaging now recommended as the imaging modality of choice. current international recommendations state that t2-weighted, fast - spin - echo (fse) or turbo - spin - echo (tse) sequences to enable gross tumour to appear as high signal intensity masses should be taken in the para - axial, para - coronal, and para - sagittal planes when possible, for the ease of clinical target volume (ctv) visualisation. ideally, ctv contouring and applicator reconstruction should be performed on the same image sequence to reduce uncertainties in image fusion and patient / organ motion between different sequences. however, it has been acknowledged that there are often difficulties accurately visualising the applicator in t2-weighted mr images , particularly those with large slice thicknesses. in those instances, additional sequences such as a t1-weighted mr scan, a ct scan or even x - ray images may be required. these recommendations are based on studies conducted in brachytherapy treatment centres using low (0.1 - 0.5 tesla) and high (1.0 - 1.5 t) magnetic resonance imagers, with acknowledgement that these studies contained no experience with higher (3 t or higher) magnetic resonance imagers. several diagnostic imaging studies have shown that 3 t mr image of the female pelvis provides better image contrast and signal to noise ratio in the uterine cervix and vagina , and there is continuing work in the evaluation of 3 t mr scans for brachytherapy imaging. brachytherapy, along with surgery and external beam radiotherapy (with or without chemotherapy) remains a significant part of the pattern of care for stage i - iva cervical cancer. hdr brachytherapy is generally performed post external beam irradiation, which provides greater initial tumour shrinkage, allowing the brachytherapy boost to sterilise any residual tumour while sparing bladder, rectum and small bowel from excessive dose. cervical cancer has a high - signal intensity on t2 weighted mr images. evaluation of t2 weighted mr images, taken at diagnosis, during treatment (external beam), after external beam (for brachytherapy), and up to 6 months follow up are often a good indication of how the tumour is responding to treatment. unfortunately, any inflammation, fibrosis and oedema ing from the external beam irradiation may also exhibit intermediate to high signal intensity on t2 weighted images. gec - estro have recommended these areas of intermediate to high signal intensity be incorporated into either the high or intermediate risk clinical target volume (ctv). this makes mr image quality an important consideration in brachytherapy planning images and image resolution a major issue in accurate brachytherapy applicator and ctv determination. in mr imaging, some important considerations are the signal - to - noise ratio (snr) and image resolution. a 3 t mr image has approximately double the snr of a 1.5 t mr image. by reducing slice thickness of the 3 t mr scan, it is possible to maintain similar snr to a 1.5 t mr image, whilst increasing the resolution. snr is a central factor in hr ctv contouring and whilst resolution is critical in both hr ctv contouring and applicator reconstruction. the purpose of this study was to determine a suitable 3 t mr t2-weighted image sequence for 3d planning of hdr cervical cancer brachytherapy treatments. the increased image quality from a 3 t mr image was hoped to produce a single image sequence for contouring tumour and organs at risk, applicator reconstruction, and treatment planning. a single image set will be more useful not only for treatment planning purposes but also a time - advantage to those departments that do not have immediate access to an mr scanner and may need to transfer a patient from theatre to radiology for treatment imaging. the study was performed on 20 cases referred to the radiation oncology department for the treatment of cervical cancer with brachytherapy. all patients had mr imaging performed for brachytherapy planning after plastic - based, mr compatible ring applicator (ring ct / mr applicator, nucletron, an elekta company) placement under ultrasound guidance and general anaesthesia. the age range of patients was between 31 - 88 years with a mean age of 54.3 years and all patients were rated figo (international federation of gynaecology and obstetrics) stage iib to iiib. the range of contoured hr ctv for all patients was between 47.9 and 9.7 cm with a mean of 22.6 cm. the range of hr ctv dimensions (l - r, s - i and a - p) were between 4.5, 4.6 and 5.5 cm and 1.9, 1.7, and 1.8 cm, respectively. all patients underwent external beam radiation therapy (ebrt) between 45 and 52.2 gy in 1.8 or 2 gy fractions. hdr brachytherapy (24 gy in 8 gy fractions prescribed to the 100% isodose line) was commenced either within the final week or within a week of finishing ebrt. each insertion was given 1 week apart, with the applicator removed after each insertion and mr - based treatment planning occurring for each fraction. all patients had cervical hdr brachytherapy applicators inserted under general anaesthetic, underwent mr scanning in radiology after leaving recovery and were then transported to the department of radiation oncology for treatment. a 3 t (skyra, siemens healthcare ag, germany) mr imaging system with combination of an 18 channel body matrix and 32 channel phase array spine coil was used for generating hdr cervix brachytherapy planning images. patients were positioned in supine head - first orientation with a body matrix coil placed over the pelvis. a small foam wedge was placed between the pelvis and coil to help reduce anterior abdominal wall motion. the pre - emptied bladder was filled with 100 ml of saline for adequate distension of the bladder during imaging and reproducibility of bladder volume at treatment. an anti peristalsis agent was administered intravenously (hyoscine butylbromide 20 mg in 1 ml) to minimise ghosting artefacts from gross peristaltic movements during data acquisition. scanning sequences that were part of the standard radiation therapy scanning practice in our department were considered for assessment. in collaboration with a radiologist and mr imaging specialist, a parallel imaging technique, generalized auto - calibrating partially parallel acquisition (grappa) algorithm (siemens healthcare ag, germany) with r factor of 2 spatially selective saturation bands were used superiorly and anteriorly on all sequences to suppress the signal from inflowing spins and reduce motion related artefacts. t2 weighted turbo spin echo (tse) sequences were selected without the application of iv contrast media in keeping with international recommendations. both 3d t2-weighted sampling perfection with application - optimised contrast and different flip - angle evolutions (space, siemens healthcare ag, germany) (3d t2) sequences and contiguous 2d t2-weighted tse sequences (2d t2) scans were performed for comparison in sagittal, axial, and oblique planes tilted to correspond to the treatment applicator. firstly, a 3-plane half - fourier acquisition single - shot turbo spin - echo (haste, siemens healthcare ag, germany) localizer was taken. this haste scan was used for planning the axial and oblique - axial sequences oriented to correspond to the treatment applicator. initially, a 2d t2 weighted tse (2d t2) sequence was obtained in the sagittal plane between the obturator muscles, and a 2d t2 sequence was positioned and acquired in a true axial plane and reviewed for both applicator and tissue delineation. a 3d t2 volumetric sequence was also performed in a true axial plane with an isotropic 3d t2 volumetric sequence acquired in the sagittal plane. both a 2d t2 sequence and a 3d t2 volumetric sequence were planned and acquired para - axially with the scanning plane aligned parallel to the ring surface from the sagittal haste scan (axial oblique, see fig . another 2d t2 sequence was then obtained with the scanning plane parallel to the ring surface, based on both the sagittal and the coronal reconstructions of the haste localiser ( axial double oblique, see fig . 2). axial - oblique plane, aligned parallel to the plane of the ring in the sagittal scan axial double - oblique additional plane, aligned parallel to the plane of the ring in the coronal localizer scan all scan volumes included the uterine fundus superiorly and the distal end on the treatment applicator inferiorly. all scanning sequences were designed to have slice thicknesses less than 2 mm to aid in 3d reconstruction in the brachytherapy treatment planning system. turbo spin echo, space sampling perfection with application - optimised contrast and different flip - angle evolutions, tr relaxation time, te echo time, fov field of view, nex number of excitations after importation into the treatment planning system (oncentra brachy version 4.2 and 4.3, nucletron, an elekta company, sweden), the images were initially evaluated by an experienced medical physicist with regards to the ability to reconstruct the treatment applicators. an applicator library (applicator modelling module in oncentra version 4.2 and 4.3, nucletron, an elekta company, sweden) was used in the reconstruction of each applicator. in this module, a minimum of 3 pre - defined points (anchor points) are required in order to reconstruct the applicator. successful if, after placement of the anchor points and any additional manual manipulation, the reconstructed applicator was clearly seen to lie within 2 mm of the visualised applicator in the mr images. this value was chosen as it represents the tolerance placed on applicator reconstruction defined by the aapm task group 56. each image set was then assessed for total scanning time and usefulness in tumour localization via inter- and intra - observer analysis of high - risk clinical target volume (hr ctv) contouring. hr ctv contours were generated based on gec - estro recommendations for each data set by three radiation oncologists experienced in gynaecological brachytherapy, who were blinded to the scanning technique. anatomical changes and geographical shifts between imaging sequences were also examined, along with visual differences between the contouring and planning modules of the brachytherapy treatment planning system. intra - observer analysis (comparing contours from a single radiation oncologist between different imaging sequences) was performed by determining the relative volume difference from the mean in hr ctvs between image sets for the same patient. these values were then averaged across all patients in the study. inter - observer analysis (comparing contours from all three radiation oncologists for a single image sequence) was performed not only by determining the relative volume difference in hr ctvs, but also by applying a conformity index (ci) based on common and encompassing volume for all 3 observers. the ci was determined based on the work by kouwenhoven et al. that allowed a ci to be determined independent of the number of observers. the ci is given by the following equation:1ci=k=1kk(k-1)vk2(k-1)k=1kkvk-k=1kk(k-1)vk where: k total number of observers, v k volume encompassed by k observers. the study was performed on 20 cases referred to the radiation oncology department for the treatment of cervical cancer with brachytherapy. all patients had mr imaging performed for brachytherapy planning after plastic - based, mr compatible ring applicator (ring ct / mr applicator, nucletron, an elekta company) placement under ultrasound guidance and general anaesthesia. the age range of patients was between 31 - 88 years with a mean age of 54.3 years and all patients were rated figo (international federation of gynaecology and obstetrics) stage iib to iiib. the range of contoured hr ctv for all patients was between 47.9 and 9.7 cm with a mean of 22.6 cm. the range of hr ctv dimensions (l - r, s - i and a - p) were between 4.5, 4.6 and 5.5 cm and 1.9, 1.7, and 1.8 cm, respectively. all patients underwent external beam radiation therapy (ebrt) between 45 and 52.2 gy in 1.8 or 2 gy fractions. hdr brachytherapy (24 gy in 8 gy fractions prescribed to the 100% isodose line) was commenced either within the final week or within a week of finishing ebrt. each insertion was given 1 week apart, with the applicator removed after each insertion and mr - based treatment planning occurring for each fraction. all patients had cervical hdr brachytherapy applicators inserted under general anaesthetic, underwent mr scanning in radiology after leaving recovery and were then transported to the department of radiation oncology for treatment. a 3 t (skyra, siemens healthcare ag, germany) mr imaging system with combination of an 18 channel body matrix and 32 channel phase array spine coil was used for generating hdr cervix brachytherapy planning images. patients were positioned in supine head - first orientation with a body matrix coil placed over the pelvis. a small foam wedge was placed between the pelvis and coil to help reduce anterior abdominal wall motion. the pre - emptied bladder was filled with 100 ml of saline for adequate distension of the bladder during imaging and reproducibility of bladder volume at treatment. an anti peristalsis agent was administered intravenously (hyoscine butylbromide 20 mg in 1 ml) to minimise ghosting artefacts from gross peristaltic movements during data acquisition. scanning sequences that were part of the standard radiation therapy scanning practice in our department were considered for assessment. in collaboration with a radiologist and mr imaging specialist, a parallel imaging technique, generalized auto - calibrating partially parallel acquisition (grappa) algorithm (siemens healthcare ag, germany) with r factor of 2 was incorporated in all sequences to reduce scan times to tolerable limits. spatially selective saturation bands were used superiorly and anteriorly on all sequences to suppress the signal from inflowing spins and reduce motion related artefacts. t2 weighted turbo spin echo (tse) sequences were selected without the application of iv contrast media in keeping with international recommendations. both 3d t2-weighted sampling perfection with application - optimised contrast and different flip - angle evolutions (space, siemens healthcare ag, germany) (3d t2) sequences and contiguous 2d t2-weighted tse sequences (2d t2) scans were performed for comparison in sagittal, axial, and oblique planes tilted to correspond to the treatment applicator. firstly, a 3-plane half - fourier acquisition single - shot turbo spin - echo (haste, siemens healthcare ag, germany) localizer was taken. this haste scan was used for planning the axial and oblique - axial sequences oriented to correspond to the treatment applicator. initially, a 2d t2 weighted tse (2d t2) sequence was obtained in the sagittal plane between the obturator muscles, and a 2d t2 sequence was positioned and acquired in a true axial plane and reviewed for both applicator and tissue delineation. a 3d t2 volumetric sequence was also performed in a true axial plane with an isotropic 3d t2 volumetric sequence acquired in the sagittal plane. both a 2d t2 sequence and a 3d t2 volumetric sequence were planned and acquired para - axially with the scanning plane aligned parallel to the ring surface from the sagittal haste scan (axial oblique, see fig . another 2d t2 sequence was then obtained with the scanning plane parallel to the ring surface, based on both the sagittal and the coronal reconstructions of the haste localiser ( axial double oblique, see fig . axial - oblique plane, aligned parallel to the plane of the ring in the sagittal scan axial double - oblique additional plane, aligned parallel to the plane of the ring in the coronal localizer scan all scan volumes included the uterine fundus superiorly and the distal end on the treatment applicator inferiorly . all scanning sequences were designed to have slice thicknesses less than 2 mm to aid in 3d reconstruction in the brachytherapy treatment planning system . magnetic resonance scanning parameters used for image acquisition tse turbo spin echo, space sampling perfection with application - optimised contrast and different flip - angle evolutions, tr relaxation time, te echo time, fov field of view, nex number of excitations after importation into the treatment planning system ( oncentra brachy version 4.2 and 4.3, nucletron, an elekta company, sweden), the images were initially evaluated by an experienced medical physicist with regards to the ability to reconstruct the treatment applicators. an applicator library (applicator modelling module in oncentra version 4.2 and 4.3, nucletron, an elekta company, sweden) was used in the reconstruction of each applicator. in this module, a minimum of 3 pre - defined points (anchor points) are required in order to reconstruct the applicator. successful if, after placement of the anchor points and any additional manual manipulation, the reconstructed applicator was clearly seen to lie within 2 mm of the visualised applicator in the mr images. this value was chosen as it represents the tolerance placed on applicator reconstruction defined by the aapm task group 56. each image set was then assessed for total scanning time and usefulness in tumour localization via inter- and intra - observer analysis of high - risk clinical target volume (hr ctv) contouring. hr ctv contours were generated based on gec - estro recommendations for each data set by three radiation oncologists experienced in gynaecological brachytherapy, who were blinded to the scanning technique. anatomical changes and geographical shifts between imaging sequences were also examined, along with visual differences between the contouring and planning modules of the brachytherapy treatment planning system. intra - observer analysis (comparing contours from a single radiation oncologist between different imaging sequences) was performed by determining the relative volume difference from the mean in hr ctvs between image sets for the same patient. inter - observer analysis (comparing contours from all three radiation oncologists for a single image sequence) was performed not only by determining the relative volume difference in hr ctvs, but also by applying a conformity index (ci) based on common and encompassing volume for all 3 observers. the ci was determined based on the work by kouwenhoven et al. that allowed a ci to be determined independent of the number of observers. the ci is given by the following equation:1ci=k=1kk(k-1)vk2(k-1)k=1kkvk-k=1kk(k-1)vk where: k total number of observers, v k volume encompassed by k observers. all sequences were able to be imported into the brachytherapy treatment planning system, however, those sequences that contained oblique planes required any automatic image fixing to be disabled. due to the use of plastic - only treatment applicators, image distortion effects that are known to be present particularly in 3 t mr imaging were negligible. visualization and eventual reconstruction of the applicator by the medical physicist ed in agreement between the applicator model and the mr images of 4 mm at first pass, and less than 2 mm after manual manipulation for all imaging sequences. table 1 indicates the scanning times for each of the sequences tested. a double oblique axial t2 3d scan was not used in this study as the scanning time the double oblique axial t2 2d scan had the fastest scan time after the t2 2d sagittal scan, however the sagittal scan had a much larger slice thickness (2.5 mm compared with 1.8 mm). for intra- and inter - observer hr ctv contouring are shown in table 2. for intra - observer hr ctv analysis, both 2d and 3d sagittal image sequences had the worst consistency across all radiation oncologists with an average difference of 19.8% and 15.1%, respectively. the best volume consistency across all radiation oncologists was for 2d axial double oblique and 3d axial oblique image sequences, with an average difference of less than 1%, and there was an average difference of 3.1% across all 2d and 3d axial image sequences. intra- and inter - observer differences for different 3 t t2 mr scanning sequences when comparing volume sizes only for inter - observer hr ctv analysis, the mean relative sd across all cases and sequences was 24.2%. the 2d axial double oblique image sequence had the best consistency with a mean relative sd of 9.2% and the 2d sagittal the worst with a mean relative sd of 37%. the 2d axial double oblique image sequence had the highest conformity index (mean 0.80, range 0.72 - 0.91), while the 2d axial image had the worst (mean 0.55, range 0.54 - 0.75). problems may arise with reconstruction from mr imaging scans as it is very difficult to visualize the internal structure of the applicator to confirm the source path. whilst markers and applicator libraries can be effective in detailing the source path, there are uncertainties in reconstruction that relate directly to the slice thickness of the imaging scans used. reconstruction uncertainties have been noted to be greater in the direction perpendicular to slice orientation which is commonly linked to the large pixel size or slice thickness in this direction. it is recommended that slice thickness be as small as possible without increasing mr image acquisition time too greatly as this increases the risk of patient motion during the scan. reducing the mr slice thickness to less than 2 mm can help minimize random error in applicator reconstruction. this type of scan is only achievable in high strength (3 t) mr scanners, as the loss of snr, increased scanning time, and increased potential for patient motion would be too great for lower strength mr scanners. the gec estro recommendations for 3d image guided brachytherapy of the cervical cancer are acknowledged as being a well - defined method for creating target and organ at risk volumes with which to optimize a patient treatment. even with these guidelines, there are still uncertainties and different interpretations of clinical and pathological disease, implying that it is not possible to have complete agreement between observers. there have been several studies into inter - observer differences for target volume delineation in cervical cancer brachytherapy. performed a multi - institutional study with 10 observers and 6 cases. the inter - observer relative sd for the hr ctv was up to 42% with an average conformity index of 0.74. in 2008, petri et al. investigated hr ctv contour differences between axial and para - axial (oblique axial) image sets for a 0.2 t mr scanner. the inter - observer conformity index for the axial and para - axial sequences was given as 0.79 and 0.78, respectively. there was a large difference between the 2d t2 axial oblique and 2d t2 axial double oblique, 30.6% mean relative sd dropping to 9.2% mean relative sd. this indicates that all scanning planes must be carefully and individually designed before a scan takes place and highlights the importance of testing multiple imaging planes, when setting up a mr imaging program for cervical cancer brachytherapy. the dosimetric impact of inter - observer variability has been studied, with levels of uncertainty in hr ctv contouring causing variation of 5 gy in d90 over the total course of radiotherapy (external beam and brachytherapy) or an uncertainty of 9% for a single intracavitary brachytherapy fraction. this study used images from mr scanners with magnet strength 0.2 t and 1.5 t. the mean relative sd of the contoured hr ctv was 19% (across 10 observers and 6 cases). interestingly, the patients scanned on the 0.2 t mr scanner had lower mean sd than the patients scanned on the 1.5 t (17% vs. 21%). this is compared to 9.2% for 3 t mr 2d axial double oblique scans in the present study. in any case , it is ideal to eliminate as much as possible any potential causes of observer contouring variation. there have been several studies that have looked into ways to reduce this variation. in these they recommended multiple imaging modalities, the latter two recommendations can be addressed using the gec - estro contouring criteria , while the second recommendation can be achieved by optimizing the choice of scanning sequence and the quality of the imaging system. studied the differences in contours between 2d multi - planar scanning sequences (as recommended by gec estro) and a 3d single scanning sequence for a 1.5 t mr scanner and found little difference in target contours for 2 observers and 14 patients. the of our study also indicate that there is minimal difference in hr ctv contours when completed on 2d or 3d image sets using a 3 t mr scanner. several diagnostic studies have also indicated little difference in image quality between 2d t2 weighted and 3d space t2 weighted images. the main benefit of using a 3d space t2 image set was defined as the time saving over using multi - planar 2d t2 image sequences. if the 2d multi - planar image set could be reduced to a single image set without compromising image quality in 3-dimensional reconstructions, the reduction in scanning time could be even further improved over the 3d t2 sequence. this study has examined 7 different 3 t mr scanning sequences in order to determine which single image set provides the best consistency for target delineation, both intra- and inter - observer. whilst a limitation to this study is the size of the number of observers (n = 3), this work still highlights the variability that the choice of scanning sequence can produce. the organ at risk (oar) contour variation was not performed in this study. further work investing the possible changes to oar volume definition for varying 3 t mr sequences will be carried out. patient shifts sometimes seen between scanning sequences can vary the position of the applicator relative to the ctv and oar, ing in inconsistencies if using multiple scanning sequences to contour and plan. this would be a particular concern if one sequence was used for contouring and another sequence used for planning as the relationship between source position and ctv or oar may have changed between scanning sequences. sagittal and coronal images as recommended by gec estro are currently mostly used for verification purposes as treatment planning software does not allow users to contour a single structure across multiple imaging planes. a standard sagittal 2d t2 tse sequence scan can also be performed if time permits, and there appears to be little patient motion during the para - axial scan. this scan can then be a useful tool in single plane verification of the ctv and oar contours if required. using the 2d axial double oblique scanning sequence, the total scanning time for each patient can be reduced to less than 7 minutes. even with a bladder filling protocol and administration of anti - peristalsis agent in the scanning room , patients can be in and out of the mr scanning room in less than 15 minutes. this has multiple benefits to the patient with less time isolated inside the scanning room and less time required to be lying compliant on the scanning table. this is particularly important in smaller centres that do not have the capability to generate treatment planning images, whilst the patient is under a general anaesthetic. the rapid turnaround of patients also allows a more efficient use of staff time and equipment resources. different mr scanning sequences can greatly affect the disparity in hr ctv contours between radiation oncologists. intra and inter - observer variation in hr ctv contouring suggests a 2d t2 tse axial sequence oriented in all planes to the treatment applicator (double oblique) on a 3 t mr scanner, allows more accurate and uncomplicated tumour determination in brachytherapy treatment planning of cancer of the cervix. applicator visualization is sufficient for planning requirements and the thinner slice thickness reduces the expected uncertainty in applicator reconstruction. the ability to contour, plan, and optimize the brachytherapy treatment using a single image set that is orientated in the most useful plane for these tasks is a great benefit. this imaging sequence was tested on 20 cases, but has since been successfully applied to another 35 cases, including patients using an interstitial / intracavitary applicator (interstitial ring ct - mr applicator set, nucletron, an elekta company, sweden). minimizing scanning time through elimination of excess scanning sequences implies greater patient throughput via reduced demand on the mr scanner and lessens patient discomfort via reducing the time required to be on the mr scanning table. this is a critical factor in departments that do not have access to an mr unit whilst the patient is under general anesthetic.

purposethe superior image quality of 3 tesla (3 t) magnetic resonance (mr) imaging in cervical cancer offers the potential to use a single image set for brachytherapy. this study aimed to determine a suitable single sequence for contouring tumour and organs at risk, applicator reconstruction, and treatment planning.material and methodsa 3 t (skyra, siemens healthcare ag, germany) mr imaging system with an 18 channel body matrix coil generated hdr cervical cancer brachytherapy planning images on 20 cases using plastic - based treatment applicators. seven different t2-weighted turbo spin echo (tse) sequences including both 3d and contiguous 2d scans based on sagittal, axial (transverse), and oblique planes were analysed. each image set was assessed for total scanning time and usefulness in tumour localization via inter- and intra - observer analysis of high - risk clinical target volume (hr ctv) contouring. applicator reconstruction in the treatment planning system was also considered.the intra - observer difference in hr ctv volumes between 2d and 3d axial - based image sets was low with an average difference of 3.1% for each observer. 2d and 3d sagittal image sets had the highest intra- and inter observer differences (over 15%). a 2d axial double oblique sequence was found to produce the best intra- (average difference of 0.6%) and inter - observer (mean sd of 9.2%) consistency and greatest conformity (average 0.80).there was little difference between 2d and 3d - based scanning sequences; however the increased scanning time of 3d sequences have potential to introduce greater patient motion artifacts. a contiguous 2d sequence based on an axial t2-weighted turbo - spin - echo (tse) sequence orientated in all planes of the treatment applicator provided consistent tumour delineation whilst allowing applicator reconstruction and treatment planning.

imatinib, a specific tyrosine kinase inhibitor has shown excellent efficacy in management of chronic myeloid leukemia (cml). there may be almost complete remission hematologically in the chronic phase of management of cml due to this drug. most commonly reported adverse events are maculopapular eruptions, periorbital edema, and the less commoner ones include steven johnson syndrome - toxic epidermal necrolysis (sjs / ten), acute generalized exanthematous pustulosis, hypopigmentation, lichenoid reaction, pityriasis rosea, and sweet's syndrome. a 52-year - old male presented to the medical outpatient department with complaints of low grade fever and lump on the left side of abdomen since one month duration. bone marrow examination revealed 2% blast cells, serum lactate dehydrogenase was 1134 iu / l, and an ultrasound of the abdomen revealed splenomegaly with span of 19 cm. the case was diagnosed as cml in chronic phase and patient was prescribed imatinib mesylate 400 mg once daily. ten days later, he developed redness and scaling involving dorsum of both hands which increased to involve his entire body over a period of 20 days. there was no history of any other drug intake, or history of fever, jaundice, chest pain, palpitation and dyspnoea on exertion. imatinib was continued and the rash worsened. when the patient was referred to us, he had generalized skin rash of 40 days duration. general physical examination was unremarkable while systemic examination revealed splenomegaly, 3 cm below costal margin. dermatological examination revealed generalized involvement of the body in form of dusky, blanchable erythema, and diffuse fine scaling. lichenification was noted in flexures viz groin, axillae and neck. investigations revealed hemoglobin of 14.5 gm%, total leukocyte count 11,500/mm, and differential count was polymorphs- 50, lymphocytes- 8, monocytes- 2, and eosinophils 40. urine examination, blood sugar, renal, and liver function tests were within normal limits. extensive scaling of (a) neck (b) axillae and upper chest involvement of (a) lower limbs and (b) both forearms imatinib was immediately stopped and patient was prescribed on tablet prednisolone 40 mg / day, which was maintained at full dose for two weeks, tapered by 10 mg every week, and stopped after a total of five weeks. supportive measures including high protein diet, appropriate temperature control, fluid, and electrolyte balance were provided. improvement was noted with decreased erythema on fifth day and significant reduction in scaling by day 11. the patient was changed to an alternative anti - cml medication (cyclophosphamide based) and rechallenge or desensitization was not resorted to, as erythroderma is considered a severe form of drug reaction. despite common occurrence of cutaneous adverse event with imatinib, severe adverse cutaneous drug reactions are uncommon and seen in 5% of cases. acute generalized exanthematous pustulosis, epidermal necrolysis, and steven johnson syndrome have been reported previously. other rare reactions mentioned include mycosis fungoides like eruption, follicular mucinosis, porphyria cutanea tarda, neutrophilic eccrine hidradenitis, eccrine squamous syringometaplasia, and panniculitis. there are only six probable cases of exfoliative dermatitis due to imatinib reported in literature. exfoliative dermatitis generally occurs 1 - 3 wks after starting therapy on initial exposure and within hours to days on rechallenge. mechanism of development of rash after imatinib administration is not known, however, hypersensitivity reaction as a mechanism has been postulated. in this case causality assessment using naranjo scale showed that imatinib was the probable cause for the adrs (score 7). most of the rashes due to imatinib are self limiting and do not require discontinuation of treatment. desensitization therapy can be used by administering increasing doses of drug in cases of mild reactions. erythroderma necessitating stoppage of therapy is considered as grade 4 toxicity according to national cancer institute / national institutes of health (nci / nih) common toxicity criteria. it is important to recognize the cutaneous adverse effects of this drug, so that severe and potentially life threatening adverse reaction can be identified early and remedial action can be taken early. it is equally important to continue therapy in minor cutaneous adverse events so that patient can get benefit from treatment with this gold standard drug for cml.

imatinib, a specific tyrosine kinase inhibitor is a newer anticancer agent, which has shown excellent efficacy in managing chronic myeloid leukemia. it is generally well tolerated with few side effects. most commonly reported adverse events are maculopapular eruptions and periorbital edema. severe adverse reactions are seen in 5% of patients. exfoliative dermatitis has been very rarely reported with this drug. we report a case of a 52-year - old male who initially presented with a maculopapular rash and developed erythroderma on continuation of the drug.

the prevalence of childhood - onset type 2 diabetes mellitus has increased dramatically over the past two or three decades in japan. several nation - wide surveys have been conducted, but epidemiological and clinical data remain limited. it is well known that life style modification and weight loss are the most important interventions that may realistically be expected to prevent disease progression and complications in type 2 diabetes mellitus in adults. if the treatment goals with nutrition education and exercise are not met, pharmacologic therapy is indicated for children with type 2 diabetes. indeed, many pediatricians realize the necessity of medical therapy for type 2 diabetes in children, and research on oral anti - hyperglycemic agents for children and adolescents is warranted. the uk prospective diabetes study (ukpds) reported that intensive blood - glucose control with sulfonylureas (sus) or insulin substantially reduced the risk of complications but not macrovascular disease in adult type 2 diabetes patients. the ukpds also reported that, since intensive glucose control with metformin appears to decrease the risk of diabetes - related endpoints in overweight diabetic patients, and is associated with less weight gain and fewer hypoglycemic attacks than are insulin and su, it may be the first - line pharmacological therapy of choice for these patients. metformin has been recommended as a first choice for the treatment of type 2 diabetes in children and adolescents by the american diabetes association (ada). metformin improves glycemic control by reducing hepatic glucose production, increasing insulin sensitivity, and reducing intestinal glucose absorption, without increasing insulin secretion. in the u.s. , metformin was approved by the fda in 2000 for the treatment of diabetes in children based on from a randomized controlled trial. in japan, no oral anti - hyperglycemic agents, including metformin, have been approved for children with type 2 diabetes by the ministry of health, labour and welfare. furthermore, information on the efficacy, safety and problems associated with the use of anti - hyperglycemic agents for children is insufficient. this survey was conducted to elucidate the current use of antidiabetic medication and the efficacy, safety and problems associated with these agents in children and adolescents with type 2 diabetes mellitus in japan. a questionnaire survey targeted councilors of the japanese society for pediatric endocrinology (jspe) and members of the japanese study group of insulin therapy for childhood and adolescent diabetes (jsgit) in february 2003, in advance of our main survey of current medical treatments for childhood - onset type 2 diabetes mellitus. seventy - two doctors currently managed childhood - onset type 2 diabetes patients, and the total number was estimated to be 530 patients. clinical data sheets were sent to the 70 members of the jspe and jsgit, who signed up to participate in this survey, in june 2003. clinical data on 259 children (younger than 18 yr of age ; 121 males and 138 females) with type 2 diabetes treated at 42 medical centers throughout japan were received between june 2003 and september 2003, and were then analyzed. the percent overweight was determined on the basis of the japanese standard body weights for height by age and sex: (actual body weight the study was approved by the institutional ethics review board of tokyo women s medical university and informed consent was obtained from the patients and/or their parents . the mann - whitney u, and fisher exact probability tests were used for statistical analysis . the mann - whitney u, and fisher exact probability tests were used for statistical analysis . the ages of the 259 recruited patients were 11.9 2.1 yr ( 616 yr) at diagnosis and 14.4 2.0 yr (917 yr) at survey. their hba1c were 8.8 2.8% (4.418.4%) at diagnosis and 7.0 2.2% (4.114.2%) at survey. sixty - nine percent (n=174) of 253 patients had a family history of diabetes: father, mother, siblings and/or grandparents. 1 the percent overweight distribution of patients at the time of type 2 diabetes mellitus diagnosis. the percent overweight was determined on the basis of the japanese standard body weights for height by age and sex: (actual body weight standard weight)/ standard weight 100 (%). seventy - eight % (n=93) of the boys and 63% (n=86) of the girls were obese (percent overweight 20%) at the time of diagnosis. shows the distribution of percent overweight at diagnosis of type 2 diabetes. seventy - eight percent (n=93) of the boys and 63% (n=86) of the girls were obese (percent overweight 20%) at the time of diagnosis. thirty - nine percent (n=47) of the boys and 23% (n=31) of the girls had percent overweight values 50%. the percent overweight distribution of patients at the time of type 2 diabetes mellitus diagnosis. the percent overweight was determined on the basis of the japanese standard body weights for height by age and sex: (actual body weight standard weight)/ standard weight 100 (%). seventy - eight % (n=93) of the boys and 63% (n=86) of the girls were obese (percent overweight 20%) at the time of diagnosis. one - hundred and seventy subjects (66%) were diagnosed through school - age screening for glycosuria. hba1c levels of patients identified by school - age glycosuria screening were 8.4 2.6% and most were asymptomatic. eighty - nine patients were found by means other than school - age screening, and their hba1c levels were 9.5 3.0%. hba1c levels of patients identified by school - age glycosuria screening were significantly lower than those of the others (p<0.005). eighty - seven (34%) of 259 subjects were treated without medication by adjusting their diet and physical activities. one - hundred and seventy - two subjects (66%) were treated using anti - hyperglycemic agents, including -glucosidase inhibiter (-gi), insulin, metformin, sulfonylureas (sus) and nateglinide. table 2table 1 prescribed medication for 172 type 2 diabetes patients in this surveytable 2 clinical characteristics of medicated or unmedicated patients with type 2 diabetes mellitus shows clinical characteristics of medicated and unmedicated subjects with type 2 diabetes. percent overweight at diagnosis was higher in unmedicated (46.0 32.4%) than in medicated (34.9 31.8%) patients (p<0.005). hba1c levels of unmedicated patients were lower than those of medicated patients at both diagnosis (7.3 2.1% vs. 9.5 2.8%, p<0.0001) and survey (5.9 1.2% vs. 7.4 2.4%, p<0.0001). the frequency of a family history of diabetes was significantly higher in medicated (75.9%) than in unmedicated (55.2%) patients (p<0.005). -gi was the most commonly used anti - hyperglycemic agent, followed by insulin, metformin, sus, nateglinide and a combination of -gi and metformin (table 3table 3 age, hba1c and percent overweight at the time of diagnosis according to prescribed medications). figure 2fig. 2 distribution of hba1c at the time of diagnosis according to prescribed medication. mainly -gi was prescribed for children with a lower hba1c level, while mainly insulin was prescribed for children with a higher hba1c level. shows the distribution of hba1c at the time of diagnosis according to the prescribed medication. -gi was prescribed mainly for children with a lower hba1c level (8.1 2.5%), while insulin was prescribed mainly for subjects with a higher hba1c level (11.3 2.6%) at the time of diagnosis (table 3, fig . 2). metformin and sus were given mainly to subjects with moderate levels of hba1c, while metformin was prescribed mainly for subjects with a higher percent overweight (49.9 29.9%) in contrast to sus in subjects with lower percent overweight (21.4 28.1%) at the time of diagnosis (table 3, fig . 3fig . 3 distribution of percent overweight at the time of diagnosis according to prescribed medication . metformin was prescribed mainly for subjects with a higher percent overweight, while sus was given to those with a lower percent overweight at the time of diagnosis .). mainly -gi was prescribed for children with a lower hba1c level, while mainly insulin was prescribed for children with a higher hba1c level. metformin and sus were mainly prescribed for children with a moderate hba1c level. metformin was prescribed mainly for subjects with a higher percent overweight, while sus was given to those with a lower percent overweight at the time of diagnosis. table 4table 4 age, hba1c and percent overweight at the time of survey according to prescribed medications shows age, hba1c and percent overweight at the time of survey according to the prescribed medication. many patients who were initially treated with a single agent eventually required combination therapy with an additional medication. twenty - three (38%) of 61 patients who started with -gi alone remained on monotherapy with -gi. however, 10 (16%) of 61 patients who started with -gi were given insulin therapy, 14 (23%) were also given metformin, 9 (15%) had sus added to their treatments, and for 8 (13%) nateglinide was added (fig . 4fig . twenty - three ( 38%) of 61 patients who started on -gi alone remained on -gi monotherapy. however, 10 (16%) of 61 patients who started on -gi were also given insulin therapy, metformin was added in 14 (23%), cases sus in 9 (15%), and nateglinide in 8 (13%). ). the addition of another agent to -gi treatment for 59% of patients suggests that their diabetic control had not changed for the better or had deteriorated during the course of treatment. twenty - three (38%) of 61 patients who started on -gi alone remained on -gi monotherapy. however, 10 (16%) of 61 patients who started on -gi were also given insulin therapy, metformin was added in 14 (23%), cases sus in 9 (15%), and nateglinide in 8 (13%). two (8%) of 24 patients who started with metformin alone were given insulin therapy (fig . two ( 8%) of 24 patients started on metformin alone, and insulin therapy was later added. ). on the other hand, 13 (25%) of 51 patients who started with insulin alone were able to discontinue insulin therapy, suggesting recovery of endogenous insulin secretion (fig . thirteen ( 25%) of 51 patients who started on insulin alone were able to discontinue insulin therapy, suggesting the recovery of endogenous insulin secretion. ). two (8%) of 24 patients started on metformin alone, and insulin therapy was later added. thirteen (25%) of 51 patients who started on insulin alone were able to discontinue insulin therapy, suggesting the recovery of endogenous insulin secretion. fourteen subjects from 8 centers were treated with metformin alone (table 5table 5 changes in hba1c and percent overweight in 14 type 2 diabetes patients treated with metformin alone). regarding the metformin dosage, 7 of the 14 received 500 mg or less, 5 were given 750 mg, and 2 subjects received 1000 mg. the hba1c level of the 14 subjects who received only metformin decreased significantly from 9.1 1.9% to 6.7 1.3% (p=0.001) without a significant improvement in obesity (table 5). three cases with adverse events were reported (table 6table 6 three cases with adverse events reported in this survey), but causal relations with metformin were not clear. school - age screening for glycosuria has proven useful for finding diabetes mellitus in japan, but a follow - up system for the diabetic patients identified has not yet been established in many regions of japan. furthermore, the precise number of children and adolescents with childhood - onset type 2 diabetes in japan is unknown. the nation - wide survey in 1999 by ohki et al. recruited 812 patients less than 18 yr of age with type 2 diabetes mellitus in japan. they estimated that the prevalence of type 2 diabetes in japan was 3.25: 100,000 children. in 2000, 1,019 patients with type 2 diabetes and 3,740 with type 1 diabetes were registered with the official fund for chronic diseases in children and adolescents in japan. in our survey, about 530 patients with type 2 diabetes were estimated to be under the management of pediatric endocrinologists in 2003 in japan and clinical records of 259 patients were obtained and analyzed. the present survey may represent one half to one quarter of the population with childhood - onset type 2 diabetes in japan. this survey revealed the clinical characteristics of childhood - onset type 2 diabetes and current medical treatment for type 2 diabetes by pediatric endocrinologists in japan. this survey also revealed that 78% of the boys and 63% of the girls were obese (percent overweight 20%) at the time of their diagnosis. obesity was confirmed to be one of the main factors associated with a childhood - onset type 2 diabetes in japan. it was notable that the percent overweight distribution was similar between male and female patients except for a high frequency in non - obese female patients with 010% of percent overweight (fig . no individual in this non - obese female population ( n=25) had positive gadab titers. the frequency of a family history of diabetes in this non - obese female population was slightly higher (76%) than that of the obese population (68%), suggesting that this non - obese female population may be associated with stronger genetic factors including mody. in total, 172 subjects (66%) were treated with anti - hyperglycemic agents. the frequency of anti - hyperglycemic agent usage has doubled, as compared to the approximately one third of patients who were on medication in the survey of 1999 by ohki et al. mainly -gi, insulin and metformin are currently prescribed for childhood - onset type 2 diabetes patients. -gi is used mainly for children with a lower hba1c level, in contrast to insulin which is mostly prescribed for subjects with a higher hba1c level at the time of diagnosis (table 3, fig . 2). metformin and sus were prescribed mainly for subjects with moderate levels of hba1c, metformin mainly for those with a higher percent overweight in contrast to sus given to subjects with a lower percent overweight at the time of diagnosis (table 3, fig . the choice of these medications in this survey is considered to be reasonable, given the functional mechanism of each agent ( table 1). after the start of treatment with a single medication, combination therapy with an additional agent increased in many patients, suggesting that their diabetic control had not changed for the better or had deteriorated during the course of treatment. further study to clarify the clinical characteristics of patients with poor diabetic control during the course of treatment may be necessary. medication for children with type 2 diabetes has not been approved by the ministry of health, labour and welfare. anti - hyperglycemic agents except for insulin are all off - label pharmaceuticals for children with diabetes in japan. the american diabetes association consensus statement in 2000 was, of note is the fact that efficacy and safety data are not available for children nor are any of the oral drugs fda approved for use in children. if treatment goals with nutrition education and exercise are not met, pharmacologic therapy is indicated. the first oral agent used should be metformin. in the u.s. , metformin was approved in 2000 by the fda for use in children (older than 10 years of age, maximum dose : 2000 mg / day), based on the of a randomized controlled trial. in the ukpds report, metformin similarly improved hba1c level as did sus and insulin therapy but did not induce weight gain. in meta - analysis metformin lowers blood glucose and hba1c significantly, compared with placebo, and the body weight is significantly lower after metformin treatment compared with su treatment because of an increase in body weight after su treatment. our survey reveals that the hba1c level of the 14 subjects who received only metformin decreased significantly without a remarkable change of percent overweight (table 5). lactic acidosis is a life - threatening condition characterized by low arterial ph (< 7.35) and elevated arterial lactate levels (> 5.0 meq / l). lactic acidosis is precipitated by phenformin, a biguanide formerly used to control hyperglycemia in type 2 diabetes. metformin is now the only biguanide in general use, since it has a 1020-fold lower risk of lactic acidosis than phenformin, and is regarded as a safe drug provided it is not used in at - risk patients, such as those with renal failure. there is no evidence to date that metformin therapy is associated with an increased risk of lactic acidosis or with increased levels of lactate compared with other antihyperglycemic treatments if the drugs are prescribed under study conditions, taking into account contraindications. three cases with adverse events were reported (table 6), but causal relations with metformin were not clear, and there were no reports of lactic acidosis among 51 patients treated with metformin in this survey. these provide very useful and important information for us. in , mainly -gi, insulin and metformin have been prescribed for childhood - onset type 2 diabetes patients in japan. this survey suggests that metformin is safe and effective for the treatment of type 2 diabetes with obesity in children. additional studies on the efficacy and safety associated with a higher dosage (10001500 mg) of metformin in japanese children may be required. furthermore, we hope that a medication for children with type 2 diabetes will be approved by the ministry of health, labour and welfare in the near future.

the prevalence of childhood - onset type 2 diabetes mellitus has increased dramatically over the past two or three decades in japan, but epidemiological and clinical data remain limited. this survey was conducted to elucidate the current use of antidiabetic medications and the efficacy, safety and problems associated with the use of these agents. clinical data on 259 children (younger than 18 yr of age ; 121 boys and 138 girls) with type 2 diabetes treated at 42 medical centers throughout japan between june and september 2003 were analyzed. sixty - nine percent of all the type 2 diabetic patients (78% of the boys, 63% of the girls) were obese (percent overweight 20%) at the time of diagnosis. overall, 172 subjects (66%) were treated using anti - hyperglycemic agents, including -glucosidase inhibitors (-gi), insulin, metformin and sulfonylureas (sus). many patients who were initially treated with a single medication eventually required insulin alone or in combination with an additional agent, suggesting that their diabetic control had deteriorated during the course of treatment. the hba1c level of the 14 subjects who received only metformin decreased significantly without an improvement in obesity. three cases with adverse events were reported, but causal relations with anti - hyperglycemic agents were not clear. in , mainly -gi, insulin and metformin have been prescribed for childhood - onset type 2 diabetes patients in japan. the of this survey suggest that metformin is safe and effective for the treatment of type 2 diabetes with obesity in children and adolescents.

statistics demonstrate that, in the past 20 years, with the development of industry and aggravation of air pollution, the morbidity rate and mortality rate of lung cancer are increasing most quickly in all malignant tumors in china. in cities, the mortality of lung cancer is 27.5/1 000 000, accounting for 22.4/100 of all death in malignant tumors. the five - year survival rate is 14/100 in america, less in china. besides the reasons of environment pollution, smoking, heredity, lack of early diagnostic tools is also the important factor which contributes to that bad situation. in the recent years, with the development of biology and imageology, for example, spiral ct, cytological examination of sputum and endoscopy of bronchus, and so on , positive rate of early diagnosis is increasing but there are related limitations in all kinds of methods. the sensitivity of fine needle aspiration biopsy (fnab) in diagnosis of malignant nodes is 70/100100/100, but it may destroy some normal tissues and bring about some complications, for instance, pneumatothorax and hemoptysis. blood plasma is the most easily controlled and acquired specimen. so making use of serum sample to detect special markers is the most perfect method in diagnosis of clinical lung cancer. the reasons why we choose the blood plasma as the pathfinder of proteome research are as follows. (i) serum proteome is the most complicated proteome, including inferior proteome in different tissues. (ii) it is easy to obtain enough serum proteins as research sample and it is easy to be standardized. (iii) there is a large range of dynamic state in property of serum proteins. (iv) there is no strict linear relationship between the expression levels of the mrnas and proteins. the modification after translation, such as glycosylation and phosphorylation, is more and more popular, so it is necessary to globally analyze the secreted proteins expressed by cells or tissues. in the research of neoplastic serum proteomics, the related proteomics technology is principally used to fathom the change of serum proteins in the course of forming tumor, to screen and discover the tumor markers and potential drug targets which could be used for classification, early diagnosis, therapy, and intervention of the tumor. nowadays, two technical modes are often applied in the research of serum proteomics: (i) surface - enhanced laser desorption and ionization take - of - flight mass spectrometry (seldi - tof - ms), (ii) matrix assisted laser desorption / ionization time - of - flight mass spectrometry (maldi - tof - ms). the new generation of maldi - tof - ms could quickly, exactly identify polypeptides and proteins with high sensitivity and high throughput. there are many advantages: (i) small needed quantity (fmol), (ii) short time of analysis (35 minutes), (iii) high accuracy of quality (1/10000), (iv) the immunity to the effect of n - terminal close, (v) making use of situ - enzymolysis - technology could directly identify the protein spots which are cut from gels. so it reduces the loss of specimen, (vi) it could identify a single protein in protein mixture and directly identify the modification after translation. so it develops quickly and becomes the preferred way of analyzing polypeptides and proteins in laboratory. in our research , we made the rats our lung cancer model which was induced with mca, used the method of 2de combined with maldi - tof - ms, made compared serum proteomics research in lung cancer, analyzed the changes of serum proteins before and after the formation of lung cancer in same body, identified serum proteins which related to lung cancer for offering serum proteomic evidence to probe the pathogenesis of lung cancer as well as providing new style to probe hematological diagnostic method of lung cancer. 20 wistar rats (10 male, 10 female) were purchased from experimental zoology department at tongji medical college in huazhong technology university. they ranged in weight from 180 g to 200 g. 3-methylcholanthrene (mca) was the product of sigma (sigma - aldrich, inc, saint louis, missouri, usa), immobilized ph gradient dry strip was purchased from amersham pharmacia biotech company, sodium dodecyl sulfate (sds), dithiothreitol (dtt), tris(hydroxymethyl) aminomethane (tris), bromophenol blue (bpb), ammonium persulfate (aps), glycine, acrylamide, methylene bisacrylamide (bis), tetramethylethylenediamine (temed), ipg - buffer, drystrip cover fluid, agarose, carbamide, protein - markers were purchased from a - pharmacia co., coomassie brilliant blue r-250, 3--1-propanesulfonate (chaps) were purchased from sigma co., glacial acetic acid, methanol, glycerol, paraform, alcohol were national analytical pure, sequencing - grade modified trypsin was purchased from american roche co., -cyano-4-hydroxycinnamic acid (cca) (sigma co.), acetonitrile (acn) (fisher chromatographic pure), tri - fluoro - acetic - acid (tfa) (sigma co.). ipgphor isoelectric focusing electrophoretic apparatus was purchased from swedish amersham pharmacia biotech co., proteanxi cell vertical electrophoretic apparatus was purchased from bio - rad co., image scanner was purchased from swedish amersham pharmacia biotech co., centrivap centrifugal concentrators and cold traps were purchased from labconco co., spotcutter apparatus was purchased from bio - rad co., and autoflex maldi - tof - ms mass spectrometry apparatus was purchased from germany bruker co. image master ide software and mascot software are found in http://www.matrixscience.com/search_form_select.html. the rats were kept in cozy (1525c), quiet and avoided of highlight for 58 hours before making the experiment. the rats were kept for 14 days after the whole blood was collected by heart puncture. then we made the model of lung cancer induced with mca in iodized oil by intrabronchial installation with minor modifications. the details were as follow: iodized oil 0.1 ml containing mca 10 mg was dissolved at 7678c for 24 hours. as the basal anesthesia, ketamine hydrochloride 0.12 ml was injected into the muscle of right thigh (0.6 ml / kg), at the same time, penicillin 20 000 u and phytomycin 50 mg per rat were injected into the muscle of left thigh once a day for a week for infection prevention. after about 2 - 3 minutes, early drugged state was showed up, and then the rats were anaesthetized with diethylether for relaxing the muscle of laryngeal. the anaesthetized rats were put on a consple with 45c inclined plane and the upper jaw incisor teeth were fixed by a retention suture on superior extremity of table top. the throat was exposed with gripping tweezers and bended tongue spatula with elastic metals. in a frontal mirror, at the moment of inspiration, the injection needle with blunt end in 1 ml syringe was inserted in glottis until reached the crotch of trachea, then turned left bottom to lobar bronchi in inferior lobe of left lung, and then iodized oil 0.1 ml with mca 10 mg was injected. take pictures with dsa after installation for a day to find the location of iodized oil with mca. sacrificed the animals after installation for 180 days dissected the rats systematically and took the tissues where pathological changes were obvious and all organs, metastasis, lymph nodes with suspected of invasion, fixed them with paraform, embeded them with paraffin wax, sliced them, and stained with he. the rats were raised with skins of hind neck in order that the limbs could downward sagging after they were anesthetized with diethylether. the injector that contains heparin was used to pump blood (13 ml) on the left chest wall where the heart beats most powerfully (1.52 cm higher than the lower margin of ensiform cartilage). the blood was kept in centrifuge tube for 30 minutes at room temperature, centrifuged at 2000 rpm for 15 minutes. the plasma was taken out and aliquoted into sterile ep tubes, stored at 70c until used. 50 ul plasma was taken out, respectively, before and after the formation of lung cancer, then combined and quantified with bradford method, followed by taking 1 mg protein used for isoelectric focusing. all measurements were performed as described previously with minor modification. in the first dimension, proteins were separated by ief with ipg strip (ph310, nonlinear gradient and ph47, linear gradient, both 180 mm 3 mm 0.5 mm). of the total proteins, 1 mg was dissolved in lysis buffer (8 m urea, 0.02 chaps, 0.02 m dtt, 0.05 ipg buffer) to obtain a total volume of 350 ul per strip. focused ipg were rehydrated at 30 v for 6 hours, at 500 v for 1 hour, at 1000 v for 1 hour, and at 8000 v for 10 hours. after ief, ipg strips were washed with milli - q water and then incubated in equilibration buffer (30% glucerin, 6 m urea, 2% sds, 50 mm tris - hcl ph 8.8, trace bromophenol blue) containing 20 mm dtt on rocking bed for 15 minutes, followed by 15 minutes incubation in the same equilibration buffer containing 100 mm - iodoacetamide on rocking bed. equilibrated ipg strips were placed on top of a 13% homogeneous sds - polyacrylamide gel. a scrap of filter paper was immersed in 10 ul protein marker and put on top of one side of the gel. 13% sds - page gels 75 ml contained 30% acr/0.8% bis 33.5 ml, 4 tris - hcl 19.5 ml ph 8.8, milli - q water 24.5 ml, 10% aps 0.25 ml, temed 0.05 ml. gels were run in parallel at constant power (40 ma for 40 minutes, 60 ma for 5 hours) in running buffer until bpb reached the bottom line of each gel. the gels were put into 0.1% coomassie brilliant blue solution (methanol 484 ml, glacial acetic acid 92 ml, milli - q water 454 ml, coomasie brilliant blue r-250 1 g) and stained on rocking bed for 3 hours. after pouring out the coomassie brilliant blue solution, we decolored them in depigmenting agent solution (methanol 50 ml, glacial acetic acid 75 ml, milli - q water 875 ml) for 24 hours until the of gel pieces became clear and transparent. spots detection, quantification, matching and comparison of 2de protein pattern were done with image master ide software. in the protein matching, serum protein gel maps of normal controls were regarded as referenced gel, artificial matched spots were built up, and the analysis of different expression was performed by applied software. the individual spot volumes were normalized by dividing their intensity values by the total intensity values of all the spots present in the gel and expressed as % vol. if the ratio of relative content in different group was more than 2 or less than 0.5, we could assess there were differences. it was carried out by the procedure described in detail previously with minor modification. of all the spots, 7 protein spots were overexpressed, 4 protein spots were underexpressed, and 2 protein spots were new in the plasmas of lung cancer. the chosen protein spots in 2de maps were excised with spot - cutter apparatus and transferred into 1.5 ml ep tube, followed by washing them thrice with 1 ml water for 10 minutes. gel pieces were discolored in 50100 ul solution (50% acetonitrile, 25 mmol / l ammonium bicarbonate) for 20 minutes with constant oscillation, followed by discarding the solution, repeated 1 - 2 times until blue color disappeared, dried them in vacuum centrifugal dryer for 30 minutes. then 510 ul of 0.01 ug / ul trypsin diluted in 25 mmol / l ammonium bicarbonate was added to each sample tube for digesting the proteins and kept the tubes in freezer at 4c for 2030 minutes. after all digestion solution was ingested, 510 ul of 25 mmol / l ammonium bicarbonate was added to it. stored it at 37c for 15 hours. the digested polypeptides were eluted twice with 5% tfa 50100 ul at 40c for 1 hour and 2.5% tfa, 50% acn 50100 ul at 30c for 1 hour, supernatants were collected, combined, vacuum - dried by centrifugation until used for mass spectrographic analysis. 5 ul of 0.5% tfa was added to dried sample tubes for dissolving the peptides mixture. the sample and ground substances (cca added to 30% acn or 10% aceton to obtain saturated solution) were combined with 1:1 volume and spotted onto the target plate, then air - dried. peptide mass fingerprinting spectrum was evaluated with a reflector positive ion model in following setting: accelerating voltage, 20 kv; reflected voltage, 1.12 kv; nitrogen laser wavelength, 337 nm; pulse width, 3 nanoseconds; delayed extraction time, 100 nanoseconds; the degree of vacuum, 4 10 torr; each spectrum was obtained by accumulating 50 laser shots and averaging them. a matrix peak and the peptide mass fingerprinting of each protein was searched from ncbinr database using mascot software. the conditions of searching are as follows (i) the peptides mass were controlled between 800 da and 4000 da. (ii) the sphere of error in apparent pi and apparent mr was + 0.5ph and 20%. (iii) the largest error in molecular weight of peptide fragment was controlled in the range of 0.3 da to + 0.3 da. (iv) the trypsin was chosen as the enzyme and the incomplete choice of enzymolysis fragment was 1 or 2. lung cancer models were made successfully after installing mca in iodized oil. it was demonstrated that iodized oil was present in the inferior part of left pulmonary lobes by scissors shadow with x - ray. only in ten rats of 18 rats (two rats died naturally in 20 rats), tubercles in bottom left of pulmonary lobes were observed in naked eye. by pathological examination, it was proved that they were squamous cell carcinomas. after he staining, by light microscope, we could observe that carcinoma nests were different in size and shape, the horny pearl was obvious, cell nucleus were very big, part of them were vacuous, the chromatins in cell nucleus were rough, there were many karyokinesis, and nucleoli were big and localized to the edge (figure 1). the electrophoresis of plasma in 10 animal models was performed before and after forming lung cancer, respectively. the protein spots were separated better using ph47 ipgief (figure 2(b) ) than ph310 ipgief (figure 2(a) ). the protein spots in the range of pi46 and mr1065 kd were distributed most intensively. when making an analysis of 2de patterns which were separated with ph47 ipg strip (figure 3), we found that 455 spots on the normal plasmas 2de patterns and 716 spots on the lung cancer plasmas 2de patterns were detected. when we matched the lung cancer plasmas 2de patterns with the normal plasmas 2de patterns, it was demonstrated that the number of matched protein spots was 295 and matched rate was 50.38%. 141 protein spots were differently expressed significantly in plasmas 2de patterns, 82 spots overexpressed in the lung cancer and 59 spots overexpressed in normal controls. we chose 13 differently expressed proteins spots with clear margin and high quantity of proteins for maldi - tof - ms analysis. of all the protein spots, 7 protein spots were overexpressed (numbers 1, 2, 3, 4, 7, 8, 9), 4 protein spots were underexpressed (numbers 10, 11, 12, 13), 2 protein spots were new in the plasmas of lung cancer (numbers 5, 6) (figures 4 and 5). the peptide mass fingerprinting maps were obtained successfully by analysis of maldi - tof - ms after digesting the chosen protein spots. finally, 3 proteins were identified through the ncbinr database search by mascot software in chosen 13 protein spots. the 3 proteins were heptoglobin (overexpressed protein), transthyretin (underexpressed protein), and tumor necrosis factor receptor superfamily member 8 (new protein). the figures were the mass spectrum of identified proteins spots and detailed of database retrieval (figures 6, 7, 8, 9, 10, and 11). mca is an indirect chemical carcinogen, which belongs to polycyclic aromatic hydrocarbon with 3, 4-benzpyrene, and is responsible to air pollution. its major effect is to make epithelium mucosae cell of bronchiole, respiratory bronchiole squamous metaplasia and gradually progress to squamous carcinoma cell. we had proved that there were a series of changes of genetic materials and related genes in the process of variation from normal bronchi epithelium to lung squamous carcinoma cell by immunohistochemistry staining and molecular pathology. for example, the different positive expression rate of p53 protein was 69.57% in rat lung cancer tissue. the positive expression rate of proliferative cell nuclear antigen (pcna) took up 20% in normal bronchi epithelium tissue, and it advanced to 82.61% in infiltrating lung cancer tissue. in addition, the number of strong positive expressed cells was increasing obviously, even all carcinoma nests were positive expressed, it hinted the changes of activity of cell proliferation during the formation of lung cancer. thus making use of the model of rat lung cancer to carry out serum proteome research was effective and feasible. haptoglobin, one of acute phase proteins, is an acid glycoprotein, which belongs to 2-globulin and extensively exists in human plasmas and other body fluid. most of hp is synthesized in liver. in addition, hp mrna is also expressed in human endometriotic lesions, intestinal epithelium and mouse liver, lung, adipose tissue cell, skin. friedrichs et al. found that hp mrna was detected in some special cells of adernal gland, submandibular gland, ovary, ureter. further proved that hp was considered as a secretory protein, however, hp synthesized in thp-1 monocytic cells was largely retained within cells. and the content of hp in plasmas often greatly increases in pathologic state, for example, carcinoma, inflammation, infection, myocarditis, and so on. the main factor stimulates the composition of hp is il-6. on the condition of inflammation, injury, il-1, tnf are secreted by macrophage, leading to the increase of il-6, ing in the composition of hp. in addition, darmiento proved that lps also could stimulate the expression of hp mrna. proved protein kinase - c - delta could stimulate haptoglobin secretion as well. by forming the high - affinity complexes, , these hp - hb complexes could bind to the cd163 scavenger receptor on macrophages with high specificity, leading to endocytosis and subsequent intracellular degradation. so hp - hb complex formation is considered to lessen the loss of free hb through glomerular filtration and it is helpful for supporting the recycling of iron. moreover, because free hb is a source of iron, which may otherwise enhance bacterial growth and virulence, so it is important that hb released from erythrocytes could be cleared promptly. finally, free hb could release heme and iron which may participate in generating reactive oxygen species (fenton reaction) and promote the injury of tissue as well. in a word, hp works indirectly as a bacteriostatic agent and an antioxidant in the way of facilitating the immediate clearance of free hb by macrophages. haptoglobin polymorphism correlates to the prevalence and clinical evolution of many inflammatory diseases, for example, infections, atherosclerosis, and autoimmune disorders and that leads to the generation of two distinct alleles, hp2 - 1, hp1 - 1, and hp2 - 2. hp2 - 2 has been reported to be associated with the risk of atherosclerosis and coronary heart disease. vormittag et al. proved that hp2 - 2 was a potentially new risk factor for spontaneous venous thromboembolism as well. there are many researches about hp correlate with injury, inflammation, metabolic disease. more and more documents report that the level of hp in plasmas goes up in many malignant tumors, such as ovarian cancer, breast cancer, renal cell cancer, colon carcinoma, acute leukemia, and lung cancer. our research were consistent with them. in our research, we found hp greatly increased in lung cancer (the content of protein adds up to 8.328 times). though maybe it does not belong to specific protein in lung cancer, owning to greatly increase level of hp in the active stage of carcinoma and high sensitivity of immunology method, it could become an important check index for lung cancer diagnosis and monitoring and improve the sensitivity and specificity of early diagnosis in lung cancer when combining with other diagnostic indexes. as for its function, ttr could transport the thyroid hormone t and retinol through binding to the retinol binding protein. in addition , ttr has been established as a cryptic protease able to cleave apoa - i in vitro. some scholars have reported that ttr is involved in preventing a - beta fibrillization by inhibiting and disrupting a - beta fibrils, with consequent abrogation of toxicity. costa et al. further confirmed that as a protective mechanism in alzheimer's disease (ad), ttr could serve as a protective molecule in this disease and prompted a - beta proteolysis. it may be proved that ttr is a useful therapeutic agent in preventing or retarding the formation of cerebral amyloid plaque implicated in ad pathology. in the past , someone confirmed that in familial amyloidotic polyneuropathy (fap) which was associated with the abnormal extracellular tissue deposition of mutant transthyretin (ttr), inflammatory and apoptotic pathways both are triggered in the presymptomatic stages of the disease on the condition that nonfibrillar ttr deposits are present. meistermann et al. also reported that transthyretin could serve as a biomarker for gentamicin - induced nephrotoxicity in rat. conversion of ttr tetramer to monomer may relate to the development of beta - cell failure / destruction in type 1 diabetes. so we could see that ttr expression was associated with diabetes. at the same time, bai et al. gussed that the concentration of ttr maybe relates to the level of blood pressure, however, the concrete mechanism is still unclear. the content of ttr quickly decreased when the proteins were insufficient and increased obviously when the amount of proteins gone up. therefore, ttr was often used as an index of evaluating nutritional state, especially the nutrition and immunity state after operation. so far, reports about ttr relate to tumors mainly concentrate on liver cancer, ovarian cancer, leukemia. gu et al. first reported that the gene of ttr had close correlation to liver cancer, they found that the gene of ttr was expressed most highly in normal liver. the level of its mrna greatly went down in liver cancer which the parenchyma was destroyed little. ttr was also considered as potential serum marker in early ovarian cancer. in children with leukemia, the content of ttr and rbp in plasmas was greatly less than that in normal children. that difference also existed in different race, the melanoderm children were much less, and the lower level related to bad prognosis. some scholars reported that ttr was overexpressed in blood serum in lung squamous cell carcinoma compared with normal lung. our research also demonstrated that the content of ttr in plasmas greatly declined in the formation of lung cancer, it was consistent with their . the amount of proteins in normal ones was 14 times bigger than that in lung cancer. tnfrs8 also named cd30, because ki-1 monoclonal antibody was used in identifying it, therefore it named ki-1 as well. as a marker of activation of t and b lymphocytes, cd30 is a 120 kda surface phosphorylated glycoprotein and it is predominantly overexpressed by the tumor cells (hodgkin 's and reed - sternberg cells) of classical hodgkin's lymphoma (chl) and those of anaplastic large cell lymphoma (alcl) in classical hodgkin lymphoma. it is well known that cd30 expression is not unique to hodgkin lymphoma and anaplastic large cell lymphoma (alcl) because of its expression in mediastinal large b cell lymphoma, a subset of nodal diffuse large cell lymphoma, large cells in follicular lymphoma, nodal and cutaneous diffuse large b cell lymphoma, peripheral t cell lymphoma, embryonal carcinoma, and other nonlymphoid cells and neoplasms. the expression of cd30 was also associated with pregnancy. kusanovic found that the content of maternal serum soluble cd30 increased in normal pregnancy, however, it decreased in preeclampsia and it is small for gestational age pregnancies. at the same time, lambropoulou et al. proved that during the second trimester, cd30 was expressed highly in many medullary thymic epithelial cells (tecs) and hassall's corpuscles, but small numbers of cd30 tecs were showed during the late first trimester. large number of researches proved that if cd30 crosslinked with cd30l, activated signal may be issued. so it promoted the transmission of intracellular information. because that information could be positive or negative signal, consequently, it ed in cellular growth was inhibited or cellular apoptosis, cellular hyperplasia or differentiation, secretion of cytokine, and so on. wright et al. further confirmed that in alcl cells, the stimulation of cd30 elicits p21 (waf1)-mediated arrest through the canonical but not the alternative nf - kappab pathway. reported that there were many cd30cd4, cd30cd8 cell in joint synovial fluid of patient with ra, large amount of scd30 existed in peripheral blood and affected joint synovial fluid, especially in active stage, it had direct relation with the titre of raf. it hinted that there was relationship between cd30 cell, scd30 cell, and th2 reactive disease. in addition, the increase of cd30 cells and scd30 cells was detected in acute stage of th2 reactive illness, such as sle, scleroderma, specifical scytitis. many researches showed the amount of scd30 increased in active stage of chronical viral hepatitis b and there were striking differences compared with normal controls and virus carries, hinting the activity of th2 cytokine in active stage of hepatitis. in hodgkin's disease, the level of serum scd30 abnormally increased in active stage in nontreated patients. the extent of increase related to the stage of illness and general symptoms. it could be an independent prognosis index, the higher of scd30, the worser of prognosis. in alcl, the great increase of serum scd30 there was not scd30 in normal controls, indicating that the amount of scd30 went up indeed in forming lung cancer, so far, the mechanism of increase is not clear. hargreaves et al. guessed that it was possible that the function of scd30 brought into full play by blocking the cross - link of cd30 and cd30l. recently, researches in lung cancer demonstrated that a series of complicated molecular events involved in carcinogenesis, such as activation of oncogene and inactivation of antioncogene. proteomics aim at the large - scale analysis of protein in tissues, cell, and plasmas, including protein expression, posttranslational modification, and interaction of proteins. proteomics has become a hot spot in lung cancer research. by applying the proteomics technology , many researchers had found a large number of proteins as the candidate tumor markers from blood plasmas in lung cancers. by comparing protein expression levels among 93 lung adenocarcinomas and 10 uninvolved lung samples, chen et al. found that nine different enzyme proteins were identified by the method of 2d - page and maldi - ms or peptide sequencing. all of them increased obviously in the lung adenocarcinomas, including the antioxidant enzyme aoe372, atp5d, b4galt, cytosolic inorganic pyrophosphatase (ppase), grp58, gstm4, p4hb, tpi, and ubiquitin hiolesterase. identify proteins that commonly induce an antibody response in lung cancer by making use of proteomic approach; protein gene product 9.5 (pgp 9.5) was detected in serum. when they took advantage of a549 lung adenocarcinoma cell line, they have proved that pgp 9.5 existed at the cell surface as well as secreted. therefore, the discovery of pgp 9.5 antigen and/or antibodies in serum of patients with lung cancer showed that pgp 9.5 may be used in lung cancer screening and diagnosis. chatterji and borlak found that eight proteins were also identified in serum though they were identified in tissue of lung tumor bearing mice. a total of 50 proteins were identified in serum, some of which were specifically regulated or exclusively expressed either in tumor bearing or wild - type mice. what is more, they revealed that alpha-1-antitrypsin (a1at) and alpha-2-macroglobulin (a2 mg) were upregulated in the serum proteome of 12 months old mice. in cancer tissue, hemoglobin subunit alpha went up in serum samples of lung tumor bearing mice (12 months). they discovered the expression of transthyretin to be gone up in 1 month and repressed in 12 months old tumor bearing mice. thus they suggested that their in - depth validation could become biomarker candidates for the identification of lung adenocarcinomas. serological proteome analysis (serpa) of ten hlsc tissues was performed by li et al. to identify the tumor - associated antigens. the showed 36 8 differential proteins reactive with patients' autologous serum, 14 proteins of them were identified. in addition, six of the 14 proteins, alpha enolase, pre - b cell - enhancing factor precursor, triosephosphate isomerase, phosphoglycerate mutase 1, fructose - bisphosphate aldolase a, and guanine nucleotide - binding protein beta subunitlike protein were also increased in hlscs in the comparative proteomic research, therefore, it indicated that these 6 hlsc - associated antigens can be applied in diagnosis and therapy of hlsc. autoantibodies against triosephosphate isomerase (tim) and superoxide dismutase (mnsod) were detected by yang et al. in serum from over 20% patients with lsc but none from the normal controls. their suggested that autoantibody and antigen of tim and mnsod may also be potential application for screening and diagnosis of the lung squamous carcinoma. at the same time, lisa et al. found that five truncation forms of serum amyloid a protein precursor and serum amyloid a1 isoform 2 as a part of the components of the serum proteomic biomarkers of lung cancer. these indicated that it was possible that the serum proteomic signature of lung cancer was the part of the of specific proteolytic activity in the serum and/or in lung tumors. built up the protein configuration in the plasmas of lung adenocarcinoma using the method of the seldi - tof - ms technology and discovered 5 special protein peaks which highly expressed in lung adenocarcinoma. in addition, liao et al. captured serum proteins in lung adenocarcinoma by the technology of wcx2 chip and found 10 different expressed proteins in which 6 overexpressed proteins and 4 underexpressed proteins. however, a large number of lung cancers and normal people were essential to prove these differently expressed proteins and special expressed spectral patterns, to identify and show their functions in the formation of tumor. due to direct examination of the molecular machinery of cell physiology, including protein expression, sequence variations and isoforms, posttranslational modification, and protein - protein complexes, proteomics has the self - evident advantages over genomic - based assays. nonetheless, there are certain disadvantages as well, for instance, (i) sample collection, preparation, and analysis need rigorous requirement, (ii) there are no amplification procedures which similar to pcr that could enable assay to improve making use of the limited biological starting material, (iii) we are short of purification strategies which could enrich samples for intended work (e.g., phosphoprotein analysis), and (iv) necessary costs are insufficient for staffing and equipping shared resources or clinical laboratory which can be able to perform the required assays. though there are some limitations in the technology of proteomics, with the maturation of proteomic technique and improvement of sample pretreatment and preparation technique. we believe its application in analysis of blood plasmas will become more and more popular and become a new method for clinical diagnosis and illness monitoring. in addition, the screening and identification of tumor markers will step into a new stage. in a word, the research tactics in proteomics of tumor markers will bring about far - reaching extensive effects for lung cancer diagnosis and treatment. it will quicken the process of researches in lung cancer as well as making a great contribution to win a victory in human being's flight with tumor. in our experiment, we made the rat plasmas derived from lung cancers and the normal controls to be our targets of observation and comparison. by regulating and optimizing all conditions of 2de technique, we finally obtained satisfactory 2de patterns in which the 700 serum proteins were detected from the lung cancers and the normal controls. in addition, the 141 differently expressed protein spots were acquired by image screen and image analysis. the 82 spots were overexpressed in lung cancer and the 59 spots were overexpressed in normal controls. at the same time, we successfully identified three proteins including hp, ttr, and tnfrs8 by making use of the maldi - tof - ms to analyze the 13 differently expressed protein spots. the increase of hp, which belongs to app, hinted the appearance of stress reaction in the process of forming lung cancer. in addition, the decrease of ttr, which shows the nutritional state, hinted the changes of nutritional state. as a member of tumor necrosis factor super family, tnfrs8 could regulate the function of immunity and hinted a series of changes of immunity state in the process of forming lung cancer. in a , (i) the plasmas proteins were separated successfully for proteome research of lung cancer by the optimized 2de technique. (ii) there were the differently expressed proteins between the normal plasmas and the lung cancer plasmas. (iii) the differentially expressed proteins in plasmas of rat lung cancer provided the evidence for keeping searching the markers for lung cancer diagnosis and therapy.

lung cancer remains the leading cause of cancer - related mortality worldwide. early detection of lung cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. to determine the differently expressed proteins in the serum of lung cancer and figure out the function of the proteins, two - dimensional electrophoresis (2de) and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry (maldi - tof - ms) were used to screen the serum proteins of lung cancer model induced by 3-methylcholanthrene (mca). from optimized 2de image, 455 spots in the normal sera and 716 spots in the lung cancers sera were detected. among them, 141 protein spots were differentially expressed when comparing the serum from normal rat and serum from lung cancer model, including 82 overexpressed proteins and 59 underexpressed proteins. changes of haptoglobin, transthyretin, and tnf superfamily member 8 (tnfrs8) were confirmed in sera from lung cancer by maldi - tof - ms. proteomics technology leads to identify changes of haptoglobin, transthyretin, and tnfrs8 in serum of rat lung cancer model and represents a powerful tool in searching for candidate proteins as biomarkers.

many cam modalities afford relief from pain, each in its own way, or according to its own terminology. comparison of different cam modalities in a simple phenomenology of pain centered around the idea that pain may be associated with blockages of the flow of energy in the system of nadis / acupuncture meridians.

human papillomavirus (hpv) is the most common newly acquired sexually transmitted infection (sti) in the united states, with an estimated 20 million people infected. furthermore, incidence of hpv infection has increased during the past two decades, with approximately 6.2 million newly diagnosed cases annually (figure 1). hpv infection has a very high prevalence rate in adolescent girls and young women. one study showed that 36% of women 25 years of age or younger are hpv - positive compared with less than 3% of women 45 years of age and older. hpv is the etiologic agent of several genital epithelial lesions including genital warts (condylomata acuminata), cervical intraepithelial neoplasia (cin), and cervical cancer. more than 100 different types of hpv have been identified (table 1). low - risk types, hpv 6 and hpv 11, are the most common types implicated in causing genital warts. furthermore, due to their ability to cause low - grade cervical lesions, infection with low - risk hpv types is often associated with abnormal papanicolaou (pap) test . although genital warts are medically benign, and low - grade cin can spontaneously regress, diagnosis of genital warts or an abnormal pap smear can cause emotional distress and impose an economic burden. in contrast to infection with hpv types 6 and 11, infection with high - risk hpv types 16 and 18 can lead to anogenital cancers. hpv types 16 and 18 cause 70% of all cases of cervical cancer, with half of all cervical cancers caused by type 16 alone. persistent infection with high - risk hpv types is implicated in 99.7% of cervical cancers. preventing infection with the most common low - risk and high - risk hpv types would prevent the majority of cases of genital warts and cervical cancer, respectively. the transmission typically occurs through the skin - to - skin anogenital contact. increased risk for acquiring hpv has been associated with multiple sexual partners, younger age of sexual debut, failure to use condoms, and sex with uncircumcised males. however , one study reported that 20% of women became infected with only one lifetime sex partner, suggesting that both partners must be sexually nave to prevent infection. several studies have shown that the risk of infection increases substantially when initiating a new sexual relationship. the transmission of hpv infection can be blocked by latex condoms if the infected area is physically covered. nonetheless, hpv often manifests on external anogenital sites not covered by a condom, and so the latter does not prevent all infections. however, the use of a condom may reduce hpv persistence and therefore aid in the regression of hpv - associated lesions. annual cervical cancer screening is expensive. in a study of women enrolled in a usa health care plan, it was estimated that an average of $ 26,415 per 1000 women was spent on annual cervical screening and treatment for hpv - related diseases. when these data are extrapolated to the general usa population, it can be estimated that $ 3.4 billion is spent annually on diagnosis and treatment of hpv infection and its associated cervical diseases. approximately 90% of the estimated cost can be attributed to strategies for prevention of cervical cancer, such as treatment of precancerous lesions and routine pap tests, whereas the other 10% is spent on treatment of cervical cancer (figure 2). hpv infection is most prevalent in adolescents and young adults, and this group also incurs the majority of hpv - associated health care costs. when it comes to hpv - related health care, women in the 2029 year age group incurred an annual cost of $ 51,863 per 1000 women. the estimated lifetime total medical cost of hpv infection for men and women aged 1524 is $ 2.9 billion, which makes hpv the second most expensive sti after hiv. in addition, when the cost of treating genital warts is analyzed by itself, it becomes apparent that this too causes a substantial economic burden. based on an incidence of 500,000 cases of hpv infection per year, the annual total direct medical cost for treatment of anogenital warts in all age groups for the year 2000 was $ 167.4 million. assembly of infectious virus, a necessary step in the hpv life cycle, involves the formation of the capsid, or outer layer, of the virion. the capsid is composed of two proteins, l1 and l2, which are expressed later in infection. the major capsid protein l1 comprises the outermost layer of the capsid and is necessary for virus structure. hpv vaccine development has been considerably advanced due in part to the production of virus - like particles (vlps). hpv - like vlps, which mimic the structure of the hpv virion but do not contain genetic material and can be manufactured by exogenous expression of l1 in a variety of cell types, including bacterial, yeast, insect, and mammalian cells. vlps are noninfectious and nononcogenic, making them ideal candidates for use in hpv vaccine production. vlps are purified, concentrated, distributed into aliquots, and combined with an adjuvant. early studies with a monovalent vaccine against hpv 16 have shown that vlp vaccines induce a strong immune response against l1 in animal models and humoral immunity in humans. other trials demonstrated that booster doses of vlp vaccines induced protective levels of antibodies. furthermore, in a proof - of - principle study, the hpv 16 vlp vaccine was safe, well tolerated, and induced antibody titers to levels significantly higher those produced in response to natural infection. although this study was not sufficiently powered to assess vaccine efficacy in preventing clinical disease, vaccine recipients developed fewer cervical lesions than placebo recipients. in addition, the vaccine was 100% effective in preventing persistent infection, suggesting that vlp vaccines may help reduce the incidence of cervical cancer precursors and invasive cervical cancer. similar have been reported with other monovalent vlp vaccines. immune responses to hpv infection are type - specific; therefore, vaccine efficacy can be greatly improved by combining vlps from several types of hpv into one multivalent vaccine. multivalent vaccines that offer protection against the most common disease - causing hpv types are in late stages of clinical development. currently, a bivalent vaccine that protects against 2 high - risk hpv types, and a quadrivalent vaccine that protects against 2 high - risk and 2 low - risk hpv types are being tested. to determine the efficacy, immunogenicity, and safety of a bivalent vaccine containing hpv types 16 and 18, a double - blind, placebo - controlled phase 2 trial was conducted on 1113 women (1525 years old) with no prior history of hpv infection and normal cervical cytology. in this study, women received intramuscular injections of vaccine (20 g of each vlp plus adjuvant) or placebo (adjuvant alone) on day 1, at month 1, and at month 6, and then followed for at least 17 months. the bivalent hpv 16/18 vaccine was well tolerated, produced no vaccine - related serious adverse events, and induced a major humoral immune response to both hpv types. furthermore, the vaccine was 90% efficacious at reducing incident infection and 100% efficacious at preventing persistent infection. including additional hpv types in vaccines would be expected to cumulatively reduce hpv - associated disease burden by preventing additional hpv infections. a quadrivalent vaccine has been developed to protect against hpv types 6, 11, 16, and 18. a double - blind, placebo - controlled phase 2 safety and efficacy trial women who were enrolled in the trial received either the quadrivalent vaccine (20 g each of hpv 6 and 18 vlp, and 40 g each of hpv 11 and 16 vlp plus adjuvant) or placebo (adjuvant alone) on day 1, month 2, and month 6, and then were followed for 36 months. of this trial showed that the quadrivalent vaccine was well tolerated, produced few serious adverse events (none of which were judged to be related to the vaccine), and stimulated the production of antibodies directed against all four hpv types. furthermore, the vaccine reduced persistent infection in vaccine recipients by 89% and prevented 100% of clinical disease associated with hpv types 6, 11, 16, and 18 (figure 3). similarly high efficacy were reported for a cohort of women who did not adhere completely to the study protocol. recently, data from the phase 3 females united to universally reduce endo - ectocervical disease (future ii) clinical trial were presented. the quadrivalent hpv vaccine was 100% effective at preventing cin 2/3, ais, and cervical cancer associated with hpv 16 or 18 infection during two years of follow - up. the vaccine was well - tolerated and there were no vaccine - related serious adverse events. vaccines that provide protection against the most common disease - causing hpv types would be expected to significantly reduce the incidence of hpv - associated diseases. reducing hpv - associated disease burden may also reduce the health care costs associated with these diseases. although hpv vaccines are not currently available to the public, their potential public health benefits have been reported in several mathematical modeling studies. sanders et al reported that if a hypothetical vaccine that was 75% efficacious at preventing high - risk hpv infection were administered to approximately two million 12-year - old girls, it would prevent 224,255 hpv infections, 3317 cases of cancer, and 1340 cervical cancer - associated mortalities in the girls' lifetimes. another study predicted that an hpv 16/18 vaccine would reduce the number of cervical cancer cases associated with the hpv 16 and hpv 18 by 95%. mathematical modeling and sexual transmission data have also suggested that both sexes should be vaccinated to provide the greatest reductions in hpv infections. for example, one population - level study predicted that a female - only hpv vaccination program would be only 68% as effective in reducing hpv prevalence as a program aimed at vaccinating both men and women. in fact, diagnosis with genital warts is often the most anxiety - provoking outcome of hpv infection. because vaccination has shown promising in the reduction of the disease burden associated with hpv infection, it would be expected to reduce some of the psychosocial and emotional burden as well. a number of obstacles will need to be overcome to maximize the public health benefits of hpv vaccination. vaccination programs must identify appropriate candidates for vaccination, establish booster requirements, and overcome potential individual, parental, and social barriers to hpv vaccine acceptance. for example, individuals may view acceptance of hpv vaccines as admitting to risky sexual behavior. furthermore, research has shown that knowledge about hpv, an infection that many people know little about, is directly correlated to vaccine acceptance. parents may feel that their child is not at risk, or that vaccination would support teenage sex or encourage risky sexual behavior (ie, reduced condom use). societal and cultural issues may include beliefs that sexually transmitted diseases are a deterrent or punishment for non - marital sexual behavior. each of these obstacles can be overcome by widespread efforts to educate individuals and society about the prevalence of hpv and the risks associated with forgoing vaccination. administering hpv vaccines to populations prior to initiating sexual activity will yield the greatest health benefit. because preadolescent and adolescent children are generally sexually nave and develop robust immune responses to vaccines, vaccinating young adolescents is predicted to significantly reduce the incidence of hpv infection and hpv - associated diseases. in order to foster broad acceptance of hpv vaccine, public health initiatives will need to educate parents / caregivers as well as health care professionals about the risks associated with hpv infection and the benefits of vaccination. vaccination has been widely accepted as an effective means by which infectious diseases can be prevented or eliminated. vlp vaccines that protect against infection with the most common disease - causing hpv types are currently in clinical development and early reports have suggested that hpv vaccines are highly efficacious in preventing hpv infection and hpv - associated disease. hpv vaccines will be most effective when administered prior to initiation of sexual activity and vaccination initiatives will most likely target preadolescent and adolescent populations. * average age adjusted to the 1998 us female population; all cost estimates were converted to 2002 dollars; asc = atypical squamous cells; agc = atypical glandular cells; lsil = low - grade squamous intraepithelial lesion; hsil = high - grade squamous intraepithelial lesion. incidence of infection or disease associated with hpv 6, 11, 16, or 18 after vaccination with a quadrivalent vaccine versus placebo (* reported as incidence per 100 women - year at risk).

human papillomavirus (hpv), a sexually transmitted infection and the etiologic cause of genital warts and cervical cancer, is highly prevalent in sexually active men and women. although cervical screening procedures have significantly reduced the disease burden associated with hpv infection, they are expensive and abnormal cause significant emotional distress. therefore, prevention may be an effective strategy for reducing the economic, psychosocial, and disease burden of hpv infection. multivalent vaccines are now in clinical development. a bivalent vaccine that protects against hpv 16 and 18, and a quadrivalent vaccine which protects against hpv types 6, 11, 16, and 18, have been shown to significantly reduce the occurrence of incident and persistent hpv infections in phase 2 clinical trials; phase 3 trials are currently underway. hpv vaccines will be most effective when administered prior to initiation of sexual activity, and vaccination campaigns should aggressively target preadolescent and adolescent populations.

raised body mass index (bmi) is common in patients with cardiovascular disease (cvd) and obesity (bmi 30) has been known as a risk factor for hypertension, type 2 diabetes, functional capacity (fc), dislipidemia, and generally for coronary heart disease (chd) that is the most frequent cause of death. the prevalence of obesity in patients with coronary artery disease (cad) moves toward 40%. noticeably overall 80% of patients that referred to cardiac rehabilitation program (crp) were overweight and obese. crp is known as a way for enhancement and maintenance of cardiovascular health through individualized programs, designed to optimize physical, psychologic, social, vocational, and emotional status by improving coronary risk factors management. regarding the gender differences, the risk of developing cvd is recognized to be obviously different between men and women. according to previous studies , cad presents in women approximately 10 years later than in men; and, the incidence of coronary events was 60% higher in men than in women. nevertheless, some studies revealed that women have similar improvements in fc and risk factors compared with men after crp. also, there are many contradictory about similarity and dissimilarity between men and women. kazuhiro et al confirmed gender differences in physiologic outcomes, whereas in another study both the groups benefited in a similar way from crp in most aspects. by paying attention to these investigations, so, the purpose of this survey was to explore the effect of crp on fc and risk factors, such as obesity indexes, lipid profiles, and fasting blood sugar (fbs) in obese men and women patients with chd; also, to know whether obese men and obese women equally achieve the benefits of crp or not. in an observational study between 2000 and 2011, a total of 156 eligible patients were identified as obese patients with chd who were referred to phase ii cardiac rehabilitation of isfahan cardiovascular research institute. it should be mentioned that phase ii has taken the form of a structured and supervised exercise program in a hospital setting with educational and psychologic support and risk factors modification. the patients cardiac diagnoses were defined as a history of at least one of the following: myocardial infarction, coronary artery bypass grafting, percutaneous coronary intervention, and chronic stable angina. for patients to enter a crp, written informed consent was obtained from all the patients and then their heights and weights (after they had fasted overnight) were measured by a nurse in the morning and bmi was calculated by weight / height(kg / m). also, blood samples were taken after 12 - 14 h of fasting for measuring fbs, serum lipids, including triglycerides (tg), total cholesterol (tc), high - density lipoprotein (hdl) using enzymatic methods, and low - density lipoprotein (ldl) were calculated. these patients used usual angina treatment and the dose of those medications were not altered during the program. exercise test which was performed by each patient under the supervision of a cardiologist and a nurse, provided an opportunity to identify body reaction by monitoring hr and blood pressure and observing the electrocardiogram. in fact, it determined fc by indicating the changes in hemodynamic responses and metabolic equivalent. all the patients were divided into 2 groups: obese men and obese women; and they began crp for 2 months, which consisted of 24 sessions and 3 times a week. crp included exercise training sessions, nutritional and psychologic consultation, and risk factor management. exercise sessions were similar in each group and exercise training consisted of combined aerobic and resistance training and it was performed in cardiac rehabilitation center under the supervision of a physician, a nurse, and an exercise physiologist by using treadmill, ergometer, stair climbing, rowing, step, jogging, and by using some resistance devices. each session lasted 90 min, 10-min warm - up followed by 60 min aerobic and resistance training, and finally 20 min cooldown and relaxation. the intensity of training was established according to the clinical condition and calculated between 60% and 85% of maximum heart rate. in addition, personal information of each patient and their history of disease were registered by a nurse and were archived. also, each patient had individual dietary instruction by a visiting dietitian during the program. after 2 months and completing the rehabilitation program, the tests were accomplished by each patient, again. the research protocol was taken under the medical ethics standards and approved by the isfahan cardiovascular research centre ethics committee, which is a member of the office for human research protections, u.s. independent sample t tests were used to identify baseline differences and comparing the changes between the 2 groups. for investigation about the changes between baseline and completion of crp, we used paired t tests. were expressed as mean and standard deviation (sd) and the level of significance was set at p < 0.05. they were separated into obese men group and obese women group. independent sample t tests were used to identify baseline differences among obese men and obese women patients. at baseline obese men had higher fc (p = 0.001) and weight (p = 0.00); but, tc was higher in women (p = 0.01). both groups did not have significant differences in other risk factors, such as tg, ldl cholesterol (ldl - c), hdl cholesterol (hdl - c), and fbs. comparison between obese women and obese men at baseline also, for examination of the changes between baseline and completion of crp, we used paired sample t tests. obese women had significant improvements in all risk factors except tc and fbs, and obese men had positive improvement in weight, bmi, fc, and tc but we did not see any significant improvement in other risk factors. again, for comparing the changes between the 2 groups, independent sample t tests were used. these data did not show any significant differences in fc and most evaluated risk factors except hdl - c and ldl - c / hdl - c, which showed more significant improvement in obese women. it is proved that obesity is a major cause of mortality and morbidity and it is associated to cvd and its risk factors, including hyperlipidemia and diabetes mellitus. because the vast majority of patients entering into cardiac rehabilitation are overweight and limited data are available regarding to the effects of crp and exercise training in obese men and women with chd, the present study was aimed at finding out the benefits of crp in obesity indexes, fc, and some lipid profiles on obese men and women. our baseline finding showed women who participated in the crp were 3 years older than men in comparison with 10 years in another study; and, bmi was greater in women than in men, in spite of women s lower weight; in this regard we can explain that heavier skeleton mass in men can be a cause of men being heavier compared with women. after crp, both groups attained significant improvement in weight and bmi; but comparison of the 2 groups did not show any significant differences after intervention. considerably, there are many studies which implied that crp and exercise training had significant effects in obesity indexes in obese patients with chd. on the contrary, kiat et al. observed no weight reduction in patients with cad after 2 years of crp. on the other hand, obesity is associated with lower exercise performance, which in reduced fc after coronary events. in the present study, obese women had lower fc than obese men at baseline, but similar relative improvements were seen (32.49% and 29.41%, respectively) as a of crp. the difference between the 2 groups was not significant but data showed greater improvement in women. in this regard, it can be noted that lower baseline fc values may interpret this greater improvement in obese women. like our several studies suggested that women had lower fc than men and; also, there were statistical similarities in improvement between men and women. generally we can explain that weight reduction after crp may be a cause for affirmative effects on fc and fitness level in both the groups. in our , at baseline, no considerable differences were seen in lipid profiles between two groups except tc, which was higher in obese women than in obese men. after crp, obese women had favorable changes in tg, hdl - c, ldl - c, and ldl - c / hdl - c; but, tc did not have any significant differences in them. according to a pervious study, there was a significant relationship between tc and obesity, which is due to an increase in cholesterol production with increased weight. conversely, in obese men improvement was seen just in tc but other lipid factors did not have any significant improvement. also, comparing the 2 groups showed that among lipid profiles, substantial differences were seen only in hdl - c and ldl - c / hdl - c. in relation to tg , we can note that obese women had higher tg value at baseline that was the strongest predictor of improvement in tg level when compared with men after crp. in addition, there are many investigations that have similarity or dissimilarity to our finding. lavie and milani showed significant improvement in hdl - c and ldl - c / hdl - c in obese patients but other lipid profiles did not have any changes during crp. also, another study suggested improvement only in hdl - c in women after comprehensive crp. williams et al. did not demonstrate significant reductions in ldl - c levels with obesity intervention, exercise training, and dietary therapy. some studies proposed weight reduction with exercise training, and dietary limitation has significant effects on increasing levels of hdl - c and reducing tg. another study showed that patients had improvement in tc, tg, hdl - c, ldl - c, and ldl - c / hdl - c after cardiac rehabilitation and exercise training. also, sarrafzadegan et al. reported that neither men nor women patients experienced a significant overall improvement in tc, ldl - c, and tg after the 3-month exercise training alone. the present study did not show significant improvement in fbs in obese men and women after crp. brochu et al. confirmed our , but ades et al. mentioned exercise and weight reduction prevent type ii diabetes and increasing fbs. about some disparities between our study and other investigations , we can mention that lower age difference between our groups and also the range of patients age and even the kind and level of exercise training and finally variation in their diets may be the cause for dissimilarities. in general, it is understood that at baseline women had worse cvd risk factors than men. after crp, both the groups achieved significant improvements in fc and most evaluated risk factors. and comparing the 2 groups demonstrated no significant differences between them except hdl - c and ldl - c / hdl - c so, it should be noted that both obese men and obese women can benefit from crp without any attention to gender differences. the first limitation was short - term intervention, which consisted of 24 sessions of crp. it would be desirable to document long - term intervention changes in physiologic outcomes and risk profiles in patients with chd. the second limitation was the lack of control group who did not participate in crp after cardiac events, and comparing them to our groups. the third limitation was that a detailed evaluation of dietary adherence and exercise training outside of the formal crp was not available. we can suggest for future researches to study, whether considerable weight reduction after crp in improved clinical outcomes in patients with chd or not. we conclude that crp, which is accomplished under attendance and supervision of a physician, a nurse, and an exercise physiologist is an important step initiating the process of risk reduction and restoration of fc in obese men and women with chd. it is an acceptable management for enhancement and maintenance of cardiovascular health through individualized programs designed to optimize physical condition and manage related risk factors, such as obesity indexes and lipid profiles.

: obesity is common in patients with cardiovascular disease (cvd) and the vast majority of patients entering into cardiac rehabilitation program (crp) are obese. regarding the gender differences, the risk of developing coronary heart disease (chd) is recognized to be different between obese men and women. so, the purpose of this study was to explore the effect of crp in functional capacity (fc) and risk factors, such as obesity indexes, lipid profiles, and fasting blood sugar (fbs) in obese men and women with chd.marterials and methods: in an observational study between 2000 and 2011, we evaluated a total of 156 obese men and women patients with chd who were referred to cardiac rehabilitation of isfahan cardiovascular research institute. before and after crp, fc and risk factors were assessed and all the participants completed this period. data were analyzed with spss software version 15. for comparing the mean of outcomes, independent t tests and paired sample t tests were used.:data revealed, after crp, obese women had significant improvement in most evaluated risk factors except total cholesterol (p = 0.05) and fbs (p = 0.09); and obese men had favorable changes in weight (p = 0.00) and body mass index (p = 0.00), fc (p = 0.00) and total cholesterol (p = 0.02); in spite of no significant differences in other lipid profiles. comparing the 2 groups did not show any significant differences unless high - density lipoprotein cholesterol (p = 0.01) and low - density lipoprotein cholesterol / high - density lipoprotein cholesterol ratio (p = 0.02) had greater improvement in obese women.:we concluded that crp is an important step initiating the process of risk reduction and restoration of fc in obese men and obese women with chd under attendance and supervision of physician, nurse, and exercise physiologist.

middle ear acquired cholesteatoma is a pathological condition associated with otitis media , and as the cause of otorrhea, hearing loss, and occasionally complications such as facial palsy, brain abscess, and meningitis. middle ear acquired cholesteatoma is morphologically characterized by epithelial cell proliferation and granulation tissue formation. unfortunately, despite many studies our understanding of the mechanisms underlying the pathogenesis of cholesteatoma is limited. metaplasia theoryin 1873, wendt suggested that metaplasia of the mucosa of the middle ear into the keratinizing epithelium led to cholesteatoma. proposed that chronic irritation can cause the mucosal lining of the middle ear to convert to a keratinizing epithelium. in 1873 , wendt suggested that metaplasia of the mucosa of the middle ear into the keratinizing epithelium led to cholesteatoma. proposed that chronic irritation can cause the mucosal lining of the middle ear to convert to a keratinizing epithelium. immigration theorybezold in 1890 and habermann described immigration theory. friedmann and tumarkin have been more contemporary supporters of this aspect, which proposed that squamous epithelium migrates through a defect in the tympanic membrane in an effort to cover areas of inflammation in the middle ear. friedmann and tumarkin have been more contemporary supporters of this aspect, which proposed that squamous epithelium migrates through a defect in the tympanic membrane in an effort to cover areas of inflammation in the middle ear. hyperplasia theoryredi presented evidence that supported the basal cell hyperplasia (papillary proliferation) theory first published by manasse et al. in 1917. as the of inflammation of the middle ear, the proliferation of epithelial cones in the basal layers of the keratinizing epithelium of shrapnell's membrane leads to cholesteatoma formation. redi presented evidence that supported the basal cell hyperplasia (papillary proliferation) theory first published by manasse et al. in 1917. as the of inflammation of the middle ear , the proliferation of epithelial cones in the basal layers of the keratinizing epithelium of shrapnell's membrane leads to cholesteatoma formation. retraction pocket theorybezold in 1890 first described this currently most - accepted theory that proposes that acquired cholesteatoma develops from retraction pockets. a retraction of shrapnell's membrane as a of chronic dysfunction of the eustachian tube might progress into cholesteatoma formation.4(a)habitual sniffing theory was described as under the heading of retraction theory. habitual sniffing associated with closing failure of the eustachian tube is believed to be closely related to the etiology of retraction - type cholesteatoma. it seems that such sniffing induces a high negative pressure in the middle ear and may sometimes promote the development of cholesteatoma or its recurrence after surgery. bezold in 1890 first described this currently most - accepted theory that proposes that acquired cholesteatoma develops from retraction pockets. a retraction of shrapnell's membrane as a of chronic dysfunction of the eustachian tube might progress into cholesteatoma formation.4(a)habitual sniffing theory was described as under the heading of retraction theory. habitual sniffing associated with closing failure of the eustachian tube is believed to be closely related to the etiology of retraction - type cholesteatoma. it seems that such sniffing induces a high negative pressure in the middle ear and may sometimes promote the development of cholesteatoma or its recurrence after surgery. habitual sniffing associated with closing failure of the eustachian tube is believed to be closely related to the etiology of retraction - type cholesteatoma. it seems that such sniffing induces a high negative pressure in the middle ear and may sometimes promote the development of cholesteatoma or its recurrence after surgery. currently, the retraction pocket theory has many supporters following clinical observation, and there is clinical evidence for the retraction and proliferation theory on the pathogenesis of cholesteatoma. sudhoff and tos suggested the proliferation of epithelial cells in the retraction pocket was altered by inflammatory stimuli of the subepithelial connective tissue and that this excessive proliferation may finally lead to cholesteatoma formation. they proposed a four - step concept for the pathogenesis of cholesteatoma that combined the retraction and proliferation theories: (a) the retraction pocket stage, (b) the proliferation stage of the retraction pocket, subdivided into cone formation and cone fusion, (c) the expansion stage of attic cholesteatoma, and (d) bone resorption (figure 1,). but, there was a lack of explanation for the transition from a retraction pocket to cholesteatoma. animal models are very important for studying the pathogenesis of acquired cholesteatoma. chinchillas, guinea pigs, mongolian gerbils, meriones unguiculatus, and rats have been used to make animal models of cholesteatoma. in several studies, chinchillas were used because their auditory apparatus is similar to that of humans. on the other hand, pigs were used for temporal bone pneumatization models because they have a mastoid (figure 2). the gerbil is the only nonhuman animal known to spontaneously develop aural cholesteatomas. the ultrastructure of the epithelial and subepithelial linings of the gerbilline middle ear are similar to that of the human, and destructive characteristics of the gerbilline cholesteatoma closely mimic human cholesteatoma. chole et al. indicated that aural cholesteatoma were found to arise spontaneously in 45.7% of gerbilline ears studied. henry et al. indicated that both the prevalence of spontaneous cholesteatoma and the ant peripheral auditory evoked potential threshold increased from 6 to 18 months of age. stage i is an accumulation of keratin debris on the outside surface of the tympanic membrane; stage ii has a medial displacement of the tympanic membrane into middle ear without contact with the middle wall of the bulla; stage iii, the cholesteatoma is in contact with the prominence of the cochlea; in stage iv, cholesteatoma fills the bulla; in stage v, the cholesteatoma extends intracranially. most of the affected 6- and 12-month - old gerbils (younger gerbils) had stage i or ii cholesteatomas. more than half of the 18- and 24-month - old gerbils (older gerbils) had stage iii or stage iv cholesteatomas. some of the oldest gerbils were at stage v, with the cranial cavity being invaded. also, in 2006, tinling and chole investigated the migration rate and patterns for keratin on the tympanic membrane of the gerbil and guinea pig in comparison to human data and indicated that the gerbil was an appropriate model for cholesteatoma because gerbils quite closely resemble humans in rate and pattern of epithelial migration. in this paper, although the spontaneous gerbilline cholesteatoma is useful for studying the pathogenesis of cholesteatoma, the experimental ligation technique of the external ear canal in gerbils is more valuable in predicting the stage of the cholesteatoma. mongolian gerbils between 2 and 6 months of age were used in this experiment. under anesthesia, some 69 months after ligation of the external auditory canal, these cholesteatomas are in contact with the bone of the middle ear. these induced cholesteatomas were seen to erode bone and displace soft tissue structures, as is typical of human aural cholesteatomas (figure 4,). from a surgical point of view, this model is easy to handle, with a high percentage of success. this animal model has been proved to be useful and informative regarding retraction pocket formation and cholesteatoma development. kim and chung examined the distribution of cytokeratin and the binding patterns of lectin in experimental cholesteatoma specimens. they concluded that the origin of aural cholesteatoma may be the external auditory canal epidermal cells, and the characteristics of these cells do not change once the cholesteatoma develops. they also suggested that cholesteatoma have a different biological nature from that of normal epithelial cells, especially basal cells. larsson et al. performed the acoustic admittance measurements and morphological analysis in experimental cholesteatoma and tympanic membrane. they indicated that the thickness of the fibrous layer was almost doubled, mostly because of an increased amount of collagen fibers, and the acoustic stiffness was significantly increased in all cholesteatoma ears. park et al. investigated the immunohistochemical study of cell proliferation using brdu labeling and the expression of plg - gamma1, ligand - mediated signal transduction for cell proliferation on the tympanic membrane, external auditory canal, and induced cholesteatoma in gerbils. according to the , the induced aural cholesteatoma showed a more active proliferation center of the epithelial cell and more intense immunolabeling of plg - gamma1 protein than the eardrum and external ear canal of the normal gerbil. tinling and chole also indicated the hyperproliferation of kertinocytes to be a causative factor in the development and progression of spontaneous and experimental cholesteatomas in this gerbilline model. the cornerstone of the retraction pocket theory is that eustachian tube obstruction leads to negative middle ear pressure, middle ear effusion, and retraction of the pars flaccida into the epitympanum, and subsequent cholesteatoma. to evaluate the evidence of this theory, the eustachian tube blocking model was developed by wolfman and chole in 1986. bilateral eustachian tube obstruction by electrocauterization of the nasopharyngeal portion was performed in gerbils. mongolian gerbils between 6 to 10 weeks of age were used in this experiment. after each animal was anesthetized, they attempted to gain exposure of the tubal orifice by slitting the soft palate in the midline (figure 5(a), ). a cautery tip was then inserted through the soft palate in the midline and rotated so as to contact the right and left nasopharyngeal walls, respectively (figure 5(b), ). the of their study were as follows (figure 6,). at two weeks, all animals had bilateral serous effusions and retraction pockets. at four weeks, four of eight ears had middle ear fluid, retractions, and cholesteatomas. after eight weeks, five of eight ears had middle ear effusions, and four of these had cholesteatomas; one ear had total atelectasis with a cholesteatoma filling the bulla. by 16 weeks this study provides experimental evidence that aural cholesteatomas may arise by retraction of the tympanic membrane. in 2001 , they suggested that the expression pattern of cytokeratin in retraction pocket cholesteatoma is different from that in normal skin and that the transmigration and hyperproliferation process of squamous epithelium occurs in areas adjacent to the aural cholesteatoma. using this model, wilmoth et al. suggested that the elevation of tumor necrosis factor - alpha and matrix metalloproteinases associated with progressive tympanic membrane atelectasis indicated a possible role for these inflammatory mediators in the pathogenesis of cholesteatoma it was known that chemical injection into the middle ear led to the occurrence of inflammatory change and cholesteatoma formation. an animal model of cholesteatoma was made by using these chemicals in guinea pigs or rats. placing a mixture of talcum powder and fibrin in the bulla of a guinea pig in a typical cholesteatoma. it originates from the epidermal basal cells of the tympanic membrane and migrates into the middle ear. schmid and hellstrom induced the cholesteatoma formation in rats by using dimethyl - benzanthrancene (dmba). after perforating the upper quadrant of the tympanic membrane of a rat, the perforation was exposed to dmba four times at weekly intervals. propylene glycol (pg) has been shown to cause epithelial migration and cholesteatomatous chronic otitis media in north - american chinchillas using optic drops (cortisporin) containing pg (10%). increasing the dose of pg in topical preparations injected transtympanically yields cholesteatomas in a progressively higher percentage of rats, reaching 100% at a concentration of 90%. several studies were done in another to establish new therapies as alternative to surgery for middle ear cholesteatomas using these models. after producing a cholesteatoma in chinchillas by using pg (60%), 5-fluorouracil was used to inhibit growth of the cholesteatoma with satisfactory . hyaluronic acid was applied to the external ear of chinchillas in an attempt to inhibit pg - induced cholesteatoma development with poor . the local use of the transretinoic acid is effective in inhibiting the induced formation of cholesteatomas in guinea pigs. cholesteatoma is a squamous cell cyst, characterized by keratinizing epidermal tissue that can migrate and erode to adjacent structures frequently found in temporal bones. an animal model of cholesteatoma was made by using dermal tissues in the middle ears of animals. after each animal was anesthetized, a retroauricular skin incision was made following abrasion of the graft in the opposite ear. the dorsal bulla was opened, and the whole of the mucosal lining was carefully removed. the graft, taken from the external ear canal or from other parts of the body, was then placed so that the subepithelial plane faced the exposed bone. the opening of the bulla was covered with a piece of temporalis muscle and the wound sutured. according to the , full - thickness skin grafts transplanted into the middle ear with superimposed infections induced expansive growth and cholesteatoma development. to circumvent limitations of the previous models and their unpredictable degree of bone resorption, chole et al. developed a new model of bone resorption in mouse calvarias using keratin particles. under anesthesia, murine keratin collected from the nails and fur of mice was implanted onto the dissected calvarium of mature mice. the caused an activation of osteoclasts in the adjacent bone in a manner similar to that seen in human cholesteatoma and in particle - induced osteolysis (figure 7). this model was useful in investigating the pathological bone remodeling related to cholesteatoma in a genetically well - defined animal. many of the genes and their products that control the inflammatory process are well characterized in this mouse model. based on their findings, sudhoff et al. also used this dermal implanting model to investigate bone resorption observed in middle ear cholesteatoma. they concluded that the dermal implant tissue remained viable and produced a robust, localized inflammatory osteolytic response on the adjacent calvarial surface and that osteoclasts were predominantly found on the surface of the calvarium with the greatest osteoclast density under the increased expression of osteoprotegerin (opg), opg ligand, and macrophage - colony stimulating factor. to investigate the origin of the epithelial cells of cholesteatoma, whether from the epithelial cells of the external auditory canal or the tympanic membrane, in a previous study, we described a new animal model named local hybrid ear model and used in situ pcr, which can detect a few copies of genes within a cell in the section by amplifying the target gene. cholesteatomas were induced in gerbils with transplanted tympanic membranes using the ear auditory canal ligation method. after the pars flaccida of the tympanic membranes were completely removed from male gerbils, corresponding portions of tympanic membranes obtained from the ear of female gerbils were transplanted to the area of defect in the tympanic membranes. we then ligated the external auditory canal of the hybrid - model group. as a control group, the ear auditory canal of normal male and female gerbils was ligated without previous myringoplasty. the origin of cholesteatoma cells was analyzed by the identification of male (xy) or female (xx) cells in the tissue section. thus, in situ pcr was performed to detect the mouse x - chromosome - linked phosphoglycerate kinase-1 (pgk-1) gene on the paraffin sections. as a , one pgk-1 spot in the epithelial nuclei was detected in male cholesteatoma (figure 8(a) ), and one or two pgk-1 spots were detected in female cholesteatoma (figure 8(b) ). on the other hand, in the hybrid - model group, we detected not only one but two pgk-1 spots in the epithelial nuclei of cholesteatoma (figure 8(c) ) and one pgk-1 spot was detected in the cells of the ear auditory canal (figure 8(d) ). the percentage of the number of cells having one pgk-1 spot or two pgk-1 spots of cholesteatoma in the hybrid model was almost the same as that of female cholesteatoma. these indicated that all cholesteatoma cells in the hybrid model have xx chromosomes that were female tissue origin. the strongly demonstrated evidence that the origin of epithelial cells in cholesteatoma is the tympanic membrane, not residential middle ear epithelial cells or the skin of the external ear canal, in this hybrid model of cholesteatoma. animal model studies on the pathogenesis of cholesteatoma have led to an improved comprehension of this disease. mongolian gerbils have a remarkable propensity for the development of aural cholesteatoma; canal cholesteatomas develop spontaneously in aged animals. cholesteatomas were produced by five different methods of induction: ligation of the external ear canal, eustachian tube blocking, chemical or free skin graft injection into the middle ear, the autologous dermal implantation model, and a local hybrid - ear model of experimentally induced cholesteatoma. as shown in table 1, we summarized the advantages and disadvantages of each model. depending upon the purpose of studying the pathogenesis of cholesteatoma gerbilline cholesteatomas induced by these three methods (method 1~method 3) were compared by kim and chole. these animal models proved to be useful and informative regarding retraction pocket formation and cholesteatoma development. as a , the appearance rates of cholesteatoma in each group were almost the same, but the patterns of epithelial hyperplasia, keratin accumulation, thickening of the tympanic membrane, and adhesions of the tympanic membrane were different among the three groups. also, in 2002, kim et al. detected prominent changes in the expression of markers for migration and hyperproliferation in gerbilline cholesteatomas produced by three methods compared with that in the tympanic membrane, and their supported the epidermal migration theory. as in their previous study, the expression patterns of epithelial markers in gerbilline cholesteatomas produced by the three methods were not similar. they concluded that each of the three methods of inducing cholesteatoma may be helpful in investigating different clinical aspects of this disease. on the other hand, in 2007, choufani et al. performed the quantitative comparison of eight biological markers involved in inflammation, cell differentiation, and cell adhesion / apoptosis between sections of the ligated external ear canal animal model (method 1) and clinical specimen. their indicated that the majority of staining parameters was statistically significantly different between sections of the animal model and clinical specimen from the panel of the above markers. they concluded that from a surgical point of view, this model was easy to handle, with a high percentage of success, thereby allowing one to quickly obtain an external auditory duct cholesteatoma. however, these data did not support the concept of complete validity of the popular animal model. the problem with the previous method 1 to method 3 animal models was that the morphological aspects were almost the same as human cholesteatoma, but the immunohistochemical were not strictly the same. in the recent study, we modified the auditory canal ligation model to investigate the origin of the cholesteatoma cells. we analyzed spontaneously occurring cholesteatomas associated with a new transplantation model in gerbils, and using pgk-1 as a tracer provided evidence that transplanted tympanic membranes were the origin of the epithelial cells associated with the development of auditory canal - ligated cholesteatomas in gerbils. the strongly demonstrated that the origin of epithelial cells in cholesteatoma is the tympanic membrane, but not residential middle ear epithelial cells or the skin of the external ear canal in this hybrid model of cholesteatoma. accordingly, other studies revealed different protein and cytokeratin profiles between cholesteatoma keratinocytes and epithelial cells of the middle ear. habitual sniffing has clinically been observed to increase the risk of developing retraction pockets and cholesteatoma. recently, the habitual sniffing - simulating model in gerbils was made by von unge and dircks. the retraction of the tympanic membrane was found, but cholesteatoma formation did not appear. the retraction and proliferation theory is a pathogenesis that combines the retraction caused by otitis media or habitual sniffing and the proliferation of the epithelial cells in the retraction pocket altered by inflammatory stimuli of the subepithelial connective tissue. therefore, some improvement will be needed to investigate the retraction and proliferation theory using this habitual sniffing model. based on the of inflammatory changes in clinical cholesteatoma specimens , we would like to develop a new animal model in the future. in our previous study , we indicated that keratinocyte growth factor (kgf)/fibroblast growth factor-7 plays an important role in cholesteatoma formation. kgf is a mesenchymal cell - derived paracrine growth factor that specifically stimulates epithelial cell growth and is supposed to be secreted from fibroblasts mainly in stroma binding to kgf receptor, which has only been detected on the surface of epithelial cells. on the other hand, direct in vivo plasmid dna transfer to the skin via injection has been reported previously , and transfer of dna using electroporation has been demonstrated as a useful procedure for the short - term delivery of gene therapy. we will use kgf - cmv-14 vector and cause overexpression of kgf by electroporatively transfected kgf cdna in the cells of epithelial tissues in vivo. a single injection of kgf cdna - expressive vector coupled with electroporation will enhance inflammatory reaction and increase keratinization of epithelium in the auditory canal. this model can possibly be used to investigate the effects of various cytokines and growth factors in cholesteatoma formation in vivo. the goal of the experiments using animal models is to analyze the pathogenesis of cholesteatoma. of course, we know that biological differences between animal models and human cholesteatoma would make it difficult to understand the pathogenesis of human cholesteatoma.

middle ear acquired cholesteatoma is a pathological condition associated with otitis media, which may be associated with temporal bone resorption, otorrhea and hearing loss, and occasionally various other complications. cholesteatoma is characterized by the enhanced proliferation of epithelial cells with aberrant morphologic characteristics. unfortunately, our understanding of the mechanism underlying its pathogenesis is limited. to investigate its pathogenesis, different animal models have been used. this paper provides a brief overview of the current status of research in the field of pathogenesis of middle ear acquired cholesteatoma, four types of animal models previously reported on, up - to - date cholesteatoma research using these animal models, our current studies of the local hybrid ear model, and the future prospect of new animal models of middle ear cholesteatoma.

a loss through suicide is a dreadful experience to those who are left behind.1 these persons likely consult their gp when they feel the need for help. appropriate care meets an unmet need; 2688% feel that help is required2,3 while evidence - based services are scarce. relatives of suicide victims are at increased risk of adverse mental health consequences of the loss, e.g. complicated grief (cg).35 cg is characterized by avoidance, disbelief, numbness, detachment and excessive irritability and anger. it is strongly associated with suicidal behaviour, physical morbidity and other mental morbidity.6,7 moreover, suicidal behaviour8 and completed suicide9 cluster in families, posing relatives of suicide victims at increased suicide risk. it may be effectuated by referring these high - risk individuals for effective follow - up care.10 family - based cognitive behaviour therapy (fgt) is more helpful to reduce feelings of guilt and maladaptive grief reactions than usual care.11 among relatives with suicidal feelings, fgt seems to be more effective to decrease the risk of cg confirming the benefits of grief interventions for high - risk individuals.12 gps active engagement in suicide prevention strategies is acknowledged.13 gps are identified as key persons to initiate14and ensure follow - up care for people bereaved by suicide.15 gps attitudes towards referral of suicide bereaved relatives for follow - up care are unknown. knowledge of these factors is of interest since acceptance and implementation of effective interventions need careful analyses of key factors critical in the achievement of changes in practice.16 in order to study the proposed key role of gps in referring relatives for follow - up care, we investigated the gps attitudes and management of help requests following a suicide within the framework of experimentally implementing fgt. factors associated with gps willingness to refer relatives were examined. among these factors was the gps gender as female gps have shown to be more attentive to psychosocial factors than male gps.17 the findings from the present study may be of interest for policy makers and mental health care providers deliberating the availability of effective suicide prevention measures. a cross - sectional, semi - structured questionnaire survey was sent to a random sample of 488 gps of the total number of 895 in the northern part of the netherlands18 gps exposure to patient suicide, bereaved relatives help requests, management of help requests, preference as to the type of care and gps contentment with their interventions were assessed. gps involvement in patient suicide was assessed by asking have you been exposed to a patient's suicide and if so how many times? exposure to help requests was assessed by the following item: have you been exposed to help requests for bereavement by suicide, not necessarily concerning suicide of one of your patients. if so, how many times? gps were asked to report suicides and help requests over the past 3 years. of at most the last two help requests, the time lag (in months) between the suicide and the request was assessed. gps reported whether they counselled the relatives themselves and/or referred for additional help. in case of referral, gps indicated the type of health care workers. further, it was assessed what kind of health care setting is generally preferred: mental health care, primary health care or other kinds of care. multiple services could be indicated. in the questionnaire, the aims, method, benefits and potential risks of fgt were briefly presented. gps were asked if they think fgt is useful (yes, no, i do n't know). gps were also asked if they would be willing to refer patients for fgt if it were available (yes, characteristics of the gps, their exposure to suicide and help requests, management of help requests, preferences as to types of care and gps contentment with the outcome of their proceedings were descriptively analysed . next, bivariate associations between gps exposure to suicide, exposure to help requests, gps gender, perceptions of usefulness and willingness to refer for fgt were calculated using logistic regression . using multivariable logistic regression models, it was examined whether female gender and gps exposure to suicide were independently associated with gps exposure to help requests . further, we investigated whether female gender, gps exposure to suicide and exposure to help requests are independently associated with the perception that follow - up care would be useful . finally, the associations between the aforementioned determinants as well as perception that help is useful were examined in independent association with gps willingness to refer relatives for follow - up care . were expressed as odds ratios as measures of relative risk, indicating the magnitude of associations . characteristics of the gps, their exposure to suicide and help requests, management of help requests, preferences as to types of care and gps contentment with the outcome of their proceedings were descriptively analysed . next, bivariate associations between gps exposure to suicide, exposure to help requests, gps gender, perceptions of usefulness and willingness to refer for fgt were calculated using logistic regression . using multivariable logistic regression models, it was examined whether female gender and gps exposure to suicide were independently associated with gps exposure to help requests . further, we investigated whether female gender, gps exposure to suicide and exposure to help requests are independently associated with the perception that follow - up care would be useful . finally, the associations between the aforementioned determinants as well as perception that help is useful were examined in independent association with gps willingness to refer relatives for follow - up care . were expressed as odds ratios as measures of relative risk, indicating the magnitude of associations . are presented with a 95% confidence interval . the two - sided level of significance of the 488 gps who were approached, 214 ( 44%) returned the questionnaire. the proportion of female responders was equal to the proportion in the study region (31%).18 female doctors had fewer mean (sd) years of practice experience than male doctors (11 ( 7.9) years versus 17 (9.6) years ). sample characteristics and the exposure to suicide and help requests are shown in table 1. characteristics, suicide and help request exposure of gps representing the samplea stated in n (%) unless indicated otherwise. sixty - two per cent (n = 176) indicated that follow - up care for relatives following a suicide pertains to primary health care, 47 gps (27%) to mental health care, 31 (22%) indicated that bereavement support is not a professional health care occupation; 9 (4%) did not know. seventy - four gps reported 128 help requests; 106 could be explored; 81 (76%) were presented within 6 months following the suicide; 13 (12%) were > 1 year after the suicide. table 2 shows how gps dealt with the help requests (n = 106). in 61 cases (58%), the gp was content with the of their interventions, 33 gps (31%) indicated partial and 7 gps (7%) were not content with the outcomes. gps management of help requests (n = 106) relatives were occasionally referred for multiple services. one hundred thirty - nine gps (66%) indicated that fgt might be useful; 146/214 (68%) stated that they would refer patients for fgt if it were actually available; 43 (21%) would conditionally refer patients. most noted conditions were when fgt has additional effects and when the patient wants to. bivariate relationships between determinants of gps management of help requests after a suicide are displayed in table 3. bivariate associations between determinants of gps management of help requests by relatives bereaved by suicidea values are univariable odds ratios with 95% confidence intervals. table 4 shows that gps who were exposed to a patient suicide were six times more likely to be consulted by relatives. more gps who had recently been exposed to suicide believed that follow - up care for the bereaved would be useful than those who were not. female gender was strongly associated with the perception that help is useful, independent of exposure to patient suicide and exposure to relative's help request. however, the perception that follow - up care is useful was less likely when gps had been confronted with help requests in the previous 3 years. the willingness to refer patients for follow - up care did not depend on gps gender or suicide exposure. when gps were, perceiving that follow - up care is useful, referring patients for it was four times more likely. adjusting the outcomes for the number of years of practice experience did not change the . although not statistically significant, exposure to help requests was associated with a two times reduced likelihood of the perception that follow - up care is useful and gps willingness to refer relatives for it. factors associated with gps willingness to refer relatives bereaved by suicide for follow - up care adjustment for years of practice experience of the gp did not change the . or, odds ratio; ci, confidence interval. this study demonstrates that gps think that effective follow - up care for relatives bereaved by suicide (e.g. fgt) is useful and they are willing to refer relatives if it were available. it is in line with halligan and corcoran19 who reported a 62% proportion who thinks that services to rely on in the aftermath of suicide would be helpful. although it has been suggested that female gps are more involved in patients who present with psychosocial problems than male colleagues,20 it apparently does not hold for help seeking following a suicide. the high impact of a patient suicide on gps19 may be an overriding factor in this respect. bereaved families believe that the gps should initiate contact with the family briefly after the suicide.14,15 in this study, it remained unclear whether relatives or gps were the initiators. therefore, the findings do not allow the suggestion that gps meet relatives needs sufficiently, taking into account that 57% of the help requests were referred for mental health care (table 2). possibly, gps recognize the psychiatric vulnerability of relatives of suicide victims,21 whereas an estimated 20% of the bereaved are actually at increased risk of psychiatric consequences. it implicates that the high rate of referral for mental health care may, in part, be unnecessary, but probably come along with relatives persisting need for help.2 possibly, gps may be inclined to refer relatives anyway due to gps feelings of guilt or due to a disrupted relationship with the bereaved family14,19 or when the gp is blamed for the suicide.11 however, unnecessary referral for mental health care may stigmatize relatives and should be prevented. the response rate (44%) is low, but not uncommon in studies on subjects of sensitive nature (e.g.14,22,23). probably, gps who were recently involved in suicide might be somewhat over represented in the current sample. the explanation that higher exposure is causing higher response rates in this research area is supported by a previous finding of hilligan and corcoran. they achieved a 79% response in the sample of gps, of which 86% was confronted with a patient suicide over a 10 years period, whereas dutch gps are only four times involved in a patient suicide during their professional life.24 responders in the current study may have been implicated in a randomised controlled trial (rct) to the effectiveness of fgt.11 possibly, this has affected their attitudes and proceedings regarding relatives help requests. this assumption is supported by the large proportion of gps (65%) who counselled the bereaved themselves; more often than a 50% rate found in previous studies.25 unfortunately, it remained unclear to what extent the sample represents gps who were actually involved in the rct. overall, the extrapolation of the should be applied cautiously; however, issues related to follow - up care for those bereaved by suicide principally concern gps who were actually involved and this may, in turn, support the findings validity. in countries with self - employed doctors and a referral system, gps are in a better position to provide psychosocial care.26 this may moderate the generalizabiliy of the findings to countries without a referral system. gps should be well informed of the effectiveness of follow - up care for bereaved relatives as when gps are convinced of the benefits, they are more inclined to refer relatives for it, especially male gps who previously dealt with help seeking relatives of suicide victims. however, gps are pragmatic and cautious of change27; their sense of competence regarding the management of help requests of relatives bereaved by suicide may discourage the implementation of evidence - based services.16,28 given the need for help and its effects for relatives who are liable to adverse health consequences and feelings of guilt, fgt should be available for vulnerable relatives. fgt might be provided in primary care, for instance by consultation of trained mental health nurses. this may give easier access to this kind of help than when provided in mental health care. the availability of fgt should be recommended in guidelines on the prevention of suicidal behaviours. in view of the key role for gps in suicide prevention, especially in referring people bereaved by suicide for effective follow - up care, deliberations of gps in the management of these help requests might be explored in future qualitative research. the response rate (44%) is low, but not uncommon in studies on subjects of sensitive nature (e.g.14,22,23). probably, gps who were recently involved in suicide might be somewhat over represented in the current sample. the explanation that higher exposure is causing higher response rates in this research area is supported by a previous finding of hilligan and corcoran. they achieved a 79% response in the sample of gps, of which 86% was confronted with a patient suicide over a 10 years period, whereas dutch gps are only four times involved in a patient suicide during their professional life.24 responders in the current study may have been implicated in a randomised controlled trial (rct) to the effectiveness of fgt.11 possibly, this has affected their attitudes and proceedings regarding relatives help requests. this assumption is supported by the large proportion of gps (65%) who counselled the bereaved themselves; more often than a 50% rate found in previous studies.25 unfortunately, it remained unclear to what extent the sample represents gps who were actually involved in the rct. overall, the extrapolation of the should be applied cautiously; however, issues related to follow - up care for those bereaved by suicide principally concern gps who were actually involved and this may, in turn, support the findings validity. in countries with self - employed doctors and a referral system, gps are in a better position to provide psychosocial care.26 this may moderate the generalizabiliy of the findings to countries without a referral system. gps should be well informed of the effectiveness of follow - up care for bereaved relatives as when gps are convinced of the benefits, they are more inclined to refer relatives for it, especially male gps who previously dealt with help seeking relatives of suicide victims. however , gps are pragmatic and cautious of change27; their sense of competence regarding the management of help requests of relatives bereaved by suicide may discourage the implementation of evidence - based services.16,28 given the need for help and its effects for relatives who are liable to adverse health consequences and feelings of guilt, fgt should be available for vulnerable relatives. fgt might be provided in primary care, for instance by consultation of trained mental health nurses. this may give easier access to this kind of help than when provided in mental health care. the availability of fgt should be recommended in guidelines on the prevention of suicidal behaviours. in view of the key role for gps in suicide prevention, especially in referring people bereaved by suicide for effective follow - up care, deliberations of gps in the management of these help requests might be explored in future qualitative research. funding: the impress study (primary prevention of cg in the aftermath of suicides . region - wide implementation of a brief, nurse - led, cognitive behavioural, family - oriented, primary care and evidence - based approach) was funded by zon - mw (the netherlands organisation for health research and development); http://www.zonmw.nl/en. funding to pay the open access publication charges for this article was provided by the department of general practice medicine of the university medical center groningen, the netherlands. ethical approval: the study was approved by the university medical center groningen ethics committee (metcumcg 2002/137).

. relatives who are bereaved by suicide likely consult their gp when they feel the need for professional help. gps may play a key role in establishing who is at risk for adverse consequences of the loss as they are familiar with relatives possible psychiatric vulnerabilities. the availability of evidence - based services for relatives of suicide victims is limited. successful implementation of services needs analysis of key factors considered critical in the achievement of changes. we investigated gps management of help requests of relatives bereaved by suicide and examined determinants of gps willingness to refer for evidence - based follow - up care.methods. a cross - sectional survey among 488 gps in the northern part of the netherlands.. a 44% response was achieved (n = 214) during the last 3 years, 38 (18%) were exposed to suicide, 21 (10%) to help requests without being exposed to suicide and 52 (24%) to both suicide and help requests. out of 106 requests, 69 (65%) were handled by the gp; 60 (57%) were either directly or additionally referred, principally for mental health care. suicide exposure and female gender were associated with the doctor's perception that follow - up care following a loss through suicide is useful. the perception that help is useful increased the likelihood of gps referral for evidence - based follow - up care.. gps support the availability of evidence - based follow - up care for relatives of suicide victims. to modify gps key role in referring relatives for it, gps should be well informed of its usefulness and to whom.

idiopathic parkinson s disease (pd) is often associated with sleep disorders, the most common of which are insomnia, increased daytime sleepiness, restless legs syndrome, and rapid eye movement (rem) sleep behavior disorder.1 polysomnographic studies have demonstrated that patients with pd experience increased sleep latency, reduced sleep efficiency, and reduced duration of rem sleep.2 sleep disorders affect approximately two - thirds of patients with pd,3 and these patients are likely to experience a greater severity of nonmotor pd symptoms than those without sleep disorders.4 unsurprisingly, sleep disorders in patients with pd have a negative impact on quality of life.2 assessing sleep using traditional instruments (polysomnography) is both costly and time consuming and requires specialized hospital - based settings. consequently, sleep scales are widely employed in clinical practice. in patients with pd, individual items of the parkinson s disease sleep scale (pdss) accurately distinguish patients from healthy controls, and items of the pdss have been shown to correlate with polysomnography.5 the pdss is also useful to assess the severity of sleep disorders in pd, and individual sleep scales can be used to evaluate response to treatment. the most frequently used pd medications (eg, levodopa and dopamine agonists) show little efficacy in controlling sleep disturbances.6,7 in fact, dopaminergic medication appears to adversely affect sleep continuity; low - dose dopamine agonists are associated with insomnia while higher doses are associated with day - time sleepiness.1,6 rasagiline is a selective, irreversible inhibitor of monoamine oxidase b (maob - i) that ameliorates the symptoms of pd by inhibiting striatal dopamine metabolism.8 maob - is also increase melatonin levels in the pineal gland and may therefore contribute to modulation of wakefulness / sleep patterns and circadian rhythms.9 rasagiline has been used for many years in the european union and usa as a first - line treatment for early forms of pd, as well as in combination with levodopa and dopamine agonists in more advanced forms of the disease. clinical studies with rasagiline have demonstrated that the drug has a good safety profile as monotherapy10 and in combination regimens.11 the aim of this observational study was to compare the efficacy of levodopa + rasagiline with that of levodopa alone in the treatment of sleep disorders in patients with pd. this single - center, prospective, observational study evaluated 38 outpatients with pd experiencing sleep disturbances. the study was performed over 12 weeks from january to june 2015 at the clinic of movement disorders, ii division of neurology, second university of naples, italy. the study was approved by the medical ethical committee of second university of naples and security board, and all participants provided written informed consent. patient privacy was maintained and only the investigator was able to link data to an individual via the identification number assigned at enrollment. eligible patients were aged 18 years with a diagnosis of idiopathic mild - to - moderate pd (hoehn and yahr stage 12), according to the united kingdom parkinson s disease society brain bank diagnostic criteria. patients were also required to be experiencing sleep disturbances (including insomnia, hypersomnolence, vivid dreaming, restless legs, and other sleep - related movement disorders), as reported by the patient (pdss mean score 100) and to be receiving treatment with levodopa (200300 mg / d) with or without rasagiline (1 mg / d, administered in a single dose at 7 pm). exclusion criteria included the presence of any primary sleep disorder; any neurodegenerative disease other than pd or any type of dementia (according to diagnostic and statistical manual of mental disorders, 4th edition criteria), by physician assessment; pregnancy and/or breastfeeding; and comorbidities that could in secondary sleep disorders, including congestive heart failure, other serious heart disease, severe liver disease / cirrhosis, severe renal failure / dialysis, severe respiratory failure, severe anemia, hypothyroidism, and diabetes. cutoff values for comorbidity signs and symptoms were not defined; inclusion or exclusion of a given patient was at the investigator s discretion. patients were randomly assigned to 12 weeks treatment with levodopa (200300 mg / d) or levodopa + rasagiline (1 mg / d). patient demographics and characteristics were collected at baseline and included family history, age of onset of pd, stage of disease (hoehn and yahr staging), motor symptoms (unified parkinson s disease rating scale iii scores), cognitive function (mini - mental state examination and beck depression inventory scale scores) treatments received, and the presence of comorbidities. the primary end point was improvement in sleep quality based on sleep latency (hours), number of awakenings, and total sleep time (hours) as recorded in autonomously compiled patient sleep diaries. patients were required to record, within 30 minutes of waking, information on their sleep clinical evaluations, performed at baseline and at the end of the 12-week study period, included the following: general physical, neurological and psychological examination, assessment of adherence to therapy, administration of the hoehn and yahr scale, and administration of the pdss (a decrease in pdss scores indicates improvement). given the observational nature of the study, an expected difference between the two treatment groups was not assumed a priori, and so sample size was not calculated. descriptive statistical analyses were performed using a general linear model (glm) to correct for observed variation in baseline features. data are expressed as means with standard deviation and mean standard error (se) for the continuous variables. two - tailed t - tests were applied to evaluate statistical significance within groups and significance of between - group difference was based on analysis of variance models (p<0.05). this single - center, prospective, observational study evaluated 38 outpatients with pd experiencing sleep disturbances. the study was performed over 12 weeks from january to june 2015 at the clinic of movement disorders, ii division of neurology, second university of naples, italy. the study was approved by the medical ethical committee of second university of naples and security board, and all participants provided written informed consent. patient privacy was maintained and only the investigator was able to link data to an individual via the identification number assigned at enrollment. eligible patients were aged 18 years with a diagnosis of idiopathic mild - to - moderate pd (hoehn and yahr stage 12), according to the united kingdom parkinson s disease society brain bank diagnostic criteria. patients were also required to be experiencing sleep disturbances (including insomnia, hypersomnolence, vivid dreaming, restless legs, and other sleep - related movement disorders), as reported by the patient (pdss mean score 100) and to be receiving treatment with levodopa (200300 mg / d) with or without rasagiline (1 mg / d, administered in a single dose at 7 pm). exclusion criteria included the presence of any primary sleep disorder; any neurodegenerative disease other than pd or any type of dementia (according to diagnostic and statistical manual of mental disorders, 4th edition criteria), by physician assessment; pregnancy and/or breastfeeding; and comorbidities that could in secondary sleep disorders, including congestive heart failure, other serious heart disease, severe liver disease / cirrhosis, severe renal failure / dialysis, severe respiratory failure, severe anemia, hypothyroidism, and diabetes. cutoff values for comorbidity signs and symptoms were not defined; inclusion or exclusion of a given patient was at the investigator s discretion. patients were randomly assigned to 12 weeks treatment with levodopa (200300 mg / d) or levodopa + rasagiline (1 mg / d). patient demographics and characteristics were collected at baseline and included family history, age of onset of pd, stage of disease (hoehn and yahr staging), motor symptoms (unified parkinson s disease rating scale iii scores), cognitive function (mini - mental state examination and beck depression inventory scale scores) treatments received, and the presence of comorbidities. the primary end point was improvement in sleep quality based on sleep latency (hours), number of awakenings, and total sleep time (hours) as recorded in autonomously compiled patient sleep diaries. patients were required to record, within 30 minutes of waking, information on their sleep clinical evaluations, performed at baseline and at the end of the 12-week study period, included the following: general physical, neurological and psychological examination, assessment of adherence to therapy, administration of the hoehn and yahr scale, and administration of the pdss (a decrease in pdss scores indicates improvement). given the observational nature of the study, an expected difference between the two treatment groups was not assumed a priori, and so sample size was not calculated. descriptive statistical analyses were performed using a general linear model (glm) to correct for observed variation in baseline features. data are expressed as means with standard deviation and mean standard error (se) for the continuous variables. two - tailed t - tests were applied to evaluate statistical significance within groups and significance of between - group difference was based on analysis of variance models (p<0.05). all 38 patients completed the 12 weeks treatment, and key baseline demographics / clinical characteristics are described in the supplementary information (table s1). patients had a mean age of 70.310.6 years, 56.8% were male, and mean disease duration was 55.95.5 months. almost all the patients reported difficulties in initiating and maintaining sleep, with correspondingly high pdss (104.721.5 and 103.921.8 in the monotherapy and combination therapy groups, respectively). the mean levodopa dose was 267.41 mg / d in the combination group vs 281.7 mg / d in the monotherapy group. sleep latency (figure 1a), number of awakenings (figure 1b), and total sleep time (figure 1c) all improved significantly from baseline to week 12 in patients receiving levodopa + rasagiline. in contrast, sleep latency was the only measure to show a significant improvement from baseline to week 12 (figure 1a) with levodopa alone. at 12 weeks and compared with levodopa alone, patients receiving levodopa + rasagiline had a significantly lower mean sleep latency time (figure 1a), while the improvement from baseline was significantly greater with levodopa + rasagiline (0.54740.69472 h vs 1.67891.20627 h ; p=0.001). there was no significant difference between treatment groups for the mean number of awakenings reported at week 12 (figure 1b) or change from baseline to week 12 (0.21050.41885 vs 0.15790.50146 ; p=0.728). at week 12, mean total sleep time was significantly greater in patients receiving combination treatment than monotherapy (figure 1c), and the improvement from baseline to week 12 was significantly greater with levodopa + rasagiline than levodopa alone (1.26421.62450 h vs 0.32110.69648 h ; p=0.026). applying a glm to correct for baseline features, the treatment received and the baseline value of each end point were significantly related to variation at week 12. the estimated marginal mean of delta sleep latency was 1.57 (se : 0.166 ; 95% ci : 1.906, 1.225) in the levodopa + rasagiline group vs 0.58 (se : 0.181 ; 95% ci : 0.954, 0.208) in the levodopa alone group (p=0.001); the estimated marginal mean of delta number of awakenings was 0.36 (se : 0.086 ; 95% ci : 0.535, 0.182) and 0.004 (se : 0.101 ; 95% ci : 0.203, 0.211), respectively (p=0.013), while the estimated marginal mean of delta total sleep hours was 1.35 (se : 0.315 ; 95% ci : 0.701, 1.995) vs 0.30 (se : 0.362 ; 95% ci : 0.449, 1.041), respectively (p=0.044). improvements in pdss at 12 weeks were greater with combination treatment compared with levodopa alone (figure 2). a significant reduction in pdss scores (ie, an improvement in sleep) from baseline to week 12 was observed in six of 15 items in the levodopa alone group (distressing dreams, urine incontinence, limb paresthesia, cramps, tremor, and feeling tired and sleepy) and ten of 15 items (restless limbs, fidgeting in bed, distressing dreams, distressing hallucinations, nycturia, urine incontinence, limb paresthesia, cramps, tremor, and feeling tired and sleepy) in the levodopa + rasagiline group. pdss scores at 12 weeks were significantly lower with levodopa + rasagiline vs levodopa alone for nycturia (p=0.012) and limb paresthesia (p=0.032), while the change from baseline to week 12 significantly favored levodopa + rasagiline vs levodopa alone for nycturia (p=0.002). applying a glm to correct for baseline features, the baseline value of each item was significantly related with variation for 12 of 15 items at week 12. this observational study indicates that the addition of rasagiline to levodopa improves sleep quality in patients with pd experiencing sleep disorders. after 12 weeks treatment, sleep latency and total sleep time were significantly more improved in patients receiving combination therapy than in those receiving levodopa alone. our findings support those of the randomized, double - blind actor study in patients with pd, which demonstrated that physician - assessed sleep disorders and daytime sleepiness decreased significantly more with rasagiline 1 mg / d than with pramipexole 1.5 mg / d.10 in animal models, it has been shown that inhibition of monoamine oxidase (mao) also increases melatonin levels in the pineal gland,9 and it is hypothesized that this is how rasagiline may improve sleep disorders in patients with pd. furthermore, rasagiline lacks amphetamine - like metabolites, and so may confer advantages over other maob - is in the treatment sleep disorders in patients with pd.12 although the study was prospective, it had a number of limitations including the relatively small number of patients recruited. furthermore, we restricted our study to subjective symptoms and perception of restfulness using patient sleep diaries and pdss scores. we accept that detailed assessment of the complex sleep architecture in patients with pd requires additional studies using instrumental techniques that currently are not in common clinical practice (eg, polysomnography), expanding our observations on rem behavior disorder, and periodic limb movement syndrome. this small monocentric study suggests that rasagiline added to chronic levodopa therapy might provide relevant benefit to patients with pd experiencing sleep disorders. given the high prevalence of sleep disorders in this population, prospective, randomized studies in more patients and over a longer time period are warranted. baseline characteristics and demographics note: all values are mean sd unless otherwise stated. abbreviations: bdi, beck depression inventory; mmse, mini - mental state examination; pdss, parkinson s disease sleep scale; updrs iii, unified parkinson s disease rating scale part iii; sd, standard deviation.

introductionrasagiline is a selective, irreversible monoamine oxidase b inhibitor that ameliorates the symptoms of parkinson s disease (pd) by inhibiting striatal dopamine metabolism. there is also evidence that monoamine oxidase b inhibitors increase melatonin levels in the pineal gland and may have a beneficial effect on sleep disorders, which are a common feature in patients with pd.methodsthis single - center, prospective, observational, 12-week study compared the effect of combination therapy with levodopa 200300 mg / d + rasagiline 1 mg / d (n=19) with levodopa 200300 mg / d alone (n=19) in the treatment of sleep disorders in patients with idiopathic pd.after 12 weeks treatment, mean sleep latency was significantly (p<0.001) lower and the improvement in sleep latency from baseline was significantly (p=0.001) greater in patients receiving levodopa + rasagiline than in patients receiving levodopa alone. similarly, at the end of the study, the mean total sleep time was significantly (p=0.002) longer and the improvement from baseline in mean total sleep time was significantly (p=0.026) greater in patients receiving levodopa + rasagiline than levodopa alone. there were no significant differences between treatment groups for the mean number of awakenings reported at week 12 nor the change from baseline to week 12 in mean number of awakenings.adding rasagiline to levodopa improved sleep outcomes and may be an appropriate option for patients with pd experiencing sleep disorders.

the ches is a prospective, population - based study of glaucoma and other ocular conditions of chinese americans. details of the study design and procedures are described in detail elsewhere. in brief, the examination clinic is located in the city of monterey park, california, in los angeles county, which has a large chinese - american population. the study enrollment began in 2010 and included a total of 4582 individuals of chinese descent, aged 50 years and older. ethics committee approval was obtained from the institutional review board of the university of southern california health sciences. exclusion criteria included prior intraocular surgery (e.g., history of cataract extraction, corneal transplant, incisional glaucoma surgery, and retina surgery), penetrating eye injury, or the presence of corneal disorders such as corneal endothelial dystrophy, pterygium, or corneal scars that may preclude satisfactory imaging. after a detailed ophthalmic history was obtained, participants received a complete ocular examination, including visual acuity assessment, pupil assessment, visual field exam (sita standard 24 - 2), slit - lamp biomicroscopy, goldmann applanation tonometry, gonioscopy, imaging with the eyecam, and dilated fundus examination of the optic nerve, macula, and periphery. clinical examinations were performed by two glaucoma - trained ophthalmologists (dw, cl). indirect gonioscopy was performed with a posner - type 4-mirror lens (model odpsg ; ocular instruments, inc ., the slit was minimized to a 1-mm height and width to prevent light from entering the pupil . the light was also minimized to a level that was necessary to adequately observe the angle . grade 1 was recorded for angles in which the width was judged to be 5 to 15. grade 2 was assigned for angles with a width of 15 to 25. grade 3 was used for 25 to 35 angles, and grade 4 for greater than 35 angles . subjects were placed in a supine position, and the room was darkened during testing . imaging with the eyecam was performed by a single trained technician . images were obtained from all four quadrants ( inferior, superior, nasal, and temporal quadrants sequentially) of both eyes. topical anesthetic drops (proparacaine hydrochloride 0.5% ; alcon laboratories, inc ., fort worth, tx, usa) were applied, followed by a coupling gel. if the view of the angle was blocked by a convex lens curvature, the technician was allowed to move the tip approximately 10 anteriorly along the cornea, similar to slightly tilting a gonioscopy lens. evaluation of the angle structures was accomplished by adjusting the distance of the hand piece tip from the limbus, and the illumination was adjusted by the foot pedal control. the observer was blinded to the patient's age, sex, and clinical information, including gonioscopy . parameters assessed included: image quality, graded as 1 to 3, angle grade by structures identified, pigmentation level, presence or absence of sampaolesi's line, and any abnormalities such as pigmented tumors. image quality was graded between 1 and 3, with grade 1 representing the best quality score in which the angle image was clear, grade 2 indicating a slightly blurred image in which angle structures remained distinguishable, and grade 3 a blurred image in which angle structures are difficult to identify. angle grading was according to the following: grade 4 (wide open angle) in which ciliary body band is observed for the majority of the image, grade 3 (open angle) in which scleral spur is observed for the majority of the image, grade 2 (narrow but open angle) in which the posterior tm is the most posterior structure seen for the majority of the image, grade 1 (narrow occludable angle) in which only the anterior tm is seen for the majority of the image, and grade 0 (closed / appositional angle) in which no angle structures are seen for most of the image. these angle grading categories matched the shaffer classification system used to grade the eyes clinically on gonioscopy. for both eyecam and gonioscopy, angle closure was defined as grade 0 in the classification scheme described. sampaolesi's line was graded as positive if a linear pigmented deposition was observed anterior to the schlwabe's line. abnormalities that were specifically denoted included focal peripheral anterior synechia, iris processes, patchy tm pigmentation, iris elevation, iris lesion (mass with increased pigmentation), and neovascular vessels. the right eye of each patient was used for analysis unless that eye met one or more of the exclusion criteria, in which case the left eye was used (provided it met no exclusion criteria). the mcnemar test was used to test the null hypothesis of marginal homogeneity (rater agreement) in 2 2 cross classifications of paired responses to dichotomous items (quadrant / angle closure). the simple kappa statistic was used to assess the strength of agreement between dichotomized (open / closed) variables, while the weighted was used to measure agreement for ordinal variables (the polychotomous grade variable used to quantify angle width), and to measure intra- and interrater reliability. the first - order agreement coefficient (ac1) statistic was used in addition to , as the latter statistic can yield coefficients that are paradoxically low relative to overall agreement under conditions where high trait prevalence causes a marked imbalance in contingency table marginals. receiver operating characteristic curves with calculations of area under the curve (auc) and 95% confidence intervals (cis) were used as an index of performance for identification of eyes with angle closure, using gonioscopy as the reference standard. statistical analyses were performed using sas version 9.4 (sas institute, inc ., cary, nc, usa). after a detailed ophthalmic history was obtained, participants received a complete ocular examination, including visual acuity assessment, pupil assessment, visual field exam (sita standard 24 - 2), slit - lamp biomicroscopy, goldmann applanation tonometry, gonioscopy, imaging with the eyecam, and dilated fundus examination of the optic nerve, macula, and periphery. clinical examinations were performed by two glaucoma - trained ophthalmologists (dw, cl). indirect gonioscopy was performed with a posner - type 4-mirror lens (model odpsg ; ocular instruments, inc ., bellevue, wa, usa) under dark ambient lighting. the slit was minimized to a 1-mm height and width to prevent light from entering the pupil. the light was also minimized to a level that was necessary to adequately observe the angle. grade 1 was recorded for angles in which the width was judged to be 5 to 15. grade 2 was assigned for angles with a width of 15 to 25. grade 3 was used for 25 to 35 angles, and grade 4 for greater than 35 angles. subjects were placed in a supine position, and the room was darkened during testing. imaging with the eyecam images were obtained from all four quadrants (inferior, superior, nasal, and temporal quadrants sequentially) of both eyes. topical anesthetic drops (proparacaine hydrochloride 0.5% ; alcon laboratories, inc ., fort worth, tx, usa) were applied, followed by a coupling gel. if the view of the angle was blocked by a convex lens curvature, the technician was allowed to move the tip approximately 10 anteriorly along the cornea, similar to slightly tilting a gonioscopy lens. evaluation of the angle structures was accomplished by adjusting the distance of the hand piece tip from the limbus, and the illumination was adjusted by the foot pedal control. the observer was blinded to the patient's age, sex, and clinical information, including gonioscopy . parameters assessed included: image quality, graded as 1 to 3, angle grade by structures identified, pigmentation level, presence or absence of sampaolesi's line, and any abnormalities such as pigmented tumors. image quality was graded between 1 and 3, with grade 1 representing the best quality score in which the angle image was clear, grade 2 indicating a slightly blurred image in which angle structures remained distinguishable, and grade 3 a blurred image in which angle structures are difficult to identify. angle grading was according to the following: grade 4 (wide open angle) in which ciliary body band is observed for the majority of the image, grade 3 (open angle) in which scleral spur is observed for the majority of the image, grade 2 (narrow but open angle) in which the posterior tm is the most posterior structure seen for the majority of the image, grade 1 (narrow occludable angle) in which only the anterior tm is seen for the majority of the image, and grade 0 (closed / appositional angle) in which no angle structures are seen for most of the image. these angle grading categories matched the shaffer classification system used to grade the eyes clinically on gonioscopy. for both eyecam and gonioscopy, angle closure was defined as grade 0 in the classification scheme described. sampaolesi's line was graded as positive if a linear pigmented deposition was observed anterior to the schlwabe's line. abnormalities that were specifically denoted included focal peripheral anterior synechia, iris processes, patchy tm pigmentation, iris elevation, iris lesion (mass with increased pigmentation), and neovascular vessels. the right eye of each patient was used for analysis unless that eye met one or more of the exclusion criteria, in which case the left eye was used (provided it met no exclusion criteria). the mcnemar test was used to test the null hypothesis of marginal homogeneity (rater agreement) in 2 2 cross classifications of paired responses to dichotomous items (quadrant / angle closure). the simple kappa statistic was used to assess the strength of agreement between dichotomized (open / closed) variables, while the weighted was used to measure agreement for ordinal variables (the polychotomous grade variable used to quantify angle width), and to measure intra- and interrater reliability. the first - order agreement coefficient (ac1) statistic was used in addition to , as the latter statistic can yield coefficients that are paradoxically low relative to overall agreement under conditions where high trait prevalence causes a marked imbalance in contingency table marginals. receiver operating characteristic curves with calculations of area under the curve (auc) and 95% confidence intervals (cis) were used as an index of performance for identification of eyes with angle closure, using gonioscopy as the reference standard. statistical analyses were performed using sas version 9.4 (sas institute, inc ., cary, nc, usa). ches identified a total of 5782 eligible subjects. of these, 4582 completed both a home questionnaire and a clinical eye exam. one hundred forty - one subjects refused angle assessment, leaving 4441 who underwent both gonioscopy and eyecam imaging. of those who were assessed, 292 subjects were subsequently excluded due to previous intraocular surgery, ing in an analytic sample of 4149 participants for analysis. individuals with incomplete, unavailable, or poor quality eyecam images in some quadrants were excluded from the appropriate quadrant - specific analyses; no gonioscopy cases were excluded, incomplete, or unrecorded. the sample included 1523 men and 2626 women. inter- and intraobserver agreement for eyecam (defining angle closure as 2 or more quadrants closed) was excellent (= 0.82 and 0.87, respectively). tables 1 and 2 show the agreement between gonioscopy and eyecam for various definitions of angle closure, as well as agreement between the two modalities on angle closure on a per - quadrant basis. gonioscopy and eyecam showed moderate agreement according to the statistic, and excellent agreement according to the ac1 statistic for all quadrants and angle closure definitions. agreement was best when defining angle closure as two or more quadrants on gonioscopy (= 0.60, ac1 0.90, p < 0.0001). when angle closure was examined by quadrant, the superior and temporal quadrants showed the least agreement between gonioscopy and eyecam images (= 0.52) compared with the other quadrants (nasal quadrant = 0.57, inferior quadrant = 0.54). gonioscopy identified angle closure at higher rates than eyecam when angle closure was defined as closure in two or more quadrants, three or more quadrants, all four quadrants (p < 0.0001), and in the superior and temporal quadrants. there was no change in the agreement statistics when subjects with previous lpi were removed from the analysis (data not shown). agreement between eyecam and gonioscopy in iridocorneal angle assessment using binary (open / closed) outcome measures agreement between eyecam and gonioscopy in iridocorneal angle assessment using ordinal outcome measures (angle width in degrees, categorized using the shaffer classification system) the auc was greater than 70% for all definitions of angle closure, as well as for quadrant - specific angle closure (table 3). when defined as two or more quadrants closed, angle closure was not diagnosed using eyecam despite being classified in gonioscopy in 9.0% (359/3994 eyes) of cases, while 3.4% of cases saw angle closure diagnosed in eyecam but not gonioscopy. angles graded as open on eyecam and closed on gonioscopy were attributed to partial angle closure within the quadrant (fig . a) or the presence of a pigmented schwalbe's line within the quadrant (fig . b). performance of eyecam relative to gonioscopy for each quadrant and various definitions of angle closure eyecam images classified as (a) open versus (b) closed angles. this study is the first of its kind to use eyecam in a population - based setting and reports the largest number of patients studied with eyecam to date. while prior studies have used eyecam in smaller, clinic - based populations constituting less than 200 patients, our study examined over 4000 individuals in a community setting. in our study, grading of eyecam images showed moderate to excellent agreement with gonioscopic assessment, which is consistent with prior studies in smaller clinic - based populations. our study also demonstrated excellent intra- and interobserver agreement equivalent to prior studies in a clinical setting. differences exist between our and prior studies regarding the agreement rate between gonioscopy and eyecam, which can be attributed to the different size and demographics of the populations studied, and the prevalence of angle closure within these populations. we noted a higher rate of angle closure detected by gonioscopy than by eyecam in this study. this difference can be attributed to the presence of partial angle closure within the quadrant that was not detected by eyecam but detected on gonioscopy, as well as a highly pigmented schwalbe's line that may have led to disparate interpretations of the angle structures. eyecam is unlikely to replace gonioscopy as the reference standard, and disparities in angle assessment using the two methods must be interpreted with caution. the moderate to high diagnostic performance of eyecam in detecting angle closure and its agreement with gonioscopy make it a useful screening tool in patient populations with known high rates of angle closure, such as chinese and chinese americans. in this study the utility of eyecam in populations at high risk for angle closure includes: the ability of eyecam to be operated by a trained technician rather than a physician, allowing for a wider range of angle documentation across a greater number of patients, providing a view of the angle similar to that observed with gonioscopy (apart from its inability to provide a dynamic view of the angle), allowing for 360 visualization of the iridocorneal angle, unlike the cross - sectional views afforded by traditional as - oct and ubm, and the ability to monitor the angle over time. eyecam imaging provides an unprecedented objective method of gonioscopic angle documentation that can be followed longitudinally, in a manner similar to how optic disc stereophotographs are used to track glaucomatous disease progression. these stored eyecam images can also be used in training sessions to improve agreement of gonioscopic angle grading between ophthalmologists, which remain suboptimal and subjective. furthermore, the standardization of the eyecam image acquisition process, such that identical portions of the iridocorneal angle are obtained at each exam, provides the clinician with a baseline of the gonioscopic exam for each patient and each angle quadrant that can be referenced to evaluate disease progression and changes related to treatment. in our study, 72% and 96% of eyes identified as closed and open angle using the eyecam, respectively, were given the same diagnosis using gonioscopy. separate clinicians assessed gonioscopy and eyecam images in order to prevent bias in agreement between the two methods, but there was excellent intra- and interrater reliability. there are several disadvantages to eyecam imaging, such as the inability to perform dynamic imaging, as well as its requirement for supine patient positioning. however the eyecam is not an invasive measure, and is similar to gonioscopy in this way. obtaining eyecam imaging also takes longer than gonioscopy, approximately 5 to 10 minutes per eye, which may present a challenge in employing it as a standard screening tool for all populations. this study was limited by the fact that the there was little experience with the eyecam prior to this study; additional experience may have ed in better quality images with time. in summary , our study suggests that expert grading of eyecam images of the iridocorneal angle can provide moderate to excellent agreement with gonioscopic grading in a large population - based cohort. eyecam can be an effective screening tool for the identification of closed angles or narrow angles developing creeping angle closure over time in populations with a known predisposition to angle closure. usc roski eye institute, university of southern california: rohit varma, md, mph (principal investigator); roberta mckean - cowdin, phd (co - investigator); stanley p. azen, phd (co - investigator); mina torres, ms (project director); chunyi hsu, mph (project manager); david dinh, ba (research assistant); ruzhang jiang, md (examiner); jie sun, md, phd, mph (examiner); dandan wang, md (examiner); yuping wang, cot (examiner); justine wong, ba (clinical interviewer); shuang wu, ms (statistician); rucha desai, ms (programmer). the battelle survey research center: lisa v. john, phd (recruitment director); michelle cheng, ms (field supervisor). alfred sommer, md, mhs (chair); anne coleman, md, phd; dennis han, md; craig hanis, phd; louise wideroff, phd; and terri young, md.

purposeto compare grading of goniophotographic images and gonioscopy in assessing the iridocorneal angle.methodsin a population - based, cross - sectional study, participants underwent gonioscopy and goniophotographic imaging during the same visit. the iridocorneal angle was classified as closed if the posterior trabecular meshwork could not be seen. a single masked observer graded the goniophotographic images, and each eye was classified as having angle closure based on the number of closed quadrants. agreement between the methods was analyzed by calculating kappa and first - order agreement coefficient (ac1) statistics and comparison of area under receiver operating characteristic curves (auc).a total of 4149 chinese americans (3994 eyes) were included in this study. the agreement for angle closure diagnosis between gonioscopy and eyecam was moderate to excellent (= 0.60, ac1 0.90, auc 0.760.80).detection of iridocorneal angle closure based on goniophotographic imaging shows moderate to very good agreement with angle closure assessment using gonioscopy.

dental implant treatment is very widely spread and reliable treatment that provides good clinical with high success rates over 90%. titanium is commonly used as an implant material as it has high biocompatibility and bonding ability with the bone. these characteristics were found in 1952 by the swedish scientist per - ingvar brnemark. since then, the of many studies have demonstrated that titanium has high biocompatibility. titanium has no adverse effect on the human body and bonds readily with the new bone, which penetrates into the titanium surface. implant survival rate and prognosis depends on quality of osseointegration as more direct bone - to - metal interface take place without interposition of non - bone tissue. however, differences in bonding force between the implant body and bone occur depending on the differences in surface structures of the implant. many studies have concluded that certain characteristics of the implant surface play an important role in altering the quality of osseointegration. it is commonly thought that the slightly roughened implant surface allows better osseointegration compared with the smooth implant surface. moreover nanostructured materials have shown increased cell attachment over microstructured or smooth surfaces. an essential role of osseointegration processes is played by osteoblast progenitor stem cells during recruitment, adhesion, proliferation, differentiation, and mineralized matrix deposition during bone regeneration phases. nanoporous topography tend to help the proliferation processes, acting directly on the selective adhesion of osteoblastic cells on the surface, which can accelerate the healing process around implants. low osteoblasts cell number and proliferation have been closely associated with negative when considering it to osseointegration. many studies were conducted to investigate various implant surface nanostructures and their influence on cell behaviour as proliferation, in contrast to other scopes of surface structures dimensions. however, the optimal implant surface nanostructure covering for osteoblasts proliferation is yet to be established. the aim of the present review is to compare, based on the recent available evidence, the influence of various nanostructure surface modifications of titanium for implants, on osteoblasts proliferation. protocol and registration the review is registered in international prospective register of systematic reviews prospero. the protocol can be accessed at: http://www.crd.york.ac.uk/prospero/display_record.asp?id=crd42014009436 registration number: crd42014009436. the review included laboratory research studies, in vitro studies that using cells from human or animals and in vivo studies on animals. studies published on english language between january 2009 and june 2014 with various evaluation methods for osteoblasts proliferation and various evaluation intervals between hours and days. , titanium surface with nanostructure , osteoblast. for more recent and updated information, search included only articles that were publicated from january 2009 to june 2014 to ensure sensitivity of the review. (figure 1) illustrates the flow diagram of present articles selection according to prisma guidelines. exclusion criteria inclusion criteria for the selection were: in vitro and/or in vivo studies. the search displayed 97 from which 72 abstracts were screened (figure 1). preliminary exclusion was made by the title and its relevancy, later by abstract and its relevancy. finally, articles that did not meet the inclusion and exclusion criteria, where filtered as followed: no proliferation described (n = 12), microstructured surface analyzed (n = 4), no titanium sample included (n = 3), nanostructure and morphology has not been described (n = 1), organic coating has been used (n = 5). data collection process data was independently extracted from reports in form of variables according the aim and themes of present review as listed onwards. variables on which data were sought are as follow: type of study , indicates whether it was in vivo or in vitro or both and materials respectively. sample , describes the number of particular investigated samples in the study and its singularity (e.g., a - machined, b - polished, c - acid etched). topography , describes the nanoscale topography of the nanostructures on the surface of the sample, can be interrelated with sample description of nanostructure (e.g., nanograins 100 nm). evaluation , describes evaluation methods and duration of osteoblasts proliferation cultured on the sample (e.g., histology, 24, 48 and 72 hours). , describes the impact of surface structure on osteoblast proliferation. description of nanostructure peculiarities can vary from study to study and may be located under sample column or topography or both. risk of bias assessment risk of bias (e.g., lack of information or selective reports on variables of interest) was assessed on study level. the risks were indicated as lack of precise information of interest in each individual study that can blind the reader from particular information about examined samples. the cochrane collaboration s tool for assessing risk of bias was used to assess bias across the studies that can affect cumulative evidence. relevant data of interest according stated previously variables, was collected and organized in two tables that divided according type of implant surface modification. separation of articles by their samples fabrication methods of nanostructured surfaces into two groups can provide possibility for simple comparison. numbers of samples that provide positive or negative effect on osteoblasts proliferation are assessed in each article for each modification method. in addition samples of three topography types (e.g., control, microstructured and nanostrucruted) are included in each modification methods groups for better understanding of nanostructured surface superiority. quantitative and relative comparison between examined groups of samples and illustration of their relative efficiency in cells proliferation by means of diagrams. preliminary exclusion during screening stage was made by the titles and abstracts relevancy (n = 13) and (n = 4) respectively. during eligibility stage articles that did not meet the inclusion and exclusion criteria, where filtered as followed: no proliferation described (n = 12), microstructured surface analyzed (n = 4), no titanium sample included (n = 3), nanostructure and morphology has not been described (n = 1), organic coating has been used (n = 5). additional relevant articles were added after manual selection from references according eligibility (n = 2). finely 32 studies with 122 groups of examined samples were included in present review (figure 1). study characteristics all 32 studies finally selected for the review were in vivo and in vitro studies published in english with description of osteoblast proliferation. each study conducted in vitro experiment by culturing osteoblasts on several investigated samples and controls when three studies conducted additional in vivo experiments. all authors except four, reported on conducting the experiments at least twice to ensure statistical validity. the duration of osteoblasts proliferation evaluation varied from 2 hours to 14 days across all the studies except three articles of gittens et al. the main evaluation methods for osteoblasts proliferation across the studies were: histology, mtt assay, alamarblue assay, dna assay and wst-1 or brdu marker evaluations, additional visual evaluation by scanning electron microscope (sem) was described by six authors, when tet et al. uses sem as main evaluation method. all examined studies were assessed for specific variables described previously and were further divided into two groups characterized by type of implant surface modification. the division provided better understanding of nanosurface structure characteristics and contributed to sensitivity of the review. first group direct ablative titanium implant surface nano - modifications deals with titanium implant samples that were treated by various methods directly without addition of other materials to the implant surface, include 19 studies with 70 samples (table 1). nanocomposite additive implant surface modifications deals with samples that were treated by addition of variable non organic particles, includes 13 studies with 50 samples, showed in (table 2). direct ablative titanium implant surface nano - modifications nanocomposite additive implant surface modifications risk of bias within studies only 22 from 32 studies fulfilled the expected markers of validity. the risk of bias that indicated within other 10 studies presented as lack of information values that grouped as followed: nano scale topography was not indicated , evaluation methods with timing description and significance of experiments indicated in the study (p < 0.05) (table 3). assessment of the risk of bias present review focused on describing the studies, their and qualitative synthesis rather than meta - analysis because the analyzed studies were not presented with clear quantitative of osteoblasts proliferation, furthermore examined samples, evaluation methods and duration varied markedly. overall 24 studies with 89 examined samples from which 63 are various nanostractured patterns, reporting positive effect of 33 nanostrucure modified ti samples, thus enhance osteoblasts proliferation on nanostructured features with significant differences (p < 0.05) between nanostructured surface, and microstructured or smooth control surfaces in each study. no significant difference in positive effect on proliferation of cells cultured on nanostructured samples compared to microstructured or smooth control samples, was described in 2 studies with 6 examined samples; one study reports no significant differences between all three examined samples, another study reveals equal proliferation on smooth sample and sample with micronanohybrid surface, whereas microstructured sample showed impaired proliferative activity. negative effect was described in 6 studies with 27 samples from which 15 are nanostructured samples, when vary from, microstructured surface that promote osteoblasts proliferation to smooth or control samples showed better proliferation . by type of modification method, 19 belong to direct ablative titanium implant surface nano - modifications (table 1) with 72 examined samples from which 41 are nanostructured and only 19 samples produce positive effect for cellular proliferation. 13 studies belong to nanocomposite additive implant surface modifications (table 2), with 50 examined samples from which 40 are nanostructured with 25 samples reported as having significantly positive effect on osteoblasts proliferation. of individual studies of individual studies are shown in table 1 and table 2. risk of bias across studies all studies examined did nt show numerical data on the osteoblast proliferation , what did not allow us to estimate precisely the advantage of one nanostructured sample on another. all reviewed studies except two indicated significance of their by (p < 0.05) what can be interpreted as study s quality guaranty, but as mentioned previously, absence of quantitative and calculations prevent conformation of significance and comparison across the studies by the reviewer. in addition there are 10 studies with 41 examined samples were we could not find exact information as, nanoscale topography of the specific surface structure and/or evaluation timing. review with and without inclusion of these studies found no differences in the patterns of our review but only leave the reader blinded by lack of specific features of investigated sample. the division of articles into two groups by surface modification type contributed to better understanding of nanosurface structure characteristics and provide possibility for comparison between two main nanostructure modification methods (table 4). additionally the examined groups of samples can be compared for their relative influence on cell proliferation by surface topography. percentage of groups of samples that promote proliferation in each surface topography, within nanostructures been the greatest illustrated in (figure 2). division of samples by modification and topography percentage of groups of samples that promote proliferation in each surface topography. overall, in most of reviewed articles 24 in number, nanostructured surfaces enhanced osteoblast proliferation compared to microstructured or smooth surfaces. dielectric barrier discharge (dbd) modification of ti with spherical nanoparticles of 50 - 125 nm, significantly enhance cell proliferation, adhesion and spread without negative effect on differentiation. strong evidence delivered by two different studies that acid etched microfeature and tio deposited samples with nanonodules of 100 nm, 300 nm and 500 nm increase in proliferation, with that on 300 nm being the greatest. porous ti6al4v substrate with 10 - 20 nm grains appeared to have a not only higher but also longer growth phase for cell proliferation. full contact coverage coating that was obtained by galvanotactic anodizing process in a phosphate - sulfate bath, (fcc) characterized by unique nanotopography of regular volcanoes with circular pores of 10 m and 700 nm shows greater amount of cell proliferation than micro and nanopore of 2 m and 150 nm.. stated that crystal structure of nanotube layer can override the chemistry effect and plays a main role in cell proliferation, when anatase / rutile ~80 nm nanotube layers showed significantly higher proliferation than smooth layer and amorphous nanotube layers. this statement confirmed with evidence found in another study where ~80 nm nanotube surface increase cell number when the same author reports on reduced cell number cultured on 130 nm nanotubes and in other study of same author, no difference was found between ~80 nm nanotubular pattern and polished sample. final conformation for nanotubes benefit in such dispute was found in in vivo and in vitro experiment. one of the most advantageous nanotube diameters for better cell proliferation appears to be ~30 nm. additional nanofeature with ~30 nm was reported to have largest cell response, including proliferation adhesion and differentiation, as ~30 nm saw tooth nanonetwork surface with 200 - 300 nm inter tooth distance was examined. interesting findings were observed in additional combined in vivo and in vitro studies when one reports on higher cell proliferation rate on nanoleaf feature with roughness of 228 nm than on 60 - 80 nm nanotubs and significant reduction in proliferation on nanoneedle feature with 940 nm roughness, whereas another latest in vivo and in vitro study controversially reports on enhanced proliferation on nanoneedle structure. concerning nanocomposite materials incorporated into the implants surface we saw niobium (nb2o5) doped tio2 producing nanoplate structure that promote cell adhesion and proliferation. strong evidence brought to us in two studies that reported on strontium doped hydroxyapatite with 21.6 3.7 nm grain morphology, accelerates cell proliferation. furthermore, cell proliferation can be directly regulated by sr1-ha interrod spacing, with 71.4 nm interrod space three - dimensional patterns being the greatest. controversial were noticed about incorporation of ha within nanotubes as two authors claim that there is an obvious positive change in the spreading and viability of osteoblasts on ha coated ~45 - 50 nm titania nanotubes layer comparing to cells on pure titanium or tio2 nanotubes and in contrast gu et al. reports on reduced proliferation on 90 nm nanotubules with ha compared to same nanotubes without ha. another report reveals higher cell numbers on ha - tio2 nanocomposite coated sample with 300 nm spherical tio, 20 - 30 nm in diameter and 50 - 100 nm in length ha nanorods. significantly improved bone cell proliferation on the biomimetic nano coatings compared to uncoated ti and nano - ha coated ti was reported, as nano - ha combined with both magnetically and non magnetically treated single walled carbon nanotubuls can achieve the highest osteoblasts proliferation density when diameter of swcnt is 1.19 and 1.52 nm respectively. structures with 80 nm selenium clusters incorporated onto ti implant surface, significantly increase healthy cell density compared to untreated ti, on which cancerous osteoblasts found to be prevailed. tic layer deposited on titanium sample by ion plating plasma assisted deposition, increase osteoblast growth rate as was claimed in an in vitro and in vivo studies. another nanocomposite material that succeeds to increase proliferation rate and vitality was tio2/casio3 which exists on the surface as casio3 nanocristals on 20 - 10 nm tio2 grains pattern. in contrast to 23 articles that describe positive effect of nanostructured surfaces, we are dealing with negative reports as follow. reporting superiority of microstructure surface with pore size of 1 - 2 m over nanostructured samples. yu et al. described lower proliferation rate on 80 nm nano - foveolae structure compared to smooth control sample. in three different studies describes negative for micro and nano - modified samples compared to smooth and control surfaces in regard to osteoblasts proliferation, possibly due to transcriptionally - restricted transition. transcriptionally - restricted transition between proliferation and differentiation is a process that forces osteoblasts to stop dividing once they start maturing. zhao et al. from three articles included in this work, first reporting on significant smaller osteoblasts numbers cultured on nanotubules of 130 nm than on flat ti sample, the number become even smaller when 10 - 20 nm ag particles added to the surface. second article finds no significant difference in cell numbers between polished sample, 25 nm nanonet texture and 80 nm nanotubular texture. third article reports on slightly enhanced cell number on the ~80 nm nt acid - etched/20 v anodized surface. describe relatively equivalent proliferation level on tio2 smooth and 198.5 22.3 nm tio2 micronanohybrid. after all, the most mentioned advantageous pattern is nanotubular structure with nanotube diameter of ~30 nm. other nanostructures mentioned in our review need to be further investigated and compared for most advantageous nanoscale within each particular nanostructure. furthermore this review analysed articles that present synergic effect of ablative and additive nanocomopsite surface coating to osteoblasts proliferation. most articles that were including hydroxyapatite incorporation into ti implant nanostructures describe obvious positive effect on cells proliferation especially when doped with strontium. strontium doped ha nanorods appear to be beneficial with nanoscale of ~20 - 30 nm diameter and with interrod spacing of less than 96 nm. beside strontium, nanostructures doped with niobium, selenium and casio2 nanoparticles showed promising , when selenium substrates suggesting a more favourable environment for healthy than cancerous osteoblasts. the main limitation of this overview is that the samples group types, the culture techniques and evaluation methods are not the same across studies and can not be compared. all studies examined did nt show numerical data on the osteoblast proliferation , what did not allow us to estimate precisely the advantage of one nanostructured sample on another. all reviewed studies indicated significance of their by (p < 0.05) what can be interpreted as study s quality guaranty, but as mentioned previously, absence of quantitative and calculations prevent conformation of significance and comparison across the studies by the reviewer. in addition there are 10 studies with 41 examined samples were we could not find exact information as, nanoscale topography of the specific surface structure and/or evaluation timing. review with and without inclusion of these studies found no differences in the patterns of our review but only leave the reader blinded by lack of specific features of investigated sample. from examination of selected articles we can notice marked advantage in implementation of various nanostructures onto implant surface. in our review 24 articles reporting on positive effect of nanostructured surfaces on osteoblasts proliferation, when 33 samples with particular nanostructures markedly enhance cell proliferation. most of the examined nanostructures showed obvious positive impact on osteoblasts proliferation compared to other topography scales. yet for discovering the ultimate implant surface nanostructure, further investigations of ti nanopatterns with various nanoscales need to be done, moreover for reaching the most sensitive outcome, the experiments should be statistically compared, what can be achieved only when different studies will use the same concerted evaluation method for osteoblasts proliferation.

abstractobjectivesnanothechnology found to be increasingly implemented in implantology sphere over the recent years and it shows encouraging effect in this field. the aim of present review is to compare, based on the recent evidence, the influence of various nanostructure surface modifications of titanium for implants, on osteoblasts proliferation.material and methodsa literature review of english articles was conducted by using medline database restricted to 2009 - 2014 and constructed according prisma guidelines. search terms included titanium implant , titanium surface with nanostructure , osteoblast. additional studies were identified in bibliographies. only in vitro and/or in vivo studies on nano structured implant surfaces plus control sample, with specific evaluation method for osteoblasts proliferation and at least one ti sample with nanostructure, were included in the review.32 studies with 122 groups of examined samples were selected for present review. each study conducted in vitro experiment, two studies conducted additional in vivo experiments. all studies were dispensed by type of surface modification into two major groups; direct ablative titanium implant surface nano - modifications with 19 studies and nanocomposite additive implant surface modifications with 13 studies. overall 24 studies reporting on positive effect of nanostructured surface, 2 studies found no significant advantage and 6 studies reported on negative effect compared to other structure scales.from examination of selected articles we can notice marked advantage in implementation of various nanostructures onto implant surface. yet for discovering the ultimate implant surface nanostructure, further comparable investigations of ti surface nanostructures need to be done.

there is ample evidence to prove that medical graduates are not prescribing rationally throughout the world. in many medical curricula, teaching in the clinical disciplines is focused on symptoms and diagnosis, and little or no time is given to the principles of drug treatment. unfortunately, many medical schools still do not provide a structured training in pharmacotherapy but only lectures in basic pharmacology. this approach goes a long way to explaining why many medical graduates feel insufficiently prepared to assume prescribing responsibilities after graduation, and the many hospital admissions and even deaths caused by possibly avoidable medication errors. there is also ample evidence that prescription writing by medical students, interns, and fresh graduates can be improved by proper training. the study was conducted in the department of pharmacology, of a tertiary care health center, after taking permission from institutional ethics committee. a core committee consisting of faculty and senior residents of the department was formed. in consultation with faculty and after studying the feasibility (depending on the approved sequence of topics to be covered in the semester) 24 diseases were selected for the purpose of this study. a module consisting of didactic lectures, interactive audiovisual small group sessions with evaluation methods were framed for every disease. didactic lecture, 30 min audiovisual session on case history and records, 20 min preparation of prescriptions by the small groups based on focused group discussion and available literature, 40 min discussion on prescriptions prepared by the groups, its rationale and likely adverse events of the drugs prescribed, 15 min writing of final prescription and answer of questions by every student individually, and 15 min completing the reaction questionnaire by the facilitators. the contents of the didactic lectures and audiovisual sessions were decided using standard text books, the world health organization (who) guide to good prescribing, the who teachers guide and other relevant information available on internet. the resource persons for didactic lectures and facilitators were decided, and a pilot study was conducted consisting of one full session of the module for feedback and based on that the module was finalized. october 15, 2014, and until december 31, 2014, 24 prescriptions were completed. six of the prescriptions were completed with the batch 2012 and 18 with batch 2013. a uniform question in the form of write an appropriate prescription for the given problem, write the rationale behind the drugs prescribed and write the adverse drug events (ades) that may arise because of the use of this prescription was given in every session. the questions put to the facilitators were: was the duration appropriate, were the contents of the session appropriate, was the conduct of the session appropriate, and are you satisfied with the session? the completeness of the prescription was evaluated based on the four parts of the prescription, that is: information about the patient. information about the medicines prescribed (type of formulation, name of the medicine, strength, and duration). instructions about the consumption of the medicine to the pharmacist and patient and related advise and information about the prescriber. according to the parts appropriateness of the prescriptions, the documentation of rationale of the medicines used and probable adverse events that may be caused because of the prescription were judged in three categories, that is, perfectly appropriate, appropriate but insufficient, and inappropriate. a total of 1607 response sheets consisting of prescription slip for the given health problem, the rationale of the medicines prescribed and likely adverse events because of the prescription were collected. three hundred and twenty - three response sheets were from batch 2012 as response to the six health problems, that is, duodenal ulcer, amoebic liver abscess, shigellosis, pulmonary tuberculosis, iron deficiency anemia and hypertension stage ii. rest of the 1284 response sheets were from batch 2013 as response to the 18 health problems, that is, belladona poisoning, acute organophosphate poisoning, chronic organophosphate poisoning, anaphylactic shock, benign prostate hypertrophy, motion sickness, narrow angle glaucoma, open angle glaucoma, pheochromocytoma, mild depression, major endogenous depression, posttraumatic stress, jet lag, chronic insomnia, sleep onset insomnia, generalized tonic - clonic seizures, absence seizures, status epilepticus. on evaluation, completeness score of 18% was 2, 59% was 3 and 24% was 4, that means a majority 83% of the prescriptions were almost complete with a score of three or four. similarly, 99% of the prescriptions were appropriate; 41% prescriptions were appropriate and complete, 58% appropriate but insufficient and 1% inappropriate. the rationale given for the prescription was appropriate in 92% of the cases; appropriate and complete 24%, appropriate but insufficient in 68% and inappropriate in 8%. documentation of adverse events was appropriate in 72%; appropriate and complete in 23%, appropriate but insufficient 49% and inappropriate in 28%. completeness score of prescriptions in different categories (n = 1607) appropriateness of prescriptions and documentation of rationale and adverse drug reactions (n = 1607) all the facilitators were satisfied with the duration, contents and conduct of the sessions. eighty - three percent of the prescriptions were almost complete with a score of three or four. similarly, the rationale given for the prescription was either perfectly appropriate or appropriate but insufficient in 92% of the cases, and documentation of ades was either perfectly appropriate or appropriate but insufficient in 72% cases. the figure of 28% incorrect knowledge of ades is an area of concern, and necessary modifications are required in the module. another area of concern that needs to be addressed is a large number of insufficient documentation. the reason may be, they were a part of the panel who decided the contents and implementation strategy of the sessions.

context: there is ample evidence to prove that medical graduates are not prescribing rationally and this can be improved by proper training.aims:to design and implement a prescription writing teaching module for second professional medical students.subjects and methods: a module of 3 h duration consisting of didactic lecture, interactive audiovisual small group session, and evaluation method was framed for every disease and implemented. completeness of the prescriptions was evaluated on a scale of 14. appropriateness of the prescription, knowledge about the rationale behind the drugs used and adverse events related to the drugs used was judged in three categories, that is, appropriate and complete; appropriate but insufficient; and inappropriate.:one thousand six hundred and seven response sheets to 24 health problems were collected. completeness score of 18% was 2, 59% was 3% and 24% was 4.41% prescriptions were appropriate and complete, 58% appropriate but insufficient and 1% inappropriate. the rationale behind the drugs used was appropriate and complete 24%, appropriate but insufficient 68%, inappropriate 8%. documentation of adverse events was appropriate and complete 23%, appropriate but insufficient 49%, inappropriate 28%. all facilitators were satisfied with the duration, contents and conduct of the sessions.:a module is an effective tool for teaching prescription writing to undergraduate students; modifications required in contents and strategy to emphasize the need of complete documentation.

with the advent of highly active antiretroviral therapy (haart) to treat hiv infection over the past 15 years, dramatic reductions in hiv - related morbidity and mortality have yielded improvements in quality of life and life expectancy. the risk of vertical transmission through pregnancy has also been reduced to < 1% with timely antiretroviral therapy, continuing viral suppression, delivery by caesarean section if appropriate, and avoidance of breastfeeding. women now represent 22% of canada's hiv - positive population, and with over 80% of hiv - positive women being of reproductive age, there is a need for safe pregnancy planning as more women living with hiv (wlwhiv) desire to become pregnant. while prevention of vertical transmission of hiv to the child remains a key component, the main focus of safe pregnancy planning includes prevention of horizontal transmission to an uninfected partner and treatment of infertility issues. to achieve such objectives, assisted reproductive technologies (arts) have become central to meeting the reproductive needs of wlwhiv in resource - rich countries. indeed, access to conception and fertility services including art has become an important component within the recent global movements to establish national guidelines or update existing recommendations on pregnancy planning and conception for people living with hiv (plwhiv) in canada, the united kingdom, germany, and france. furthermore, as of july 2010, the most recent american society for reproductive medicine (asrm) ethics committee guidelines also recommend that fertility clinics offer services to hiv - positive individuals and couples willing to use art for reduction of transmission risk. compared to conception through unprotected intercourse with timed ovulation, associated with a horizontal transmission risk of 4.3% for serodiscordant couples, art procedures such as sperm washing, intra uterine insemination (iui), in vitro fertilization (ivf), and intracytoplasmic sperm injection (icsi) have been found to offer significant reductions in horizontal hiv transmission or coinfection for couples wishing to conceive. as well, art is also recommended for the treatment of infertility, an issue of importance to wlwhiv as studies have indicated lower fertility rates among hiv - positive women. various studies have documented an increased rate of tubal factor infertility in hiv - positive women. previous literature on the fertility intentions of wlwhiv has been important in eliciting trends and predictors of the decision to attempt pregnancy. as well, studies exist regarding the prevalence of fertility service centers in north america receiving requests from or providing services to plwhiv. although the asrm guidelines recommend that fertility clinics offer reproductive assistance to hiv - positive patients within technical and economic feasibility, less than 3% of fertility practices registered with the american society for assisted reproductive technologies provide services to couples where both partners are hiv positive. low patient volume has been cited as the most common reason for the limitation of fertility services by american service providers. few studies have investigated the demand for or desire to use art or the trends in and predictors of conception strategies amongst plwhiv. as women are the main recipients of art, it is important to assess the current intentions, knowledge, and perceptions regarding art among the current cohort of wlwhiv in north america. the purpose of this cross - sectional study was to assess the desire and need for art among hiv - positive women of reproductive age living in ontario, canada, and to determine correlates of art knowledge desire and the knowledge and perceptions of accessibility of art by the wlwhiv in this cohort. a cross - sectional study using a survey instrument was carried out with participants who met the following inclusion criteria: hiv - positive, biologically female, of reproductive age (ages 1852), living in ontario, canada, and ability to read english or french. the upper age limit was chosen to reflect the cut - off for fertility clinic consultation in canada. recruitment was conducted from october 5th, 2007 to march 31st, 2009 through 28 aids service organizations (asos), eight primary care and specialty hiv clinics, and two community health centers (chcs) across the province of ontario. an invitation email was sent out to all ontario asos listed on the canadian aids society website (the national society for asos) and all clinics and chcs known by the investigators that care for hiv - positive women. recruitment and study qualification determination were carried out by a single research staff member at each site following a predeveloped recruitment plan (available upon request). our study determined geographic distribution of our data collection sites based on the provincial regions laid out by the provincial public health authorities with the specific regions noted in table 1. this nonrandom sampling technique was used in an effort to obtain a representative sample of hiv - positive women of reproductive age living in ontario in the absence of a registry of hiv - positive individuals. once written consent was obtained, the survey was completed at the site or at home. an amended ethics approval was obtained for this specific research analysis. a 189-item survey instrument, the hiv pregnancy planning questionnaire, was created using the methods of fowler for instrument development and has previously been described in detail. the survey consisted of 12 domains including: interest / desire to have children, intent to have children in the future, behavior related to the pursuit of fertility, menstrual, birth control, and sexual history, pregnancy and birth history, perceived support for becoming pregnant, satisfaction with providers, needs assessment, hiv medical history, demographics, anxiety and depression, and hiv stigma (full survey instrument available upon request). the survey was first developed in english and then translated into french using the back translation method. content validity was achieved by using items from 4 previously validated surveys , by developing items based on a literature search exploring factors that determined reproductive decisions in hiv - positive women and by reviewing the questions with a project advisory panel of experts including hiv specialists, obstetricians, midwives, community members, and plwhiv. face validity was achieved by initially piloting the survey with 20 hiv - positive women in 2 focus groups. the focus group participants confirmed that the survey instrument items actually appeared to be measuring the intended items related to the domains. furthermore, participants were asked to comment on each item in terms of comprehension, clarity, and relevance. further pilot testing was carried out with an initial 52 hiv - positive women that met the inclusion criteria. baseline characteristics of the study population were summarized using medians and interquartile ranges (iqrs) for continuous variables and frequencies and proportions for categorical variables. the geographic distribution of the study population was compared to the distribution of hiv - positive women living in ontario using the chi - square test. the primary outcome of interest for this analysis was desire for information about art (art knowledge desire). the question used to represent desire for art information was i would like to learn more about fertility technologies and options for people living with hiv. the response options characterized agreement with the statement on a likert - type scale. participant responses were dichotomized into yes or no with regard to desire for art. agree were classified as yes; participant answers of strongly disagree or the question used to represent previous need for art was if you have been pregnant before, did you ever experience problems or difficulties in trying to get pregnant? women who had never been pregnant and women who had been pregnant but did not answer this question were excluded from the analysis. univariate logistic regression analysis was conducted to determine the unadjusted odds ratios with 95% cis for correlates of the desire for art and need for art. the final multivariate logistic regression model determined the adjusted odds ratios with 95% confidence intervals (cis) for correlates of the desire for art and need for art. the final multivariable logistic regression models included covariates that were a priori believed to be related to desire for art and need for art, such as age, ethnic , marital status, history of hiv medication, and current contraceptive use. variables which were significant at p < 0.20 in the univariate analyses were candidates for inclusion in final multivariable logistic regression models for desire and need for art. when multiple covariates measured similar phenomenon (e.g., ethnic and country of birth), the variable representing each construct with the most statistical significance was chosen. finally, the qualifying variables were entered into the multivariable model and selected by stepwise - selection method. only the correlates with a significant effect (p < 0.05) remained in the final multivariable models. other outcomes of interest included perceptions and knowledge of art. the outcomes were reported using medians and iqr for continuous variables and proportions for categorical variables. statistical analyses were performed using sas version 9.2 (sas institute, cary, nc, usa). five hundred and four wlwhiv in ontario, canada, were recruited from 38 sites. of these participants, four did not meet the inclusion criteria (two were over the age of 52 and two were not living in ontario). of the remaining 500 participants, 10 did not answer the question used to represent desire for art. surveys from the remaining 490 participants were used for the final analysis for the primary outcome. the median age for this final study population was 38 years (iqr 32, 43). sixty - one percent of the participants were born outside of canada and 46% were of african ethnicity. within this cohort, 55% were in sexual relationships and 46% were married, common - law, or living with a partner. thirty - three percent of the women had hiv - negative partners and 24% had hiv - positive partners; 36% were currently using contraception; 88% had been pregnant previously; 74% had given birth. the demographic characteristics of the entire study population are presented in table 1. in the final study population of 490 hiv - positive women living in ontario, canada , 78% (n = 384) indicated that they would like to learn more about fertility technology options. the distribution of participant characteristics by desire for art information is shown in the logistic regression models for correlates of desire for art in table 2. in the final multivariate model, african ethnicity (or = 3.86, p < 0.0001) and current contraceptive use (or = 1.70, p = 0.05) being married, common - law, or living with a partner (or = 0.52, p = 0.04) and history of hiv medication (or = 0.34, p = 0.02) were associated with a lack of desire for art. of the 500 participants who met the study inclusion criteria, 65 did not answer the question used to represent need for art and 10 had never been pregnant. the distribution of participant characteristics by need for art is shown in the logistic regression models for correlates of need for art in table 3. for the study participants, 17% (n = 72) indicated they had difficulties getting pregnant. in the final multivariate model, annual household income of $ 20,000 to $ 40,000 (or = 2.36, p = 0.02) greater than $ 40,000 (or = 2.63, p = 0.01) and current hiv treatment (or = 2.53, p = 0.01) were associated with difficulties conceiving and two or more lifetime pregnancies (or = 0.41, p < 0.01) were associated with no difficulties conceiving. the final analysis included surveys from the 500 participants who met the study inclusion criteria and responded to these set of questions. the distribution of participant answers to the various questions representing perceptions towards and knowledge of art is shown in table 4. the majority of participants (59%) were open to medical techniques that help with conception and 39% felt they could access a sperm bank if needed. of the women surveyed, 74% agreed they could ask a physician if they needed help finding a fertility clinic. to help with the decision to become pregnant, 50% of the participants indicated they wanted information booklets and publications on becoming pregnant, 59% wanted to speak to a health professional with fertility expertise, and 50% wanted to speak to an obstetrician / gynecologist. in this study, a large majority of wlwhiv (78%) indicated a desire for information regarding reproductive technology options. correlates of desire for art, a measure of pregnancy intention, included younger age, ethnicity, residence in an urban region, and birth place outside of canada. previous publications support our findings of younger age and black / african ethnicity as significant correlates of pregnancy intention in wlwhiv. although african ethnicity has not been associated with desire to have children, women of african ethnicity may be more likely to intend pregnancy due to cultural factors and traditional gender roles that stress the importance of childbearing. history of hiv medication use was also associated with a lack of desire for art in our study. however, other studies have indicated a positive influence on childbearing decisions associated with haart. these other studies, conducted in vietnam, india, and south africa, may have reflected the more advanced disease of the study populations, where haart access is associated with better health and greater perceived ability to be a fit parent. in direct contrast to a previous study in british columbia (bc), canada, being in a stable relationship this discrepancy can be attributed to the epidemiology of hiv infection in bc, differences in sample characteristics of the study, and the location and timing of the recruitment process. these differences highlight potential regional differences in the reproductive concerns of wlwhiv, attitudes towards those seeking art, and a need for continued research in this area. in addition to desire, our study also investigated potential need for reproductive technology options among wlwhiv by assessing correlates of self - reported conception difficulty. although not a direct measure of the true infertility rates among wlwhiv, conception difficulty does provide an estimate of the prevalence of infertility concerns within the population. infertility, defined as inability to conceive 12 months after initiating attempts, affects 1015% of the general population. various previous studies have documented the lower fertility rates of wlwhiv and the percentage of wlwhiv who reported difficulties conceiving in our study (17%) was higher than that found in general population. wlwhiv with resolved primary infertility are not reflective of the population of wlwhiv and our may underestimate the true infertility rates of wlwhiv. it appears then that the potential need for art among wlwhiv may be higher than that of the general population. greater household income and current hiv treatment were both correlates of difficulty conceiving, potentially due to the association between higher income and age. the lack of association between age, a major predictor of infertility generally, and difficulty conceiving in this study may be attributed to the relative youth of the study population. the age range of study participants was 3243, and previous attempts at pregnancy may have occurred when the participants were younger (< 35 years) when age did not significantly affect fertility. current hiv treatment, while associated with increased fertility in developing countries , was associated with difficulties conceiving in our study. thus far, studies on the effects of haart on pregnancy rates have not shown conclusive evidence of any impact on fertility. more research is required to further evaluate the true effects of haart on the fertility of wlwhiv. this study also assessed the perceptions and knowledge of wlwhiv of art. in our study, at least half of the participants viewed information booklets and publications on becoming pregnant, speaking with a health care professional with fertility expertise, or speaking with a obstetrician / gynecologist as important resources needed to help with the decision to become pregnant. more than one - third of participants also indicated a desire to have access to a fertility clinic as a resource needed for making pregnancy decisions. the majority of women surveyed were open to receiving art but only 39% of participants believed they had access to art services such as sperm banks. it appears that although women are interested in pursuing art services, most do not perceive these services as being accessible. since 74% of study participants indicated they regard their physician as the first source of information for art, doctors therefore have an important role in informing their patients of their full reproductive options. with the current movement towards increasing provision of art to plwhiv for prevention of horizontal transmission and treatment of subfertility, there is an increased need for physicians to provide information and initiate discussions regarding pregnancy planning and conception to their patients who are hiv positive. the assessment of art desires of a cohort of wlwhiv in ontario, canada, has important implications for healthcare providers, policy makers, and researchers. the of this survey indicate wlwhiv of reproductive age are open to and desire access to art for the purposes of safe conception and treatment of infertility. while this desire and demand for fertility technology options is supported by asrm ethics committee guidelines for safe pregnancy planning in north america, access to fertility services although the majority of fertility clinics surveyed across canada and the us have expressed willingness to provide care to hiv - positive individuals, less than 50% have actually offered treatment. even though previous restrictive state laws that have explicitly limited the availability of art services to plwhiv did contribute to the lack of access to fertility clinics within the us, low patient volume is the most common reason given by clinics for not providing services. the perceived inaccessibility of these services by plwhiv may contribute to the low number of clinics who actually treat patients from this population. there is a need for both provider and patient education to ensure the necessary provision of safe reproductive services to hiv - positive individuals. healthcare practitioners need to provide accurate information regarding reproductive technology options and access to such services, while fertility service professionals need to be aware of the increasing demand for art among plwhiv and current recommendations regarding provision of treatment. the limitations of our study include a potential for selection bias, as the survey used for this study was provided only in english and french and potentially limited to women literate in these two languages. as the survey was self - administered to ensure privacy, the lack of added clarification by an interviewer may have contributed to certain questions being unanswered. the participants of this study were also relatively healthy, with high cd4 counts and good viral suppression on haart, and may not represent the full spectrum of wlwhiv in ontario. finally, women who desire and intend pregnancy may be more motivated to complete a survey assessing reproductive intention. however, the large sample size in the study and the matching of study recruitment to the geographic distribution of hiv - positive women living in ontario allowed for generalizability of study to the population of wlwhiv in ontario. a significant proportion of the study enrolment was also conducted using a community - based research model involving aids service organizations and wlwhiv in the recruitment coordination and process, which allowed the engagement of participants who do not usually partake in research. our findings indicate wlwhiv of reproductive age living in ontario, canada both desire and need to receive art for safe pregnancy planning and conception and view healthcare providers as the main source of information regarding reproductive options. this study continues a series of assessments intended to guide the development of canadian national guidelines on safer pregnancy planning and conception, as well as provincial and national hiv pregnancy planning and fertility programs. potential future areas of research include an exploration of healthcare provider attitudes towards fertility and pregnancy for hiv - positive individuals as well as exploration of potential regional differences in their reproductive needs. to fully assess the reproductive needs of plwhiv, similar research

the purpose of this cross - sectional study is to assess the desire, need, perceptions, and knowledge of assisted reproductive technologies (arts) for women living with hiv (wlwhiv) and determine correlates of art knowledge desire. wlwhiv of reproductive age were surveyed using the survey instrument the hiv pregnancy planning questionnaire at hiv / aids service organizations across ontario, canada. of our cohort of 500 wlwhiv, median age was 38, 88% were previously pregnant, 78% desired more information regarding art, 59% were open to the idea of receiving art, 39% felt they could access a sperm bank, and 17% had difficulties conceiving (self - reported). age, african ethnicity, and residence in an urban center were correlated with desire for more art information. of participants, 50% wanted to speak to an obstetrician / gynecologist regarding pregnancy planning, and 74% regarded physicians as a main source of fertility service information. while the majority of participants in our cohort desire access to art information, most do not perceive these services as readily accessible. healthcare practitioners were viewed as main sources of information regarding fertility services and need to provide accurate information regarding access. fertility service professionals need to be aware of the increasing demand for art among wlwhiv.

the five basic tastes comprise sweet, sour, bitter, salty, and umami. of these tastes, sour and bitter are generally unfavorable and avoided by humans, because these tastes are associated with spoiled and unripe foods as well as bitter toxins. the perception of bitterness may have evolved in humans and animals to avoid the intake of toxins. the origins and structural diversity of bitter compounds are also much greater than those of other substances. metal ions, some amino acids, peptides, and the secondary metabolites produced by plants, such as alkaloids, phenols, flavonoids, isoflavones, terpenes, and glucosinolates, are bitter. these substances are perceived by taste 2 receptors (tas2r), which are classified as g protein - coupled receptors. in human, bitter - tasting foods are not preferred in most cases; a few exceptions include coffee, beer, and wine. even though humans are averse to bitter taste, pharmaceutical compounds with physiological benefits often taste bitter. therefore, the masking of bitterness is considered to be important in food processing and pharmacology. some substances with potent tastes such as salts, acids, and sugars can suppress bitterness. cyclodextrin includes some bitter substances intramolecularly, thereby inhibiting the binding of these substances to taste receptors. phosphatidic acid (pa) and its lipoprotein derivative, formed by interactions with -lactoglobulin, are reported to suppress the bitterness of quinine sulfate. the activation pattern of tas2rs with some bitter chemicals, including quinine hydrochloride (qhcl), is now known. recently, an antagonist for several tas2r taste receptors was identified by screening a chemical compound library. this compound binds the tas2r activation pocket to inhibit ligand binding, thereby effectively suppressing bitter taste. however, these are not applicable for processed food because the safety of the antagonists is not guaranteed. to utilize these compounds for processed food therefore, it is more appropriate to screen for bitterness - masking compounds in conventional foods to meet the requirements of the food - processing industry. its unique flavors and tastes are produced by the action of microorganisms such as lactic acid bacteria and fungi. during the fermentation process, various compounds not contained in milk are produced. peptides and free amino acids are generated by the digestion of milk proteins by microbial proteinases and exopeptidases. bitter- or astringent - tasting peptides are often produced by digestion of the caseins in cheese. many kinds of tastants are present in foods; they may interact with each other to enhance or suppress the taste. the aim of this study was to purify and identify compounds in cheese with bitterness - masking properties and to reveal the mechanism of their suppression by using isothermal titration calorimetry (itc). the following cheeses were purchased from supermarkets in tokyo, japan: baraka cheese (lincet saint - julien, trappes, france), gouda (frico, wolvega, the netherlands), ricotta (galbani, milan, italy), and brie (bongrain sa, viroflay, france). chemicals were obtained from commercial sources: 9-anthryldiazomethane (adam) from funakoshi co., tokyo, japan; qhcl from nacalai tesque inc., kyoto, japan; fatty acids (fas) including myristic acid, palmitic acid, stearic acid, and oleic acid (oa), glycerides including trioleoylglycerol (tog), dioleoylglycerol (dog), and monooleoylglycerol (mog), and promethazine hydrochloride (phcl) from sigma - aldrich co., tokyo, japan; and caffeine and cdcl3 containing 1 vol % of tetramethylsilane (tms) from wako pure chemical industries, ltd. wakogel c-200 (chromatography grade, particle size = 75150 m) was purchased from wako pure chemical industries, ltd. , osaka, japan, and silica gel 60f254 (hx953368 ; 5 25 cm) for thin - layer chromatography (tlc) was from merck kgaa, darmstadt, germany. itc was performed on a microcal itc200 instrument (ge healthcare japan corp ., a kitchen knife was used to cut 150 g of cheese into small pieces after removal of the mold - covered surface, prior to the addition of 600 ml of ethanol . the mixture was homogenized at 20000 rpm for 10 min by using a polytron homogenizer ( pt1300d, da1607/2 ; ishii laboratory works co., ltd ., osaka, japan) and then centrifuged at 15300 g for 10 min at room temperature. the liquid layer was separated from the debris and re - extracted twice with 400 ml of ethanol. the liquid layers collected were then concentrated to dryness by using a rotary evaporator. the dried residue was dissolved in 200 ml of ethyl acetate, dehydrated with na2so4 anhydrate, and filtered with filter paper (advantec no . the plate was spotted with the samples, developed with a mixture of n - hexane / acetone ( 2:1), and exposed by spraying with 10% h2so4, prior to heating on a hot plate, to detect the separated samples. silica gel (80 g) was packed into a glass column (29 300 mm) and equilibrated with n - hexane. next, 510 g of the oily fraction was applied and eluted with n - hexane. the elution was performed with a n - hexane / acetone mixture, with the ratio of acetone increasing stepwise. the typical elution profile in sequence is 200 ml of n - hexane, 210 ml of n - hexane / acetone (100/5), 220 ml of n - hexane / acetone (100/10), 240 ml of n - hexane / acetone (100/20), 260 ml of n - hexane / acetone (100/30), and 280 ml of n - hexane / acetone (100/40). the ing fractions with the same tlc patterns were gathered, and the four fractions a, b, c, and d were obtained. fraction a corresponded to the n - hexane / acetone mixtures of 100/5 and 100/10, fraction b to 100/20, fraction c to 100/30, and fraction d to 100/40. the separation was performed eight times, and each pooled fraction was further purified by rechromatography, eluting using the n - hexane / acetone system. baraka, ricotta, gouda, and brie cheeses were analyzed to determine their free fa composition. the oily fraction from each cheese was prepared according to the above - described method. the free fas were then analyzed using high - performance liquid chromatography (hplc) using the adam method, as reported previously. panelists (n = 9) were selected using the difference test with five basic tastes and the discrimination test for the differences in the concentrations of four basic tastes and were trained using the methods described below. for the discernment of bitter taste, each panelist was trained with triangular tests to distinguish bitter taste at four concentration levels near the threshold value. in addition, each panelist was trained in the discernment of difference in the concentrations of bitter taste by using a ranking test involving the qhcl solution at seven concentration levels (common ratio 1.11.2). in sensory tests with panelists (n = 4), a piece of baraka cheese was placed on the tongue after peeling off the mold - covered surface and spread over the whole tongue prior to tasting 0.0080% qhcl solution. to estimate the bitterness - masking activity, fractions a, b, c, and d were each solubilized in 0.0080% qhcl containing 1% -lactoglobulin, at a concentration of 12 mg / ml. because the oily fractions dissolved poorly in water, -lactoglobulin was added as a solubilizer. -lactoglobulin (1%) alone did not possess bitterness - masking activity under these conditions (data not shown). the free fas were solubilized by the addition of equimolar naoh and stirred with a magnetic stirrer. then , 1 ml of bitter - tasting solutions containing each of the four fractions was put in the mouth, and the bitter taste intensity was evaluated on a three - level scale: bitterness equal to 0.0080% qhcl, slightly less than 0.0080% qhcl , or significantly less bitterness. panelists (n = 7) performed sensory tests by using the beer. here, a piece of baraka, gouda, brie, or ricotta cheese was put in the mouth, and a sip of beer was consumed. the bitter taste was evaluated using a four - point categorical scale: strong, medium, weak , or very weak. for evaluation of scores, the steel dwass test, a nonparametric multiple - comparison method, was applied for detecting between - sample differences. two test solutions were prepared: (a) 0.2 mm oa, 0.2 mm palmitic acid, 0.05 mm myristic acid, 0.05 mm stearic acid, and 0.0080% qhcl in 5 mm sodium phosphate buffer (ph 7.0); (b) 0.5 mm oa and 0.0080% qhcl in 5 mm sodium phosphate buffer (ph 7.0). standard solutions with seven different concentrations of qhcl, that is, 0.0026, 0.0030, 0.0035, 0.0040, 0.0046, 0.0053, and 0.0060%, in 5 mm sodium phosphate buffer (ph 7.0) were also prepared. panelists (n = 9) who could discriminate the seven standard solutions in order of concentration participated in this test. each of the standard solutions (5 ml) was put in a clear plastic cup, whereas each of the test solutions (5 ml) was in a white paper cup because the test solutions were slightly cloudy. first, the panelists tasted the three standard solutions, 0.0030, 0.0040, and 0.0053% qhcl, to remember the bitterness of each solution. then, 5 ml of the test solution was held in the mouth for 15 s prior to its being spat out. after that, the mouth was rinsed with water to remove any bitter aftertaste, and the panelist waited for 30 s before moving to the next test. an interval of 60 s was provided before and after tasting each fa solution to avoid confusing the tastes. the bitter taste intensities of test solutions a and b were estimated in comparison with seven standard solutions. panelists were allowed to repeatedly taste the standard solutions before selecting the solution that was the closest to the bitterness of the test solutions. analysis of variance (anova) was applied to detect the variation of judged sensory scores at significance level = 0.05. all statistical analyses were carried out using the computer software jmp 9.0.2 (sas institute, inc ., bitter taste intensity evaluations were performed by using the following paired difference tests : between 0.22 mm qhcl and 0.22 mm qhcl containing 0.5 mm oa ( involving 20 panelists), between 1.5 mm phcl and 1.5 mm phcl containing 0.5 mm oa (involving 6 panelists), and between 50 mm caffeine and 50 mm caffeine containing 0.5 mm oa (involving 10 panelists). statistical analysis of scores was conducted using a one - tailed binomial test (significance level = 0.05). the concentrations of oa, qhcl, and phcl used in titration were 0.5, 2.2, and 1.5 mm, respectively. they were dissolved in 5 mm sodium phosphate buffer (ph 7.0) containing 5% ethanol. the reference cell was filled with milli - q water (millipore corp ., billerica, ma, usa). each titration was carried out with initial injection (0.4 l) followed by 18 main injections (2 l each) at intervals of 120 s. the first titration (0.4 l) was excluded for the analysis. the dilution calorie of the ligand in the buffer was subtracted from the titration data of qhcl. the titration of oa with phcl was also performed except for using at 150 s intervals. although the titration of 0.5 mm oa with 2.2 mm caffeine was performed at 150 s intervals, the titration was dispersed. therefore, 50 mm caffeine containing 50 mm sodium phosphate buffer (ph 7.0) was adopted. the data were analyzed according to a model for one set of sites provided in the origin 7.0 software for microcal itc200. the dissociation constant (kd) and enthalpy change of binding (h) were obtained from the fitted curve. the entropy change of binding (s) and free energy change of binding (g) were obtained from eq 1; r is the gas constant, t, the thermodynamic temperature, and k, the association constant.1 seventy - one brands of cheeses were commercially obtained and subjected to sensory tests to identify the cheese with bitterness - masking activity. in addition, panelists were asked to consider which of the 71 cheeses had the strongest bitterness - masking activity. as a of the screening, baraka cheese was selected (supporting information). the oily fraction was extracted with ethanol and then with ethyl acetate (figure 1a), ing in a yield of 56 g from 150 g of baraka cheese. it was further separated by silica gel column chromatography by using a n - hexane / acetone stepwise elution system. four fractions, a, b, c, and d, weighing 24.3, 0.77, 0.012, and 0.17 g, respectively, were eluted in this order. tlc was performed with tog, dog, mog, and oa as the references (figure 1b). the mobilities of tog, dog, mog, and oa are indicated by their rf values of 0.96, 0.72 and 0.68, 0.63, and 0.50, respectively. the mobilities of the fractions a d were compared with those of the standard compounds. fraction a moved to the front of the plate and accounted for 96.2% of the oily fraction. the main spot of fraction b had a mobility equal to that of the dog spots, whereas that of fraction c nearly coincided with those of dog and oa. extraction and separation of oily fraction from baraka cheese: (a) fractionation scheme; (b) thin - layer chromatography (tlc) analysis. the four fractions were analyzed by tlc, developed using n - hexane / acetone (2:1), and detected by spraying with 10% h2so4 and heating on a hot plate. the rf values for each authentic sample, applied at 1 g / lane, are as follows: trioleoylglycerol (tog) (rf = 0.96); dioleoylglycerol (dog) (rf = 0.72, 0.68); oa, oleic acid (oa) (rf = 0.63); monooleoylglycerol (mog) (rf = 0.50). to identify the substances in the fractions b, c, and d, the samples were analyzed by h and c nuclear magnetic resonance (nmr) spectroscopy (varian inova 500). the methyl, methylene, and olefin signals of the fas are not shown because these signals were derived by a mixture of many fa molecules. fraction b (diacylglycerol ( dg) mixture of 1,2-isomer (53%) and 1,3-isomer (47%) ): h nmr (500 mhz, cdcl3, tms) 3.61 (2, 3-ch2 of 1,2-isomer), 3.97 (1, 2-ch of 1,3-isomer), 4.05 (4, 1-ch2 and 3-ch2 of 1,3-isomer), 4.10 (1, 1-ch2 of 1,2-isomer), 4.25 (1, 1-ch2 of 1,2-isomer), 5.00 (1, 2-ch of 1,2-isomer); c nmr (125 mhz, cdcl3, tms) 60.9 (3-ch2 of 1,2-isomer), 62.1 (1-ch2 of 1,2-isomer), 64.7 (1-ch2 and 3-ch2 of 1,3-isomer), 67.7 (2-ch of 1,3-isomer), 71.8 (2-ch of 1,2-isomer), 173.2 (2-ch o co of 1,2-isomer), 173.5 (1-ch2o co of 1,2-isomer), 173.6 (2, 1-ch2o co and 3-ch2o co of 1,3-isomer). the ratio of the 1,2- to 1,3-isomers was calculated from the integration value of the two signals corresponding to the 2-ch protons. fraction c (dg of 1,3-isomer and fas): h nmr (500 mhz, cdcl3, tms) 4.1 (1, 2-ch), 4.22 (4, 1-ch2 and 3-ch2); c nmr (125 mhz, cdcl3, tms) 62.1 (1-ch2 and 3-ch2), 68.9 (2-ch), 173.3 (1-ch o co and 3-ch o fraction d ( 1-monoacylglycerol ( mg) ): h nmr (500 mhz, cdcl3, tms) 3.60 (1, 3-ch2), 3.69 (1, 3-ch2), 3.89 (1, 2-ch), 4.15 (1, 1-ch2), 4.21 (1, 1-ch2). fractions b and d were found from their nmr spectra to include dg and mg, respectively (table 1). fraction c was determined to mainly include 1,3-dg and free fas by tlc and nmr analyses. the concentration of free fas in this fraction was 43.6%, as determined by hplc by using the adam method. the compounds containing fractions b, c, and d were identified from h and c nmr spectra. next, the bitterness - masking activity of each fraction was analyzed by sensory tests. using the examination of the wilcoxon rank - sum test and steel dwass test , fraction c had the strongest bitterness - masking activity compared to the other three fractions with a significant level of 10% (wilcoxon rank - sum test, p < 0.025 ; steel dwass test, fractions a, b, and d, p = 0.063, 0.089, and 0.089) (table 2). these suggest that free fas are the bitterness - masking compounds in cheese. sensory test i: 0.5 g of baraka cheese was put on the tongue and spread over the entire tongue prior to tasting 0.0080% quinine hydrochloride (qhcl). sensory test ii: each fraction was solubilized in 0.0080% qhcl and evaluated for bitterness. the bitterness was evaluated on a 3-score scale: equal to that of 0.0080% qhcl, slightly less bitter than 0.0080% qhcl, or significantly less bitter. each score is the average of four trials. to confirm the bitterness - masking activity of free fas the total concentration of free fas in baraka cheese was 12.15 mm, with oa present at the highest concentration (4.29 mm, 35.3%) followed by palmitic acid (3.70 mm, 30.5%), myristic acid (1.27 mm, 10.5%), and stearic acid (0.86 mm, 7.0%) (table 3). free fatty acids (fas) in the cheese samples were quantitated from their oily fractions. the molar concentration and weight percentage of free fas in the whole cheeses were calculated. nd, not detected. to examine the correlation of free fa content with bitterness - masking activity, baraka and the other three cheeses with different bitterness - masking activities were analyzed. first, the bitterness - masking activities of baraka, gouda, brie, and ricotta were estimated using beer. the bitterness - masking activity of baraka cheese was found to be significantly stronger than that of the other three cheeses by using the nonparametric steel dwass test. the significant differences between baraka and the other cheeses were as follows: baraka versus gouda, p = 0.0159; versus brie; p = 0.0423; and versus ricotta; p = 0.0077 (figure 2). the average values of the panelists answers concerning the masking activity of cheese to the bitter taste of beer are shown (n = 7). the intensity of bitter taste was evaluated using a 4-point categorical scale, as follows: strong, medium, weak , or very weak. the steel dwass test, which uses the nonparametric multiple - comparison method, was applied for detecting the between - sample differences. the baraka cheese sample showed significantly higher bitterness - masking activity than the other three samples. next, the concentrations of free fas in the other three cheeses were analyzed. the total concentrations of free fas were 1.13 mm in ricotta, 3.86 mm in brie, and 2.25 mm in gouda. thus, the total free fas are apparently high in baraka and low in the other three cheeses. these suggest that free fa content is correlated with the bitterness - masking activity. although the total concentrations of free fas in the four cheeses are quite different, their free fa compositions are almost identical (table 3). in all of the cheeses analyzed, four fas (oa, palmitic acid, myristic acid, and stearic acid) comprised up to 60% of the total free fas. a 0.5 mm solution of mixed free fas (equivalent to the average free fa composition of cheese), 0.2 mm oa, 0.2 mm palmitic acid, 0.05 mm myristic acid, and 0.05 mm stearic acid were subjected to a bitterness - masking test. a 0.0080% qhcl solution was used to determine the bitterness - masking activity of the mixed free fas. a test was constructed using seven concentrations of qhcl solution to evaluate the degree of bitterness - masking activity. wilk w test (p = 0.2123 and w = 0.932201) (figure 3a). on the basis of these , it was concluded that the bitterness - masking effect existed within the 95% confidence interval of the mean response. we found that the 0.5 mm mixed fa solution reduced the bitterness of qhcl from 0.0080 to 0.00490.0060%. however, there was a significant difference in the evaluation score between the panelists according to the f test of anova (p = 0.001). the mixed free fas apparently suppressed the bitterness of qhcl, but the data varied among panelists. this could be explained by the fact saturated fas, myristic acid, palmitic acid, and stearic acid were little dissolved and could not be suspended in buffer and, therefore, the recognition of bitter taste would be variable. two sample solutions were prepared: (a) 0.0080% quinine hydrochloride (qhcl) containing 0.2 mm oleic acid (oa), 0.2 mm palmitic acid, 0.05 mm myristic acid, and 0.05 mm stearic acid; (b) 0.0080% qhcl containing 0.5 mm oa. bitter taste intensity was evaluated by comparing the sample solutions with seven standard qhcl solutions, as follows: 1, 0.0026%; 2, 0.0030%; 3, 0.0035%; 4, 0.0040%; 5, 0.0046%; 6, 0.0053%; and 7, 0.0060%. the right panels are box plots with the smallest observation, lower quartile, median, upper quartile, and largest observation. wilk w test for non - normality showed p = 0.2123 and w = 0.932201 in (a) and p = 0.1376 and w = 0.921529 in (b). to eliminate the factor of low solubility of these fas, oa was used for the bitterness - masking test, because it can be suspended in buffer as a sodium salt and it was the predominant fa in baraka cheese. the normality of distribution of the sensory evaluation score, using the seven concentration levels of qhcl for quantitatively evaluating the degree of bitterness - masking activity, was confirmed by the shapiro wilk w test (p = 0.1376 and w = 0.921529) (figure 3b). on the basis of these , it was estimated that the bitterness - masking effect existed within the 95% confidence interval of the mean response. our findings showed that a 0.5 mm oa solution reduced the bitterness of qhcl from 0.0080 to 0.00320.0038%. in addition, there was no significant difference in the evaluation score among the panelists, as demonstrated by the f test of anova (p = 0.131). thus, in the measurements conducted using the 0.5 mm solution of oa alone, the panelists evaluations were found to be highly consistent with one another. these demonstrate that fas, especially oa, suppress the bitterness of qhcl. as already shown, the bitter taste of 0.0080% qhcl was suppressed by 0.5 mm oa. a further study involved the bitterness - masking activity of 0.5 mm oa against 0.22 mm qhcl, 1.5 mm phcl, and 50 mm caffeine. because of the sensory tests, the bitterness of qhcl and phcl was significantly suppressed by oa (one - tailed binomial test : p = 0.0059, 0.0156). on the other hand, that of caffeine was not suppressed (one - tailed binomial test : p = 0.0547). to validate the bitterness - masking activity of oa, the binding potential between oa and qhcl was examined by itc, which analyzes the interaction of two compounds by measuring the change in caloric output when they are mixed. when oa was titrated with qhcl, the reaction was exothermic, and the dissociation constant was 11 1 m (figure 4a ; table 4). the interactions between oa and phcl were also examined, and the dissociation constant was 8.8 1.6 m (figure 4b ; table 4). the interactions between oa and caffeine were also examined, but were not detected under the same conditions (figure 4c). isothermal titration calorimetry profiles of oleic acid binding to quinine hydrochloride (qhcl) and promethazine hydrochloride (phcl): (a) 0.5 mm oleic acid (oa) was titrated with 2.2 mm qhcl; (b) 0.5 mm oa was titrated with 1.5 mm phcl; (c) 0.5 mm oa was titrated with 2.2 mm caffeine. the experiments were performed at 25 c, and the titration was repeated 19 times at (a) 120 s and (b, c) 150 s intervals. in the lower panels, the area of the peak was integrated and plotted against the molar ratio of qhcl or phcl to oa. kd and h were obtained from the fitted curve according to a model for one set of sites. qhci, quinine hydrochloride; phci, promethazine hydrochloride; kd, dissociation constant; g, free energy change of binding; h, enthalpy change of binding; s, entropy change of binding. in this study, we have purified a bitterness - masking fraction in cheese, and free fas were identified as the compounds responsible for the bitterness of this fraction. the total concentration of free fas in baraka cheese was about 12 mm, which included 4.3 mm oa. a sensory test showed that both a 0.5 mm free fa mixture (oa, palmitic acid, stearic acid, myristic acid) and 0.5 mm oa alone reduced the bitterness of qhcl. the concentration of oa in baraka cheese is about 10 times larger than that of oa used for the sensory test, which is adequate for it to develop the bitterness - masking effects. several kinds of free fas other than oa were also contained in the four cheeses analyzed. although individual free fas were not examined, a mixture of these fas showed bitterness - masking activity, proving that they must possess this property. in fact, linoleic acid has been reported to mask the bitterness of caffeine. however, the concentration of linoleic acid used in the previous experiment was 70 times greater than that of the oa used in the present experiment. pa and pi exhibit a strong bitterness - masking activity against qhcl, although some other bitter substances are not similarly influenced. bitterness - masking activity can be determined by the affinity of a bitter tastant for a masking compound. on the basis of the itc analyses, the kd values between oa versus qhcl and oa versus phcl were 11 1 and 8.8 1.6 m, respectively. several studies have shown that a midmicromolar dissociation range of interaction leads to the formation of a complex between two compounds. similarly, 1-aminoanthracene interacts with horse spleen apoferritin (hsaf) with a dissociation constant of 100 m, which was shown to be appropriate for binding. when the of this study are considered comprehensively, the most likely hypothesis is that oa binds to qhcl or phcl to form complexes in aqueous solution, thereby masking the bitterness of qhcl. we speculate that a complex may form because of the interaction between alkyl chains in oa or in other free fas and the hydrophobic partial structure in qhcl and phcl. the calorimetric parameters as well as the values of h and ts are in line with the assumption that the suppression is caused by hydrophobic interactions. on the other hand the interaction between oa and caffeine was not detected by itc under the present conditions. even the higher concentrations of 0.5 mm oa and 50 mm caffeine did not show interaction. these suggest that the binding ability of oa and bitter compounds is a factor determining the bitterness - masking activity. moreover, it was suggested that the kd value was related to the strength of the bitter - masking activity. however, fermented foods contain free fas produced by the digestion of tg by the lipases secreted from microorganisms. some studies showed that short - chain fas liberated from milk lipids generate a cheese - specific flavor, although the bitterness - masking activity of free fas has not been discussed. the present study found a close relationship between the high bitterness - masking activity of baraka cheese in sensory tests and its high free fa content. to the best of our knowledge , this study is the first to report the bitterness - masking effect of foods containing free fas in general and of oa in particular. however, no convincing method for analyzing the interaction of food components at the molecular level is available. in particular, interactions among small molecules have not been detectable in the intact molecular form. in this study , we directly analyzed the interaction between a bitter tastant and its masking compound. our approach will be useful for studying the interactions concerned with tastants in foods.

bitterness - masking compounds were identified in a natural white mold cheese. the oily fraction of the cheese was extracted and further fractionated by using silica gel column chromatography. the four fractions obtained were characterized by thin - layer chromatography and nuclear magnetic resonance spectroscopy. the fatty acid - containing fraction was found to have the highest bitterness - masking activity against quinine hydrochloride. bitterness - masking activity was quantitated using a method based on subjective equivalents. at 0.5 mm, the fatty acid mixture, which had a composition similar to that of cheese, suppressed the bitterness of 0.008% quinine hydrochloride to be equivalent to that of 0.00490.0060% and 0.5 mm oleic acid to that of 0.00320.0038% solution. the binding potential between oleic acid and the bitter compounds was estimated by isothermal titration calorimetry. these suggest that oleic acid masked bitterness by forming a complex with the bitter compounds.

immunoglobulin g4-related disease (igg4-rd) is a systemic disease characterized by chronic fibrosing inflammation with abundant igg4-positive plasma cells and elevated serum igg4 levels; it tends to be mistaken for malignancy and responds well to steroids. clinical manifestations are common in the pancreas, salivary glands, hepatobiliary tract, orbit, lymph nodes, and retroperitoneum but are rare in the gastrointestinal tract. we herein report a case of igg4-rd of the ileocecal region diagnosed after surgical resection performed for a suspected malignancy. we also provide a review of the literature, with an emphasis on the treatment of this disease. a 74-year - old female presented with a two - month history of right lower abdominal pain and a slight fever. an approximately 5 cm mass in her right lower abdomen was detected by abdominal ultrasonography (us), and she was admitted for further examination. colonoscopy revealed moderate stenosis caused by edematous wall thickening of the lower ascending colon, with reddening of the mucosa (fig . since the ileocecal valve was also swollen, the scope could not be inserted into the terminal ileum ( fig . a colonic biopsy from the swollen wall showed a nonspecific inflammation with lymphoid aggregates, with no evidence of malignancy . a radiographic contrast enema indicated edematous asymmetric stenosis with erosion from the terminal ileum to lower ascending colon ( fig . 1c). an abdominal computed tomography (ct) scan indicated edematous wall thickening from the terminal ileum to the lower ascending colon (fig . positron emission tomography with f - fluorodeoxyglucose ( fdg - pet) revealed increased fdg uptake in the ascending colon (suv max : 13.3), terminal ileum (suv max : 6.9), and also in the spleen (suv max : 5.9) and paraaortic lymph node (suv max : 5.3) (fig . blood tests revealed high levels of c - reactive protein ( 27.48 mg / dl), elevated ldh (252 u / l) and elevated alp (561 u / l). the soluble interleukin-2 receptor (sil-2r) level was elevated (2305 u / ml, normal range : 122496 u / ml), but none of the other tumor marker levels were remarkable (cea : 1.0 ng / ml, ca19 - 9 : 7.1 u / ml). although a pathological diagnosis was not obtained preoperatively, she was diagnosed to have malignant lymphoma based on these findings. it seemed that passage of stool would be completely obstructed by tumor growth in the near future. consequently, she underwent a right hemi - colectomy in order to obtain a pathological diagnosis, and to avoid ileus caused by tumor growth. grossly, the segmental bowel resection included 25 cm of ascending colon and 125 cm of ileum (fig . there was irregular wall thickening with submucosal sclerosis, which was 10 cm in length from the terminal ileum to the ascending colon that accompanied the sclerosis of the mesentery of the ileum . since the sclerosing mesentery needed to be extirpated, the long ileum that received its blood supply from the mesentery was also resected . microscopically, the lesion was composed of the ileal, cecal and colonic wall with lymphoplasmacytic infiltration, lymphoid follicles and fibrosis in the subserosa and surrounding mesenteric adipose tissue ( fig . atypical lymphoid cells were not aggregated in these specimens . the elastica van gieson ( evg) staining showed obliterative phlebitis, which is one of the characteristic histological features of igg4-rd (fig . immunohistochemical staining revealed an increased number of igg4-positive plasma cells at 150/hpf, and the igg4/igg ratio was 50% (fig . the serum igg4 level was postoperatively found to be normal level at 102 mg / dl ( normal range : 6121 mg / dl). the patient's postoperative course was uneventful, and she was discharged home on postoperative day 18 tolerating a diet by mouth. she is currently doing well 10 months after the operation. during the preoperative fdg - pet examination, increased fdg uptake had been seen not only in the resected ileocecal region, but also in the spleen and paraaortic lymph node. while igg4-rd might remain in these sites thus, we decided to perform careful follow - up without any additional treatment, such as steroid therapy. many previous reports of igg4-rd have focused on pancreatic involvement, which is almost interchangeably called autoimmune pancreatitis (aip), and it has been reported that infiltration of many igg4-positive plasma cells was observed in the gastric mucosa, colonic mucosa and major duodenal papillae of some aip patients. in addition, igg4-positive plasma cell infiltration is sometimes detected in the colonic mucosa of patients with ulcerative colitis (uc). on the other hand, gastrointestinal igg4-rds without other lesions have also been reported in the esophagus and stomach. colonic igg4-rds without other lesions, such as aip or uc, have also been reported, but these have been quite rare. chetty et al. reported two cases of solitary igg4-rd at the cecum and sigmoid colon. in this case report , we presented a case of igg4-rd of the ileocecal region without any evidence of aip or uc based on the postoperative pathological examinations. the lesion was located at the subserosa of the ileum, cecum, ascending colon and mesenteric adipose tissue. a likely explanation for these findings is that the igg4-rd of the present case might have originated at the mesentery and involved the gastrointestinal tract. in fact, some recent reports have presented igg4-related sclerosing mesenteritis involving the gastrointestinal tract. since gastrointestinal igg4-rds without other igg4-rds, such as aip or uc, appear to be difficult to diagnose prior to surgical resection because of their rarity and features similar to malignancy, most of the solitary gastrointestinal igg4-rds in the previous reports were diagnosed after surgical resection, like the present case. on the other hand, a few cases that were successfully diagnosed as gastrointestinal igg4-rd by the immunohistochemical staining of preoperative biopsy specimens have also been reported. in the present case, although a preoperative colonic biopsy was performed to rule out malignancy, we did not perform immunohistochemical examination for igg4-positive plasma cells. it may be difficult to diagnose this disease based on biopsy specimens if there is sclerosis and an increased number of igg4-positive plasma cells were spread predominantly in the subserosa like the present case. according to the comprehensive clinical diagnostic criteria for igg4-rd proposed in 2011, igg4-rd is diagnosed when there is a characteristic diffuse / localized swelling or mass in a single or multiple organs with elevated serum igg4 levels, or when there are histological findings of abundant infiltration of igg4-positive plasma cells and lymphocytes, along with fibrosis. however, approximately 30% of patients have normal serum igg4 levels, despite classic histopathological and immunohistochemical findings. in our present case, although histological features of igg4-rd such as dense lymphoplasmacytic infiltration, fibrosis and obliterative phlebitis were observed, the serum igg4 level was normal. one possible explanation for this finding is that the serum igg4 level was decreased to the normal level at the point of measurement because it was measured postoperatively. although some igg4-rd patients might show false - negative serum igg4 findings, the levels should be measured prior to surgical resection in case of gastrointestinal stenosis or obstruction of uncertain cause, and where this disease might be suspected. as described above, measurement of the serum igg4 levels, immunohistochemical examinations of biopsy specimens and the use of several imaging modalities might help to diagnose the condition without the need for surgical resection. if gastrointestinal igg4-rd can be diagnosed without surgical resection, steroid therapy can be useful for the disease. however, if a malignancy can not be ruled out completely after these examinations, surgical resection as a treatment for malignancy should be performed to obtain a definite diagnosis. in addition, many patients with gastrointestinal igg4-rd may develop obstructive problems such as ileus. in such cases, surgical resection should be performed if the obstruction does not improve with conservative management. gastrointestinal igg4-rd often mimics malignancy, and a preoperative diagnosis of this disease remains difficult. the most important thing to avoid unnecessary resection is that igg4-rd in the gastrointestinal tract should be included in the differential diagnosis when marked wall thickening or a pseudotumor - like lesion is observed in the gastrointestinal region. however, surgical resection for igg4-rd may still be necessary for patients with concerns regarding malignancy or intractable gastrointestinal obstruction caused by this disease. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request. eiji oki, yoko zaitsu, koji ando, hiroshi saeki, masaru morita, hideo baba, yoshihiko maehara: writing. hidetaka yamamoto: pathologist.key learning pointsthe colonic igg4-rd is rare.we herein report the case of a 74-year - old female with igg4-rd of the ileocecal region who underwent right hemi - colectomy for suspected malignant lymphoma.the patient was diagnosed with igg4-rd by postoperative pathological examinations.the measurement of the serum igg4 levels, immunohistochemical examination of biopsy specimens and use of several imaging modalities might help us to diagnose the disease without surgical resection, and this disease can generally be treated with steroid therapy.surgical resection for igg4-rd may still be also necessary for patients with concerns regarding malignancy or with intractable gastrointestinal obstruction caused by this disease. the colonic igg4-rd is rare.we herein report the case of a 74-year - old female with igg4-rd of the ileocecal region who underwent right hemi - colectomy for suspected malignant lymphoma.the patient was diagnosed with igg4-rd by postoperative pathological examinations.the measurement of the serum igg4 levels, immunohistochemical examination of biopsy specimens and use of several imaging modalities might help us to diagnose the disease without surgical resection, and this disease can generally be treated with steroid therapy.surgical resection for igg4-rd may still be also necessary for patients with concerns regarding malignancy or with intractable gastrointestinal obstruction caused by this disease. we herein report the case of a 74-year - old female with igg4-rd of the ileocecal region who underwent right hemi - colectomy for suspected malignant lymphoma. the measurement of the serum igg4 levels, immunohistochemical examination of biopsy specimens and use of several imaging modalities might help us to diagnose the disease without surgical resection, and this disease can generally be treated with steroid therapy. surgical resection for igg4-rd may still be also necessary for patients with concerns regarding malignancy or with intractable gastrointestinal obstruction caused by this disease.

highlightsthe colonic igg4-rd is rare.we report the case of a74-year - old female with igg4-rd of the ileocecal region.the patient was diagnosed asmalignant lymphoma and underwent right - hemi colectomy.postoperative pathologicalexamination revealed igg4-rd of the ileocecal region.surgical resection for igg4-rdis necessary for cases with concerns of malignancy.

periodontitis is a disease characterized by inflammation of the gingiva that in periodontal pocket formation with loss of the supporting periodontal ligament and alveolar bone around teeth. various terminologies including scaling and root planing (srp) or open flap debridement (ofd) are conventional methods used for treatment of periodontitis. but the use of specific biomaterials was more effective than ofd in improving attachment levels in periodontal defects. in the past three decades , periodontal regenerative treatment has received increasing attention as an alternative to tooth extraction in patients with periodontal disease. implant insertion, although highly predictable, has been shown to be less predictable than saving a periodontally compromised tooth with regenerative therapy. with the advent of guided tissue regeneration (gtr), restoration of periodontium the technique using barrier was introduced by nyman in 1982, and the term gtr was coined by gottlow in 1986. gtr is based upon the biological behavior of different periodontal tissues. during 1980s, researchers published information that led to the that the periodontal ligament was the most important source of periodontal progenitor regenerative cells. current concept of gtr membrane, that is, resorbable and nonresorbable membranes act as a physical barrier to avoid connective and epithelial tissue down - growth into the defect, favoring the regeneration of periodontal tissues. the bioabsorbable membranes are the second generation gtr membranes and were developed to avoid the second surgical procedure to remove the barrier. these gtr devices fall into two broad categories, natural products (collagen membrane) and the synthetic (copolymer) materials. in an attempt to further induce attachment gain for intrabony defects, regenerative therapy with bone replacement grafts did not gain acceptance as predictable therapy until the 1980. these materials act either as osteogenic and osteoinductive or osteoconductive. among alloplastic graft materials, bioactive ceramics is a group of osteoconductive materials which include hydroxyapatite (ha), fluorapatite, bioactive glass, and tricalcium phosphate. the food and drug administration approved original composition of bioactive glass designated 45s5 was 45 mol% of sio2, 26.9 mol% of cao, 24.4 mol% of na2o, and 2.5 mol% of p2o5. this material can bond to bone through development of a surface layer of carbonated ha in situ. the calcium phosphate is layer thought to promote adsorption and concentration of osteoblast derived protein necessary for mineralization of extracellular matrix. objectives of periodontal bonegrafts are probing depth (pd) reduction, clinical attachment gain, bonefill of the osseous defect and regeneration of new bone, cementum, and periodontal ligament. to evaluate regenerative potential of the combination of gtr (periocol, eucare pharmaceuticals private limited, chennai, india) and bonegraft (grabio glascera, dorthom medi dents pvt ltd, coimbathore, india); and bonegraft (grabio glascera) alone individually with respect to clinical parameter including clinical attachment level and radiographically the bonefill, change in alveolar crest (ac) bone and defect resolutionto compare the regenerative potential of the combination of gtr (periocol) and bonegraft (grabio glascera); and bonegraft (grabio glascera) alone between each other with respect to clinical parameter including clinical attachment level and radiographically the bonefill, change in ac bone and defect resolution. to evaluate regenerative potential of the combination of gtr (periocol, eucare pharmaceuticals private limited, chennai, india) and bonegraft (grabio glascera, dorthom medi dents pvt ltd, coimbathore, india); and bonegraft (grabio glascera) alone individually with respect to clinical parameter including clinical attachment level and radiographically the bonefill, change in alveolar crest (ac) bone and defect resolution to compare the regenerative potential of the combination of gtr (periocol) and bonegraft (grabio glascera); and bonegraft (grabio glascera) alone between each other with respect to clinical parameter including clinical attachment level and radiographically the bonefill, change in ac bone and defect resolution. to evaluate regenerative potential of the combination of gtr (periocol, eucare pharmaceuticals private limited, chennai, india) and bonegraft (grabio glascera, dorthom medi dents pvt ltd, coimbathore, india); and bonegraft (grabio glascera) alone individually with respect to clinical parameter including clinical attachment level and radiographically the bonefill, change in alveolar crest (ac) bone and defect resolutionto compare the regenerative potential of the combination of gtr (periocol) and bonegraft (grabio glascera); and bonegraft (grabio glascera) alone between each other with respect to clinical parameter including clinical attachment level and radiographically the bonefill, change in ac bone and defect resolution. to evaluate regenerative potential of the combination of gtr (periocol, eucare pharmaceuticals private limited, chennai, india) and bonegraft (grabio glascera, dorthom medi dents pvt ltd, coimbathore, india); and bonegraft (grabio glascera) alone individually with respect to clinical parameter including clinical attachment level and radiographically the bonefill, change in alveolar crest (ac) bone and defect resolution to compare the regenerative potential of the combination of gtr (periocol) and bonegraft (grabio glascera); and bonegraft (grabio glascera) alone between each other with respect to clinical parameter including clinical attachment level and radiographically the bonefill, change in ac bone and defect resolution. in the present study, a total of 30 sites of systemically healthy 19 patients (14 males and 5 females) who referred to the department of periodontology and implantology of sardar patel postgraduate institute of dental and medical sciences lucknow with moderate to severe periodontitis having one or more periodontal osseous defects were treated during the period of 20092010. the other inclusion criteria were (i) pd of 5 mm (ii) clinical attachment loss (cal) of 5 mm after phase one therapy of 46 weeks and (iii) 3 mm of radiographic periodontal osseous defect. each patient exhibited at least one site chosen randomly which should be either located interproximally or involving the furcation area: (i) medically compromised patient, (ii) lactating and pregnant women, (iii) smokers and (iv) teeth with hopeless prognosis were excluded from the study. the patient had completed basic periodontal therapy including scaling oral hygiene instruction and reinforcement, srp with both hand and ultrasonic instrumentation at the time of enrollment before 46 weeks of surgeries. endodontic treatment involving root canal treatment and crown rehabilitation was done in the required cases. patient who agreed to participate signed an informed consent. using randomized parallel method the defects were randomly assigned (flip of the coin) to the two treatment groups designated as group i and group ii. group i (16 sites) was treated with bioresorbable gtr membranes (periocol) (sterile collagen periodontal membrane - fish origin 25 mm 30 mm) and bone graft in combination (grabio glascera) - containing 50% bioactive glass (, 53% calcium , 30% phosphorous) and 50% synthetic hydroxyapatite (ha) with particle size of 0.150.50 mm ). group ii - (14-sites) was treated with bonegraft alone (grabio glascera). soft tissue measurements include plaque index, (le, 1967) sulcus bleeding index, (muhlemman and son, 1971), pd and clinical attachment level which were performed with university of north carolina probe (unc-15) and hard tissue measurements (bonefill, ac bone change and defect resolution) were recorded in radiographs which were studied preoperatively and at 6 months postoperatively by single examiner. the maximum pd and clinical attachment were measured at four aspects of the tooth, that is, buccal, lingual, mesial, and distal. the radiographic measurement includes standardized reproducible intraoral periapical radiographs which were taken before surgery preoperatively (baseline) and 6 months postoperatively by the paralleling technique with rinn holder xcp system, dentsply hamm moor lane / addlestone / weybridge / surrey kt15 2se (x - ray machine - sansin dig 7 with microprocessor controlled digital timer and calibrated by national physical laboratory with settings of 70 kvp, 10 ma). radiographic measurements were made utilizing a millimeter x - ray grid, which consisted of vertical and horizontal squares, with small squares measuring 1 mm. vertical linear distances between the cementoenamel junction (cej) and the most apical extension of the defect were obtained by counting the number of squares of 1 mm in a grid. (a) measurement of preoperative, (b) six months postoperative radiograph with 1 mm grid, also showing landmarks cej, ac, and bd although the radiographic pairs were almost identical, still some amount of the vertical distortion between baseline radiographs and the 12-month radiograph was estimated. in order to get the exact values, the following measures were taken. in order to estimate this distortion, an anatomically nonvariable distance as the root length (distance from cej to root apex was measured on both the radiographs and a correction factor was calculated as follows : in case where it was not possible to measure the root length, the crown length was assessed ( distance from incisal margin of the crown to the cej). the following anatomical landmarks of the intrabony defect that is, cej, ac, and bd were identified on the radiographs based on the criteria set by bjorn et al. ac: the most coronal position of the alveolar bone crest of the intrabony defect when it touches the root surface of the adjacent tooth before treatment, thst is, the top of the crest. bd: the most apical extension of the intrabony destruction where the periodontal ligament space still retained its normal width before treatment, that is, the bottom of the defect. if the restoration was present, the apical margin of the restoration was used to replace the cej as a fixed reference point. the following linear measurements were performed with the help of grid mount: cej - bd (cej to bottom of the defect)cej - ac (cej to the most coronal extent of the interdental crest)ac - bd (depth of intabony defect at baseline was obtained by subtracting above parameters). cej - bd (cej to bottom of the defect) cej - ac (cej to the most coronal extent of the interdental crest) ac - bd (depth of intabony defect at baseline was obtained by subtracting above parameters). pre- and post - treatment radiograph pairs were analyzed in order to measure the following. (a) preoperative radiograph, (b) postoperative after 6 months with grid, (c) postoperative after 6 months without grid the radiographic bonefill after 6-month was calculated after applying the correction factor as follows: (cej - bd) (cej - bd correction factor) = bonefill (cej - ac) (cej - ac correction factor) = ac bone change if the were negative, this meant that a process of bone resorption had occurred. the radiographic defect resolution was evaluated subtracting radiographic alveolar bone crest change from radiographic bonefill. defect resolution = bonefill alveolar bone crest change. after a preoperative radiographic assessment, prior to surgery, a presurgical rinse with chlorhexidine (0.2%) (clohex plus, 0.2% w / v chlorhexidine gluconate, dr reddys, hyderabad) to maintain asepsis followed with local anesthesia 2% lignocaine containing adrenaline at a concentration of 1:80,000 was administered. intracrevicular incision along with vertical releasing incisions was performed if necessary for a better access. mucoperiosteal flaps were raised, granulation tissue was removed, roots were thoroughly scaled and planned, followed by conditioning with tetracycline. group i received combination of gtr (periocol) (sterile collagen periodontal membrane - fish origin 25 30 mm 30 mm soaked in normal saline about half an hour prior to its placement) and bone graft (grabio glascera) - containing 50% bioactive glass (17% silicon , 53% calcium , 30% phosphorous) and 50% synthetic ha with particle size of 0.150.50 mm was used and mixed with normal saline 5 min prior to the placement in the defect. whereas group ii received bone graft alone. the defect was filled in both the groups with bone graft (bioactive ceramic composite granule with particle size of 0.150.50 mm). the graft material was moistened in sterile saline for 5 min before placement into the defects, care was taken not to overfill the defect. following bone grafting, an aluminum foil template was trimmed and adapted over the entire defect so as to cover 23 mm of the surrounding alveolar bone, followed by a membrane of the same size and shape was trimmed and adapted over the entire defect so as to cover 23 mm of surrounding alveolar bone. sutures (50 vicryl resorbable), if needed, were placed to secure the membrane and to ensure stability of the graft material. finally, the mucoperiosteal flap was repositioned and sutured at the original level using 30 silk with an interrupted suture. the same surgical technique was used for group ii except the placement of the membrane, only bone graft was used. amoxicillin for 7 days ) and analgesic (ibuprofen 400 mg t.d.s for 3 days). the postoperative care consisted of 0.2% chlorhexidine rinse twice daily for 4 weeks. for clinical and radiographic measurement purposes, the patient was recalled after 6 months. soft tissue measurements include plaque index, (le, 1967) sulcus bleeding index, (muhlemman and son, 1971), pd and clinical attachment level which were performed with university of north carolina probe (unc-15) and hard tissue measurements (bonefill, ac bone change and defect resolution) were recorded in radiographs which were studied preoperatively and at 6 months postoperatively by single examiner. the maximum pd and clinical attachment were measured at four aspects of the tooth, that is, buccal, lingual, mesial, and distal. the radiographic measurement includes standardized reproducible intraoral periapical radiographs which were taken before surgery preoperatively (baseline) and 6 months postoperatively by the paralleling technique with rinn holder xcp system, dentsply hamm moor lane / addlestone / weybridge / surrey kt15 2se (x - ray machine - sansin dig 7 with microprocessor controlled digital timer and calibrated by national physical laboratory with settings of 70 kvp, 10 ma). radiographic measurements were made utilizing a millimeter x - ray grid, which consisted of vertical and horizontal squares, with small squares measuring 1 mm. vertical linear distances between the cementoenamel junction (cej) and the most apical extension of the defect were obtained by counting the number of squares of 1 mm in a grid. (a) measurement of preoperative, (b) six months postoperative radiograph with 1 mm grid, also showing landmarks cej, ac, and bd although the radiographic pairs were almost identical, still some amount of the vertical distortion between baseline radiographs and the 12-month radiograph was estimated. in order to get the exact values, the following measures were taken. in order to estimate this distortion, an anatomically nonvariable distance as the root length (distance from cej to root apex was measured on both the radiographs and a correction factor was calculated as follows : in case where it was not possible to measure the root length, the crown length was assessed ( distance from incisal margin of the crown to the cej). the following anatomical landmarks of the intrabony defect that is, cej, ac, and bd were identified on the radiographs based on the criteria set by bjorn et al. ac: the most coronal position of the alveolar bone crest of the intrabony defect when it touches the root surface of the adjacent tooth before treatment, thst is, the top of the crest. bd: the most apical extension of the intrabony destruction where the periodontal ligament space still retained its normal width before treatment, that is, the bottom of the defect. if the restoration was present, the apical margin of the restoration was used to replace the cej as a fixed reference point. the following linear measurements were performed with the help of grid mount: cej - bd (cej to bottom of the defect)cej - ac (cej to the most coronal extent of the interdental crest)ac - bd (depth of intabony defect at baseline was obtained by subtracting above parameters). cej - bd (cej to bottom of the defect) cej - ac (cej to the most coronal extent of the interdental crest) ac - bd (depth of intabony defect at baseline was obtained by subtracting above parameters). although the radiographic pairs were almost identical, still some amount of the vertical distortion between baseline radiographs and the 12-month radiograph was estimated. in order to get the exact values, the following measures were taken. in order to estimate this distortion, an anatomically nonvariable distance as the root length (distance from cej to root apex was measured on both the radiographs and a correction factor was calculated as follows : in case where it was not possible to measure the root length, the crown length was assessed ( distance from incisal margin of the crown to the cej). the following anatomical landmarks of the intrabony defect that is, cej, ac, and bd were identified on the radiographs based on the criteria set by bjorn et al. ac: the most coronal position of the alveolar bone crest of the intrabony defect when it touches the root surface of the adjacent tooth before treatment, thst is, the top of the crest. bd: the most apical extension of the intrabony destruction where the periodontal ligament space still retained its normal width before treatment, that is, the bottom of the defect. if the restoration was present, the apical margin of the restoration was used to replace the cej as a fixed reference point. the following linear measurements were performed with the help of grid mount: cej - bd (cej to bottom of the defect)cej - ac (cej to the most coronal extent of the interdental crest)ac - bd (depth of intabony defect at baseline was obtained by subtracting above parameters). cej - bd (cej to bottom of the defect) cej - ac (cej to the most coronal extent of the interdental crest) ac - bd (depth of intabony defect at baseline was obtained by subtracting above parameters). pre- and post - treatment radiograph pairs were analyzed in order to measure the following. (a) preoperative radiograph, (b) postoperative after 6 months with grid, (c) postoperative after 6 months without grid the radiographic bonefill after 6-month was calculated after applying the correction factor as follows: (cej - bd) (cej - bd correction factor) = bonefill (cej - ac) (cej - ac correction factor) = ac bone change if the were negative, this meant that a process of bone resorption had occurred. the radiographic defect resolution was evaluated subtracting radiographic alveolar bone crest change from radiographic bonefill. the radiographic bonefill after 6-month was calculated after applying the correction factor as follows: (cej - bd) (cej - bd correction factor) = bonefill (cej - ac) (cej - ac correction factor) = ac bone change if the were negative, this meant that a process of bone resorption had occurred. the radiographic defect resolution was evaluated subtracting radiographic alveolar bone crest change from radiographic bonefill. after a preoperative radiographic assessment, surgical therapy was planned. prior to surgery, a presurgical rinse with chlorhexidine (0.2%) (clohex plus, 0.2% w / v chlorhexidine gluconate, dr reddys, hyderabad) to maintain asepsis followed with local anesthesia 2% lignocaine containing adrenaline at a concentration of 1:80,000 was administered. intracrevicular incision along with vertical releasing incisions was performed if necessary for a better access. mucoperiosteal flaps were raised, granulation tissue was removed, roots were thoroughly scaled and planned, followed by conditioning with tetracycline. group i received combination of gtr (periocol) (sterile collagen periodontal membrane - fish origin 25 30 mm 30 mm soaked in normal saline about half an hour prior to its placement) and bone graft (grabio glascera) - containing 50% bioactive glass (17% silicon , 53% calcium , 30% phosphorous) and 50% synthetic ha with particle size of 0.150.50 mm was used and mixed with normal saline 5 min prior to the placement in the defect. whereas group ii received bone graft alone. the defect was filled in both the groups with bone graft (bioactive ceramic composite granule with particle size of 0.150.50 mm). the graft material was moistened in sterile saline for 5 min before placement into the defects, care was taken not to overfill the defect. following bone grafting, an aluminum foil template was trimmed and adapted over the entire defect so as to cover 23 mm of the surrounding alveolar bone, followed by a membrane of the same size and shape was trimmed and adapted over the entire defect so as to cover 23 mm of surrounding alveolar bone. sutures (50 vicryl resorbable), if needed, were placed to secure the membrane and to ensure stability of the graft material. finally, the mucoperiosteal flap was repositioned and sutured at the original level using 30 silk with an interrupted suture. the same surgical technique was used for group ii except the placement of the membrane, only bone graft was used. amoxicillin for 7 days ) and analgesic (ibuprofen 400 mg t.d.s for 3 days). the postoperative care consisted of 0.2% chlorhexidine rinse twice daily for 4 weeks. for clinical and radiographic measurement purposes, the patient was recalled after 6 months. regeneration of lost structures has become the primary therapeutic goal in periodontics over the past several decades. in the present study, the material used was bioresorbable collagen membrane of fish origin. the purpose to use this was to avoid a second surgery. before its placement, a priority was given to synthetic graft that is, bioactive glass. among the various comparative clinical trials involving the use of synthetic graft and various other types of graft, better were shown in favor of synthetic bone graft. though autogenous bone is still the gold standard in bone augmentation procedures but due to its low availability and donor site morbidity necessitates the development of alternative products for it and with allogenic graft there is a possibility of disease transfer, e.g., with freeze - dried bone allografts. clinically and statistically, a significant improvement was seen pd, cal, bonefill, crestal bone loss, and defect resolution from baseline till an observation duration of 6 months individually in both the groups. the change in pd and cal for both the groups shows a statistically significant difference between preoperative and postoperative values, that is, both the treatment modalities are effective but on comparing the net gain between group i and group ii, group i, that is, combination therapy shows slightly higher values than group ii, yet there was no statistically significant difference (p = 0.790) between the two groups. the net gain in pd and cal for combination and bonegraft were 3.94 1.81 mm, 3.57 2.21 mm and 3.94 1.81 and 3.57 2.21 mm, respectively. a similar type of study done by prathap et al., also shows slightly higher values in combination therapy than the bone graft alone and concluded that statistically no significant difference was found between both the treatment modalities. comparison of pre- and post - operative periodontal health status in group i comparison of pre- and post - operative periodontal health status in group ii comparison of pre- and post - operative periodontal health status in group i comparison of pre- and post - operative periodontal health status in group ii comparison of net pd change, net cal change, net bone fill, net alveolar bone crest change and net defect resolution in two study groups comparison of net probing depth change, net clinical attachment loss change, net bone fill, net alveolar bone crest change, and net defect resolution in two study groups the of the study with respect to mean net bonefill for group i and group ii in terms of percentage were 33.23 15.58% and 40.26 19.14% , respectively. though the value was higher in group ii than in group i statistically, there was no significant difference found between the two groups. in accordance, the present study, some other similar type of clinical trials also shows no statistically significant difference between combination and bone graft alone type of therapy. tsao et al., showed the vertical bone fill of 1.9 1.4 mm in mineralized cancellous bone allograft (mba) and 0.7 0.9 mm in combination, while keles et al. used autogenous cortical bone grafting (acb) and atrisorb as an absorbable polylactide membrane, and the values regarding the gain in radiographic alveolar bone height were 5.50 2.24 mm in the acb graft with gtr - treated group and 5.92 1.83 mm in the acb graft - treated group. both the studies show slightly higher values for the bonegraft group with no statistically significant difference between the two treatment modalities as in the case of the present study. these studies were in agreement with our study which records no additional benefit of gtr membrane together with bone graft alone. comparison of proportional change (in percentage) in net pd change, net cal change, net bone fill, net alveolar bone crest change and net defect resolution in two study groups comparison of proportional change (in percentage) in net probing depth change, net clinical attachment loss change, net bone fill, net alveolar bone crest change, and net defect resolution in two study groups however, there are certain studies which favor the combination therapy with respect to bonefill. , used autogenous bone graft + gtr and autogenous bonegraft alone and recorded probing bone level gain as 3.9 0.8 versus 1.3 0.9 mm, p = 0.034, respectively. contrary to the above study designs, there are few studies which use only bonegraft, though of different types, without the use of any membrane and showed significant improvement in defect fill. meffert et al. used hydroxylapatite as an alloplastic material for a period of 9 months and showed a defect fill of 66.89%. yukna used hard tissue replacement polymer for a period of 6-month and showed 60.8% defect fill respectively when compared with control groups. grover et al., used bioactive glass and showed a mean radiographic defect fill of 64.76% (2.49 0.5 mm) after 6-month observation. all the above studies used synthetic bone graft and showed the positive thus again favoring the use of synthetic bone grafts as a regenerative material. the net alveolar crestal bone change value was negative which showed that a crestal bone resorption had occurred in both the groups, yet there was no statistically significant difference found between both the groups (p > 0.05). this was a significant finding as many of the synthetic bone graft reported bone fill still ed in some amount of crestal bone loss. the net value was higher in group ii which showed that more crestal resorption occurred in group ii than in group i, but there was no statistically significant difference present between both the groups (p > 0.05) tsao et al. showed the 1.3 1.1 mm crestal bone resorption in mba alone and 0.7 0.9 mm in combination with gtr. the study of luepke et al. showed that when combination therapy was used, that is, bioabsorbable alone and its combination with decalcified freeze - dried bone allograft (dfdba), the crestal bone resorption seen was 0.10 1.11 mm and 0.27 1.15 mm. the of the study was almost comparable to combination group as seen in the present study. in terms of defect resolution, the mean net defect resolution in group i and group ii in terms of percentage 62.86 26.49 and 80.58 24.09, respectively, the above observation shows that the net value is higher in group ii than in group i yet there was no significant difference between both the groups. the defect resolution value was obtained after taking into consideration of the net crestal bone resorption and net bone fill. if the net crestal bone resorption would have not taken into consideration, then the values of defect resolution would have been different as compared to the actual value. thus, this error was rectified initially and after rectifying it only the net mean defect resolution was calculated. evaluated the combination of expanded polytetrafluoroethylene and dfdba versus dfdba alone but showed no statistically significant difference regarding defect resolution (81% vs. 64%) thus supporting to our study. used a combination of deproteinized bovine mineral and a collagen membrane in the treatment of intrabony defects and compared with papilla preservation flap alone and showed the radiographic resolution of approximately 60% which was almost comparable to the present study with respect to combination therapy. meffert et al., showed the radiographic resolution of 53.57 4.79% with hydroxylapatite as an alloplastic graft and 19.49 4.52% with the control group, that is, without graft. richardson et al., compared the graft, that is, bio - oss (wolhusen) and dfdba and showed the defect resolution of 77.6% for bio - oss and 59.4% for dfdba. in the present study, the were evaluated up to the end of 6-month following surgeries. had the time been permitted longer for the observation the might have been better. the radiographic outcome after the two treatment modalities should be interpreted with caution as these grafts in the radiograph are hardly distinguishable from the host bone and the graft appearing on radiographs may not necessarily be incorporated in bone. this could introduce some bias in the radiographic examination process and overestimation of radiographic bone level in both the groups. the calculation of a correction factor assessing the level of distortion between preoperative and postoperative radiograph helped to minimize errors. the correction factor for both the groups was close to one which means that the projection of pre- and post - operative radiographs was similar in both the groups. all the clinical measurements were done using unc-15 probe. since in most of the studies the measurements such as bonefill, crestal resorption, and defect resolution were done with re - entry procedure which is traumatic to the patient and on other ethical issues. within the limitations of present study, it can be concluded that the combination of gtr + bone graft and bone graft alone ed in a significant reduction in both the hard and soft tissue parameters when compared individually from baseline data to 6-month duration. on comparing both the groups together after 6 months of therapy, the were equally effective with combination of graft + membrane versus bone graft alone since no significant difference was seen between any of the parameters between both the groups. thus, both the treatment modalities are comparable and effective. taking combination of membrane and bone graft into consideration, it can be concluded that using gtr membrane with bonegraft does not have any additional benefit as compared to bone graft alone.

aim: the aim was to evaluate the bonefill in periodontal osseous defects with the help of guided tissue regeneration, bioresorbable membrane (periocol) + bone graft (grabio glascera) in combination and with bonegraft (grabio glascera) alone.materials and methods: the study involved total 30 sites in systemically healthy 19 patients. the parameters for evaluation includes plaque index sulcus bleeding index with one or more periodontal osseous defects having (i) probing depth (pd) of 5 mm (ii) clinical attachment loss (cal) of 5 mm and (iii) 3 mm of radiographic periodontal osseous defect (iv) bonefill (v) crestal bone loss (vi) defect resolution. the study involved the three wall and two wall defects which should be either located interproximally or involving the furcation area. the statistical analysis was done using statistical package for social sciences, the wilcoxon signed rank statistic w + for mann whitney u-test.:the net gain in pd and cal after 6 months for group i (+) and group ii (grabio glascera) was 3.94 1.81 mm, 3.57 2.21 mm and 3.94 1.81, 3.57 2.21 mm, respectively. the of the study for group i and group ii with regards to mean net bonefill, was 3.25 2.32 (58%) mm and 5.14 3.84 (40.26 19.14%) mm, crestal bone loss 0.25 0.68 mm and 0.79 1.19 mm. defect resolution 3.50 2.34 mm and 5.93 4.01 mm, respectively.:on comparing both the groups together after 6 months of therapy, the were equally effective for combination of graft and membrane versus bone graft alone since no statistical significant difference was seen between above parameters for both the groups. thus, both the treatment modalities are comparable and equally effective.

the thalamus is of central interest in many disorders of the nervous system (andreasen 1997 ; dom, et al . 1976 ; lee and marsden 1994 ; meador - woodruff, et al . 2003 ; speedie and heilman 1983 ; williams 1965 ; xuereb, et al . 1991). the functioning of the thalamus is crucial to many sensory, motor and cognitive systems, and therefore has also been subject to a great deal of investigation in the cognitive neurosciences (basso, et al . 2005 ; engelborghs, et al . it is in these capacities that analysis of thalamic structure and function is a continually researched theme in neuroimaging investigations, particularly using magnetic resonance imaging ( mri). analysis of volume or shape using mri techniques may provide important information with respect to the involvement of the thalamus in neurological and neuropsychiatric disorders, including generalized (du, et al . 2011 ; pulsipher, et al . 2009) and partial (gong, et al . 2008 ; pulsipher, et al . 2007) epilepsy, schizophrenia (adriano, et al . 2010), huntington s disease (douaud, et al . 2006 2008), and alzheimer s disease (de jong, et al . 2008). reliable measurement of thalamic structure is, however, notoriously difficult to achieve, particularly given the typically poor between - tissue mr contrast of the thalamic nuclei and adjacent white matter (amini, et al . it is therefore important to develop new and improve and validate existing methodologies that provide thalamic metrics . like for other subcortical brain structures, there are several approaches freely available to estimate thalamic volume from mr images . at either end of the mr image analysis spectrum, there are manual and automated approaches ; manual approaches are user - dependent, time consuming but are considered to be the gold standard of mr image analysis techniques ( bonilha, et al . 2004 ; collins and pruessner 2010 ; crum, et al . 2001 ; pruessner, et al . automated approaches remove the need of an expert anatomist, are dependent on computer algorithms, and are time efficient, but require a great deal of validation against manual methods to determine the specificity and validity of measurements ( chupin, et al . the primary goal of the present study was to evaluate the validity of thalamic volume measurements obtained from a frequently used automated approach with respect to a reputable manual approach widely used in the imaging, anatomical and histological sciences . the fully automated approach investigated in the present study was the subcortical segmentation and volume estimation techniques ( fischl, et al . 2002) incorporated into freesurfer software (http://surfer.nmr.mgh.harvard.edu/), which provide observer - independent volumes for individual subcortical nuclei from conventional mr images. similarly to other methods that automatically segment and estimate subcortical volume such as first (patenaude, et al . 2011) incorporated into fsl software (http://www.fmrib.ox.ac.uk/fsl/first/index.html), there has been a recent proliferation of studies using freesurfer methods for volumetric studies, some of which have included comparison with manual methods, most notably for the hippocampus (cherbuin, et al . to our knowledge, there has been no independent comparison between manual methods and freesurfer methods for volume estimation of the thalamus . the manual approach used to evaluated freesurfer - based thalamic volumes in the present study was the cavalieri method of design - based stereology in conjunction with point counting ( gundersen and jensen 1987 ; gundersen, et al . 2000), which is a 100 % investigator interactive technique that requires manual determination of sampling density for a given brain structure (i.e. the stereological parameters necessary to produce a reliable volume estimate) and investigator decisions on whether or not sampling probes (i.e. points) intersect the brain region - of - interest (roi). stereology requires the use of a human anatomist with expert knowledge of anatomical boundaries that divide legitimate (i.e. thalamic) and illegitimate (i.e. non - thalamic) brain tissue. manual approaches such as stereology are considered gold standard because it is assumed that human knowledge and perception is superior to computer algorithms that determine regional brain boundaries. firstly, we compared thalamic volume estimates in a sample of neurologically and psychiatrically healthy subjects. secondly, we compared the methods in their sensitivity in detecting thalamic atrophy in patients with juvenile myoclonic epilepsy (jme). jme is an electro - clinical syndrome that by definition is non - lesional and without abnormality on conventional magnetic resonance imaging (mri) (berg, et al . 2010 ; ilae 1989), but has previously been shown to be associated with thalamic structural alterations (deppe, et al . 2009), and is generally considered to be intimately associated with thalamic dysfunction (holmes, et al . we studied a neurologically and psychiatrically healthy control group that was composed of 62 adult volunteers ( 32 females, mean age 27.9 4.3 sd, range 2143), all of whom had normal neurological examination and normal mri (t1-, t2-weighted, and flair). we also studied ten patients (6 females, mean age 28.6 8.8 sd, range 1942) with jme. 2011a ). there was no statistical difference in age between patients and controls (t = 0.38, p = 0.70). all participants underwent high resolution mri t1-weighted, t2-weighted and flair imaging at 3 t (philips intera t30, t / r head coil). all mri modalities were used to exclude the possibility of brain lesions in patients and controls. for volumetric analysis, we acquired t1-weighted structural mris using a high - resolution 3d turbo - field - echo sequence (matrix 256 256 160 over a field of view of 25.6 25.6 16 cm reconstructed after zero filling to 512 512 320 cubic voxels with an edge length of 0.5 mm). prior to morphometric analyses, all mr images were intensity inhomogeneity corrected and resampled to isotropic voxels of 1 1 1 mm (256 256 160 slices) using in - house software (eval 3.0). to confirm that there was no systematic difference in head size between patient and control groups , we automatically obtained relative brain size, vscale (global tissue volumes normalized for head size) and csf volume estimates from the 3d t1-weighted images of all subjects using the sienax protocol (smith, et al . 2002) integrated into fsl software (http://www.fmrib.ox.ac.uk/fsl/siena/index.html). there were no inter - group differences in relative brain size (p = 0.40), vscale (p = 0.96) or csf volume (p = 0.95).stereology the cavalieri method of design - based stereology in conjunction with point counting (gundersen and jensen 1987 ; gundersen, et al . 2000) was used as an unbiased estimator of the volume of the left and right thalamus in all subjects. by using the cavalieri method, volume is directly estimated from equidistant and parallel mr images of the brain with a uniform random starting position. a second level of sampling is required to estimate the section area from each image by applying point counting within the roi. the mathematical justification and implementation of the methodology is simple and it can be applied to structures of arbitrary shape (garcia - finana, et al . this technique has been frequently applied to reliably estimate brain volume and surface area on mr images ( acer, et al . 2008 ; mackay, et al . 1998 ; mackay, et al . 2000 ; ronan, et al . 2006 1996), and more widely applied to study other aspects of anatomy with and without the use of mri. stereology has been shown to be at least as precise as tracing and thresholding volumetry techniques and substantially more time efficient, with validation relative to post - mortem measurements (garcia - finana, et al . windows - compatible easymeasure software ( keller, et al . 2007 ; puddephat 1999) was used for point counting on mr images. given that the borders of different thalamic nuclei are almost indistinguishable on conventional mri, the thalamus was sampled as an entire complex, including the anterior thalamic nucleus, mediodorsal thalamic nucleus, lateral dorsal thalamic nucleus, ventral lateral thalamic nucleus, ventral postero - lateral / medial thalamic nuclei, and pulvinar (duvernoy 1999). the lateral and medial geniculate nuclei were not included in measurements, similar to previous studies (natsume, et al . on axial sections, point counting began on the dorsal most section, where the area of the lateral dorsal thalamic nucleus was demarcated laterally by the corona radiata and medially by the lateral ventricles . as measurements progressed ventrally, the posterior limb of the internal capsule bordered the thalamus laterally . on ventral sections, care was taken to delineate the thalamic nuclei from the neighbouring pars medialis of the globus pallidus, and at the most ventral sections, to segregate the pulvinar and area of the ventral posterolateral nucleus from the emerging hypothalamus anteriorly, superior colliculus posteromedially, and the hippocampus posteriorly / posterolaterally . figure 1 shows point counting within the thalamic roi relative to the automated freesurfer approach described below . more information on the mri - based anatomy of the thalamus with respect to post - mortem sections can be found at http://www.psychiatry.uiowa.edu/mhcrc/pdf/papers/thalamus.pdf.fig . 1stereological and freesurfer methods used to estimate thalamic volume shown at approximately the same axial sections . both methods are shown for the same hemisphere ( the freesurfer axial sections are flipped to show maximal correspondence between techniques) of the same randomly selected subject. the sagittal mr section illustrates the approximate levels of the axial sections shown. abbreviations: di, ventral diencephalon (peach); pa, pallidum (violet); put, putamen (lilac); th, thalamus (dark green); thp, pulvinar of the thalamus (dark green) stereological and freesurfer methods used to estimate thalamic volume shown at approximately the same axial sections. both methods are shown for the same hemisphere (the freesurfer axial sections are flipped to show maximal correspondence between techniques) of the same randomly selected subject. the sagittal mr section illustrates the approximate levels of the axial sections shown. abbreviations: di, ventral diencephalon (peach); pa, pallidum (violet); put, putamen (lilac); th, thalamus (dark green); thp, pulvinar of the thalamus (dark green) the sampling density (i.e. size of points, distance between sections) were optimized to achieve a coefficient of error (ce) of less that 5 % (roberts, et al . 2000), an approach that has been adopted in our stereological analysis of the hippocampus (keller, et al . 2009a), planum temporale (keller, et al . 2007 ; keller, et al . the ce is essentially a statistical estimate of how accurate the stereological volume estimation is for each structure . separation between test points on the square grid used for point counting was 0.312 cm ( i.e., 4 pixels) and slice interval was 1 mm (singular axial mr sections). thalamic transect area was obtained by multiplying the total number of points recorded by the area corresponding to each test point. an estimate of thalamic volume was obtained as the sum of the estimated areas of the structure transects on consecutive systematic sections multiplied by the distance between sections. between approximately 400550 points were recorded on approximately 20 systematic random sections.freesurfer freesurfer software (http://surfer.nmr.mgh.harvard.edu/) was used to obtain thalamic volumes for all subjects using an observer - independent approach, which could be contrasted with the manual stereological measurements of the thalamus. thalamic segmentations are based on the assignment of neuroanatomical labels to each voxel in an mr image based on the probabilistic information automatically estimated from a manually labelled training set. the methods of the automated volumetric approach have been described in detail previously (fischl, et al . 2002), and the accuracy of automated labelling and volumetry of subcortical structures have been independently validated with respect to gold standard manual volumetric techniques, predominantly for the hippocampus (cherbuin, et al . , there has been no independent comparison of the automated thalamic volumetry offered by freesurfer and a manual volumetric method ( although thalamic tracings were compared with the performance of freesurfer in the original methods paper by fischl et al . figure 1 shows the comparison of automated labelling of the thalamus ( and extra - thalamic structures) in an individual control subject using freesurfer relative to stereological volume estimation of the thalamus in the same subject. freesurfer analyses were performed on a mac pro (version os x 10.6.6, 32 gb, 2 2.93 ghz 6-core intel xeon ( ht) ), which permitted the freesurfer recon - all function (for cortical reconstruction and brain segmentation ; http://surfer.nmr.mgh.harvard.edu/fswiki/recon-all) to complete 23 participants in less than 20 h. after the recon - all function, the neuroanatomical labels were inspected for accuracy in all patients and controls. despite that freesurfer permits manual editing to improve subcortical segmentation, no obvious errors in the automatic labelling were observed for any subject, and so all data obtained from freesurfer analyses were 100 % automated and not influenced by manual intervention. two - way mixed intra - class correlation coefficients for absolute agreement (shrout and fleiss 1979) were used to determine inter - rater agreement between two human raters using manual stereology and freesurfer for volume estimation of the thalamus in ten randomly selected controls using the statistics software spss (version 18, www.spss.com). intra - class correlations were subsequently performed between stereological volumes obtained by one human rater and freesurfer volumes for the entire sample of patients and controls (n = 72). univariate anovas were used to investigate patient - control differences in volumes, and corrected for multiple comparisons using statistica version 9.1, (stat soft . we studied a neurologically and psychiatrically healthy control group that was composed of 62 adult volunteers ( 32 females, mean age 27.9 4.3 sd, range 2143), all of whom had normal neurological examination and normal mri (t1-, t2-weighted, and flair). we also studied ten patients (6 females, mean age 28.6 8.8 sd, range 1942) with jme. 2011a ). there was no statistical difference in age between patients and controls (t = 0.38, p = 0.70). all participants underwent high resolution mri t1-weighted, t2-weighted and flair imaging at 3 t (philips intera t30, t / r head coil). all mri modalities were used to exclude the possibility of brain lesions in patients and controls. for volumetric analysis, we acquired t1-weighted structural mris using a high - resolution 3d turbo - field - echo sequence (matrix 256 256 160 over a field of view of 25.6 25.6 16 cm reconstructed after zero filling to 512 512 320 cubic voxels with an edge length of 0.5 mm). prior to morphometric analyses, all mr images were intensity inhomogeneity corrected and resampled to isotropic voxels of 1 1 1 mm (256 256 160 slices) using in - house software (eval 3.0). to confirm that there was no systematic difference in head size between patient and control groups , we automatically obtained relative brain size, vscale (global tissue volumes normalized for head size) and csf volume estimates from the 3d t1-weighted images of all subjects using the sienax protocol (smith, et al . 2002) integrated into fsl software (http://www.fmrib.ox.ac.uk/fsl/siena/index.html). there were no inter - group differences in relative brain size (p = 0.40), vscale (p = 0.96) or csf volume (p = 0.95).stereology the cavalieri method of design - based stereology in conjunction with point counting (gundersen and jensen 1987 ; gundersen, et al . 2000) was used as an unbiased estimator of the volume of the left and right thalamus in all subjects. by using the cavalieri method, volume is directly estimated from equidistant and parallel mr images of the brain with a uniform random starting position. a second level of sampling is required to estimate the section area from each image by applying point counting within the roi. the mathematical justification and implementation of the methodology is simple and it can be applied to structures of arbitrary shape (garcia - finana, et al . this technique has been frequently applied to reliably estimate brain volume and surface area on mr images ( acer, et al . 2011 ; howard, et al . 2003 ; jelsing, et al . 2005 ; keller, et al . 2008 ; mackay, et al . 1998 ; mackay, et al . 2000 ; ronan, et al . 2006 ; salmenpera, et al . 2005 ; sheline, et al . 1996), and more widely applied to study other aspects of anatomy with and without the use of mri. stereology has been shown to be at least as precise as tracing and thresholding volumetry techniques and substantially more time efficient, with validation relative to post - mortem measurements (garcia - finana, et al . 2003 ; garcia - finana, et al . 2009 ; keller and roberts 2009 ; keshavan, et al . windows - compatible easymeasure software ( keller, et al . 2007 ; puddephat 1999) was used for point counting on mr images. given that the borders of different thalamic nuclei are almost indistinguishable on conventional mri, the thalamus was sampled as an entire complex, including the anterior thalamic nucleus, mediodorsal thalamic nucleus, lateral dorsal thalamic nucleus, ventral lateral thalamic nucleus, ventral postero - lateral / medial thalamic nuclei, and pulvinar (duvernoy 1999). the lateral and medial geniculate nuclei were not included in measurements, similar to previous studies (natsume, et al . point counting began on the dorsal most section, where the area of the lateral dorsal thalamic nucleus was demarcated laterally by the corona radiata and medially by the lateral ventricles . as measurements progressed ventrally, the posterior limb of the internal capsule bordered the thalamus laterally . on ventral sections, care was taken to delineate the thalamic nuclei from the neighbouring pars medialis of the globus pallidus, and at the most ventral sections, to segregate the pulvinar and area of the ventral posterolateral nucleus from the emerging hypothalamus anteriorly, superior colliculus posteromedially, and the hippocampus posteriorly / posterolaterally . figure 1 shows point counting within the thalamic roi relative to the automated freesurfer approach described below . more information on the mri - based anatomy of the thalamus with respect to post - mortem sections can be found at http://www.psychiatry.uiowa.edu/mhcrc/pdf/papers/thalamus.pdf.fig . 1stereological and freesurfer methods used to estimate thalamic volume shown at approximately the same axial sections . both methods are shown for the same hemisphere ( the freesurfer axial sections are flipped to show maximal correspondence between techniques) of the same randomly selected subject. the sagittal mr section illustrates the approximate levels of the axial sections shown. abbreviations: di, ventral diencephalon (peach); pa, pallidum (violet); put, putamen (lilac); th, thalamus (dark green); thp, pulvinar of the thalamus (dark green) stereological and freesurfer methods used to estimate thalamic volume shown at approximately the same axial sections. both methods are shown for the same hemisphere (the freesurfer axial sections are flipped to show maximal correspondence between techniques) of the same randomly selected subject. the sagittal mr section illustrates the approximate levels of the axial sections shown. abbreviations: di, ventral diencephalon (peach); pa, pallidum (violet); put, putamen (lilac); th, thalamus (dark green); thp, pulvinar of the thalamus (dark green) the sampling density (i.e. size of points, distance between sections) were optimized to achieve a coefficient of error (ce) of less that 5 % (roberts, et al . 2000), an approach that has been adopted in our stereological analysis of the hippocampus (keller, et al . 2002a 2009a), planum temporale (keller, et al . 2007 ; keller, et al . ; keller, et al . 2009b) and insula (keller, et al . the ce is essentially a statistical estimate of how accurate the stereological volume estimation is for each structure . separation between test points on the square grid used for point counting was 0.312 cm ( i.e., 4 pixels) and slice interval was 1 mm (singular axial mr sections). thalamic transect area was obtained by multiplying the total number of points recorded by the area corresponding to each test point. an estimate of thalamic volume was obtained as the sum of the estimated areas of the structure transects on consecutive systematic sections multiplied by the distance between sections. between approximately 400550 points were recorded on approximately 20 systematic random sections.freesurfer freesurfer software (http://surfer.nmr.mgh.harvard.edu/) was used to obtain thalamic volumes for all subjects using an observer - independent approach, which could be contrasted with the manual stereological measurements of the thalamus. thalamic segmentations are based on the assignment of neuroanatomical labels to each voxel in an mr image based on the probabilistic information automatically estimated from a manually labelled training set. the methods of the automated volumetric approach have been described in detail previously (fischl, et al . 2002), and the accuracy of automated labelling and volumetry of subcortical structures have been independently validated with respect to gold standard manual volumetric techniques, predominantly for the hippocampus (cherbuin, et al . to our knowledge, there has been no independent comparison of the automated thalamic volumetry offered by freesurfer and a manual volumetric method ( although thalamic tracings were compared with the performance of freesurfer in the original methods paper by fischl et al . figure 1 shows the comparison of automated labelling of the thalamus ( and extra - thalamic structures) in an individual control subject using freesurfer relative to stereological volume estimation of the thalamus in the same subject. freesurfer analyses were performed on a mac pro (version os x 10.6.6, 32 gb, 2 2.93 ghz 6-core intel xeon ( ht) ), which permitted the freesurfer recon - all function (for cortical reconstruction and brain segmentation ; http://surfer.nmr.mgh.harvard.edu/fswiki/recon-all) to complete 23 participants in less than 20 h. after the recon - all function, the neuroanatomical labels were inspected for accuracy in all patients and controls. despite that freesurfer permits manual editing to improve subcortical segmentation, no obvious errors in the automatic labelling were observed for any subject, and so all data obtained from freesurfer analyses were 100 % automated and not influenced by manual intervention. two - way mixed intra - class correlation coefficients for absolute agreement (shrout and fleiss 1979) were used to determine inter - rater agreement between two human raters using manual stereology and freesurfer for volume estimation of the thalamus in ten randomly selected controls using the statistics software spss (version 18, www.spss.com). intra - class correlations were subsequently performed between stereological volumes obtained by one human rater and freesurfer volumes for the entire sample of patients and controls (n = 72). univariate anovas were used to investigate patient - control differences in volumes, and corrected for multiple comparisons using statistica version 9.1, (stat soft . stereology vs freesurfer : consistency between volumetric measures stereology vs freesurfer : consistency between volumetric measures figure 2 shows the comparison of the three approaches ( rater one ( r1) for stereology, rater two (r2) for stereology and freesurfer ) to estimate thalamic volume in the randomly selected ten subjects. this inter - rater / between - technique analysis indicates consistency across measures, and most notably, that freesurfer performed at least as consistent as r2 relative to r1. mean (sd) left and right thalamic volume was 7339.6 mm (567.3) and 7339.2 mm (489.3) for r1, 7456.3 mm (597.3) and 7444.3 mm (590.6) for r2, and 7365.0 mm (641.3) and 7317.6 mm (610.0) for freesurfer, respectively. measurements between r1 and r2, and between manual raters and freesurfer, achieve high intra - class correlations (all > 0.9).fig. 2inter - rater consistency in stereological volume estimation of the left (blue) and right (red) thalamus, and relation to volume estimates obtained from freesurfer. stereological volume estimates are reproducible, and freesurfer is entirely consistent with the volumes obtained with both rater one and rater two. error bars indicate the 95 % confidence intervalstable 1inter - rater intra - class coefficients for volumetric measures rater 1rater 2fsleft thalamus rater 10.9770.969 rater 20.9770.925 fs0.9690.925 right thalamus rater 10.9520.923 rater 20.9520.916 fs0.9230.916 inter - rater consistency in stereological volume estimation of the left (blue) and right (red) thalamus, and relation to volume estimates obtained from freesurfer. stereological volume estimates are reproducible, and freesurfer is entirely consistent with the volumes obtained with both rater one and rater two. error bars indicate the 95 % confidence intervals inter - rater intra - class coefficients for volumetric measures figure 3 presents the relationship between stereological (r1) and freesurfer estimates of left and right thalamus volume across the entire sample of 72 subjects investigated in the present study. intra - class correlations revealed a slightly reduced level of consistency between human and freesurfer analysis of the entire sample relative to the sub - sample of ten subjects (left thalamus = 0.812, right thalamus = 0.881). mean (sd) left and right thalamic volume was 7422.4 mm (824.3) and 7390.1 mm (805.8) for stereology and 7343.6 mm (824.3) and 7388.3 mm (844.0) for freesurfer. there was no difference between the volume of the left and right thalamus across all 72 subjects using stereology (f = 0.09, p = 0.82) or freesurfer (f = 0.10, p = 0.75) (and no differences when patients and controls were separated, p > 0.80). although there were occasional differences in the direction of inter - hemispheric thalamic volume asymmetry in individual cases between stereology and freesurfer (fig . 3, right panel), this did not represent a statistically significant group effect (f = 1.46, p = 0.23).fig. 3relationship between stereological and freesurfer volume estimates of the left a and right b thalamus in the whole study sample. the relationship between left - right asymmetries determined for each individual participant by each technique is shown in c. although stereology and freesurfer determined left - right asymmetries of the thalamus in the same direction for the vast majority of subjects (lower left and upper right quandrants), there were some disassociations (top left quadrant)stereology vs freesurfer: identification of thalamic atrophy in jme relationship between stereological and freesurfer volume estimates of the left a and right b thalamus in the whole study sample. the relationship between left - right asymmetries determined for each individual participant by each technique is shown in c. although stereology and freesurfer determined left - right asymmetries of the thalamus in the same direction for the vast majority of subjects (lower left and upper right quandrants), there were some disassociations (top left quadrant) stereology vs freesurfer: identification of thalamic atrophy in jme both techniques were equally sensitive in detecting bilateral thalamic volume atrophy in patients with jme relative to controls (fig . 4). using stereology, mean (sd) left and right thalamic volume was 6843.2 mm (746.6) and 6763.3 mm (824.0) in patients with jme, and 7507.8 mm (805.6) and 7482.6 mm (767.2) in controls, respectively. volume reduction in patients was found to be statistically significant for the left (f = 5.43, p = 0.02 ) and right (f = 6.77, p = 0.01 ) thalamus compared to controls. using freesurfer, mean (sd) left and right thalamic volume was 6803.4 mm (732.8) and 6750.0 mm (714.3) in patients with jme, and 7430.7 mm (809.9) and 7491.3 mm (822.3) in controls, respectively. freesurfer thalamic volumes were similarly smaller in patients relative to controls in the left (f = 4.76, p = 0.03 ) and right (f = 7.36, p = 0.008 ) hemispheres.fig. 4volume reduction of the left (blue circles) and right (red squares) thalamus in patients with jme relative to healthy controls using stereology a and freesurfer b. error bars indicate the 95 % confidence intervals volume reduction of the left (blue circles) and right (red squares) thalamus in patients with jme relative to healthy controls using stereology a and freesurfer b. error bars indicate the 95 % confidence intervals the volume of the thalamus is a notoriously difficult metric to estimate reliably given the low contrast between thalamic gray matter and adjacent white matter on t1-weighted mr images, which is a particular challenge for automated mr image analysis methods (amini, et al . only by comparing such automated methods with manual investigator - intensive methods can we establish the reliability of volume estimates . the present study provides important data indicating the specificity and validity of automated thalamic volume estimation using freesurfer software . in particular, further to demonstrating consistency between stereological and freesurfer volume estimates of the thalamus in healthy subjects and neurological patients, we demonstrate that the extent of agreeability between stereology and freesurfer is equal to the agreeability between two human anatomists estimating thalamic volume using stereological methods . freesurfer software is now a frequently used tool for the estimation of subcortical structure volume . at the time of writing, a pubmed search using freesurfer and volume yields 87 articles ( october 2011). the vast majority of these articles are application studies, particularly in neurological disorders, and only a few have sought to evaluate the validity of volume measurements. various levels of consistency between freesurfer and manual roi methods have been reported for the hippocampus (cherbuin, et al . ; morey, et al . 2009) and striatum (dewey, et al . dewey et al . performed a series of comparisons between the fully automated techniques of freesurfer and individual brain atlases using statistical parametric mapping (ibaspm) with auto - assisted manual tracings of the hippocampus, amygdala, putamen and caudate. the authors report that freesurfer segmentations exhibited significantly higher mean spatial overlap with auto - assisted tracings in all structures compared to ibaspm using dice coefficients. we were not in a position to perform spatial overlap analyses of the thalamus given that stereology and freesurfer are two inherently distinct mr image analysis approaches. however, this is one of the primary strengths of the data presented here, insomuch that a reliable volume estimate obtained using a gold - standard (non - voxel labelling) manual approach on mr images without automated spatial transformations (i.e. in native space) is comparable to a fully automated approach that requires spatial transformations in order to label an roi and obtain a volume. our interest was with respect to the reliability of the volume estimate of the thalamus. to our knowledge, the present study is the first to independently provide data validating the application of freesurfer to obtain automated volumes of the left and right thalamus. based on the congruence between the data obtained from freesurfer and manual stereology the latter of which is considered to represent the gold standard approach due to the requirement of an expert anatomist we recommend the use of freesurfer software for accurate volumetric quantification of the thalamus using high - resolution t1-weighted mri. the removal of an expert anatomist for volumetric analyses is cost effective and time efficient, particularly in large - scale volumetric studies. importantly, we demonstrate that the automated technique is as sensitive in detecting pathological alterations of the thalamus relative to stereology, which promotes the use of freesurfer in neurological contexts. there are two additional issues that should be highlighted. the thalamus is composed of lamellae that segregate multiple nuclei with distinct connections and functions, which are likely to be differentially affected in various neurological and neuropsychiatric disorders. for example, in disorders where the thalamus is implicated in patients also exhibiting deficits in frontal lobe functioning such as in jme (pulsipher, et al . 2009)it would be expected that anterior thalamic nuclei that project to the frontal lobe would be preferentially affected (deppe, et al . in such circumstances it will be interesting to investigate structural alterations of differential thalamic subregions, which are measures that the techniques applied in the present study can not provide . there are other techniques that may provide the basis for quantitative measurements of thalamic subregions based on dti and quantitative t1 and t2 imaging ( behrens, et al . secondly, the global estimates of thalamic volume using freesurfer in the present study was obtained on a philips intera t30 3 t mri system, requiring no additional manual edits for ( obvious) incorrect labelling of the thalamic roi after the application of our in - house image inhomogeneity and resampling algorithm. different mri systems and head coils may have different image contrast characteristics that can potentially affect the performance of automated mr image analysis techniques. however, reproducibility of freesurfer estimated thalamic volume from serially acquired mr images on the same mr system is high (jovicich, et al . 2010), and mr system manufacturer has been shown to have little effect on volume estimates (jovicich, et al . 2009). in summary, this study provides convincing evidence for the reliability of global thalamic measurements using freesurfer in healthy and damaged thalami. the use of this software is cost effective and particularly advantageous in large - scale cross - sectional studies and longitudinal investigations in neurological settings. freesurfer software is publicly and freely available from the freesurferwiki resource (http://surfer.nmr.mgh.harvard.edu/fswiki/freesurferwiki), which is developed and maintained at the martinos center for biomedical imaging (http://www.nmr.mgh.harvard.edu/martinos/noflashhome.php). easymeasure software for volume estimation using stereology is freely available from the authors of this manuscript upon request.

freely available automated mr image analysis techniques are being increasingly used to investigate neuroanatomical abnormalities in patients with neurological disorders. it is important to assess the specificity and validity of automated measurements of structure volumes with respect to reliable manual methods that rely on human anatomical expertise. the thalamus is widely investigated in many neurological and neuropsychiatric disorders using mri, but thalamic volumes are notoriously difficult to quantify given the poor between - tissue contrast at the thalamic gray - white matter interface. in the present study we investigated the reliability of automatically determined thalamic volume measurements obtained using freesurfer software with respect to a manual stereological technique on 3d t1-weighted mr images obtained from a 3 t mr system. further to demonstrating impressive consistency between stereological and freesurfer volume estimates of the thalamus in healthy subjects and neurological patients, we demonstrate that the extent of agreeability between stereology and freesurfer is equal to the agreeability between two human anatomists estimating thalamic volume using stereological methods. using patients with juvenile myoclonic epilepsy as a model for thalamic atrophy, we also show that both automated and manual methods provide very similar ratios of thalamic volume loss in patients. this work promotes the use of freesurfer for reliable estimation of global volume in healthy and diseased thalami.

we report a 17-week - old female domestic shorthair cat with lumbosacral agenesis on whole - body radiographs. lumbosacral agenesis (lsa) is a rare congenital condition found in people, where segments of the lumbar spine are missing with total or partial absence of the sacrum. the exact etiology is unknown, although several theories exist, including a potential association with maternal insulin - dependent diabetes, inherited genetic mutations and failure of certain mechanisms of embryonic differentiation of parts of the vertebral column. the purpose of this case report is to describe a clinical feline case of lsa. a 17-week - old female intact domestic shorthair cat presented on emergency for acute onset of lethargy and open - mouth breathing. the owner had adopted the cat at approximately 2 weeks of age from the local shelter. the cat was paralyzed in the hind end with severe muscle wasting in both hindlimbs and ankylotic joints. the owner reported that other than hindlimb paralysis the cat was bright, alert and responsive, and appeared otherwise normal. the cat ambulated quite well on the forelimbs, urinated and defecated regularly, and always ate well. approximately 2 days prior to presentation, the cat became lethargic and lost its appetite. the cat was taken to its regular veterinarian, who identified a left - sided inguinal hernia and the cat was given 20 ml / kg of a balanced crystalloid solution subcutaneously (sc), maropitant 1 mg / kg sc, a warm soapy enema and was scheduled for surgical repair of the hernia. the cat declined rapidly at home and presented on emergency for lethargy and open - mouth breathing. physical examination on presentation revealed dull mentation, open - mouth breathing and cyanotic mucous membranes. the cat s heart rate was 200 beats per minute, with no identifiable heart murmur and the lungs were clear. the hindlimbs were noted to be ankylotic and have severe muscle wastage; they were crossed behind the body with no attempts at ambulation. the visible external genitalia appeared normal, but there was poor anal tone and no movement of the tail. there was visible malformation of ribs 12 and 13, as well as of the first and second lumbar vertebrae. there was a large colon distended with feces with a left inguinal hernia and possible bowel entrapment. shortly after radiographs were performed the cat became agonal and had an episode of respiratory arrest. there is visible malformation of ribs 12 and 13, as well as of the first and second lumbar vertebrae. there is no evidence of any other lumbar or sacral vertebrae as indicated by the arrows. there is a large colon distended with feces with a left inguinal hernia and possible bowel entrapment. lsa is a rare malformation occurring in approximately 1 of 25,000 live births in people. it is part of a group of disorders characterized by the absence of different portions of the caudal spine, otherwise known as caudal regression syndrome. newborn children exhibit similar physical examination findings to those found in this case, with severe hindlimb muscle atrophy most common. concurrent malformations in children include hydrocephalus, myelomeningocele, kidney malformations, inguinal hernia, atresi ani, rectovaginal fistulas and congenital heart defects. concurrent orthopedic abnormalities include cervical vertebral fusion particularly at c2c3, malformed ribs, hemivertebrae and kyphosis. the cat in the case reported here had a left inguinal hernia with possible bowel entrapment, no visible kidneys radiographically, and malformed ribs 12 and 13. to the owner, the cat appeared to be neurologically normal in the cranial body but an occult neurological defect can not be completely ruled out. other intra - abdominal abnormalities may have been present but, owing to the absence of a necropsy, not identified. type ii involves partial sacral agenesis with a partial but bilaterally symmetrical defect and a stable articulation between the ilia and a normal or hypoplastic first sacral vertebra. type iii is variable lumbar and total sacral agenesis with the ilia articulating with the sides of the lowest vertebra. finally, type iv is variable lumbar and a total sacral agenesis. considering this human classification as there is no veterinary classification available, the cat presented in this report would be classified as type iv. treatment in children involves either amputation of the lower extremities and long - term prothesis or spinal pelvic fusion. many children will still have some proprioception in their lower extremities so correction of limb deformities is also pursued in some. although lsa has never been reported in any veterinary species, the malformed cat shared several important lesions with perosomus elumbis (pe), which has been documented in various species. in animals with pe the hindlimbs have severe muscle wastage and the joints are ankylotic owing to the absence of nervous innervation from the absence of the coccygeal, sacral and portions of the lumbar vertebrae. this is in contrast to lsa, where patients only have agenesis of the lumbar and sacral portions of the spine. abdominal abnormalities identified in both pe and lsa include atresia ani, cryptorchidism, renal abnormalities and hernias. however, pe can lead to altered pelvic bone structure, with the pubic bone sometimes being absent, leading to pelvic narrowing; this does not occur in people with lsa. the long - term prognosis of caudal spinal malformations is usually poor as most animals are stillborn or die shortly after birth. the cat reported herein lived longer than similar reported cases in the veterinary literature; the previous reported longest survival was 12 days in a holstein calf with pe. it is unclear what caused the sudden demise of the cat but, as in pe, it appears the identification of lsa in a cat warrants a poor prognosis. the clinical presentation in this cat is similar to pe reported in other species and, as in other species, the prognosis for caudal spinal vertebral malformations appears poor.

case summarylumbosacral agenesis is a rare congenital condition reported in children. we report a 17-week - old female domestic shorthair cat with lumbosacral agenesis on whole - body radiographs. the cat was euthanized shortly thereafter presentation. a necropsy was not permitted.relevance and novel informationthis is the first reported feline case of lumbosacral agenesis.

transmissible spongiform encephalopathies are a group of neurodegenerative disorders that include scrapie in sheep, bovine spongiform encephalopathy, chronic wasting disease in elk and deer, and creutzfeldt jakob disease in humans. the infectious agent is an abnormally folded isoform of cellular prion protein (prp), designated as prp. this misfolded protein is rich in -sheet structure, insoluble, and protease - resistant and possesses a tendency to polymerize into amyloid aggregates. these fibrils accumulate as plaques in the nervous system and have been associated with the induction of neuronal death. prp is a cell surface glycoprotein expressed throughout the body but mainly found in the central nervous system of all mammals and avian species. the n - terminal region of prp is largely unstructured, while the c - terminal region has a globular structure with three -helices and two short -sheets. the physiological role of prp has not been determined; however, it has been proposed to participate in the allosteric function, signal transduction, cell the ability of prp to bind cu in vivo has led to the proposals that it may play a role in copper sensing, buffering, and/or transport. furthermore, cellular studies show that copper or zinc binding to prp induces its endocytosis. it has been established that prp can bind up to six cu ions in its flexible n - terminal region. cu coordination to the n - terminal region of prp as well as related synthetic peptides have been extensively studied. because these copper binding sites are unstructured, the study of peptide fragments has been a successful approach to studying copper binding to prp. in the octarepeat region (or), a domain comprised of four tandem repeats of eight amino acids with the sequence phgggwgq, cu sites are populated in response to the ph and cu concentration. outside the or region , two sites have been identified; these are associated with his96 and his111. the his96 cu site can adopt a 3n1o or 4n equatorial coordination mode, with a pka value of 7.8 for conversion between these species. regarding cu binding to his111, several proposals of coordination modes have been suggested. in our previous study, cu - prp complexes were characterized using different spectroscopic techniques in combination with electronic structure calculations. this study shows a ph - dependent cu - prp coordination mode with a pka value of 7.5 (structures given in scheme 1). electrochemical measurements of copper bound to the or region have concluded that the low - occupancy copper binding mode can not reduce dioxygen to hydrogen peroxide , while the high - occupancy copper binding mode can. with respect to the n - terminal his111 site, spin - trapping and nbt / formazan experiments with the prp and prp fragments suggested that these peptides are also capable of hydrogen peroxide or superoxide production in the presence of cu, ascorbic acid, and dioxygen. while the nature of the cu coordination properties of the n - terminal region of the prp protein has been widely explored, studies of its cu binding properties have been limited. unlike the other sites, the his111 binding site contains two adjacent methionine (met) residues, which could provide good ligands for cu and promote interesting redox behavior for this site. in fact, extended x - ray absorption fine structure (exafs) studies have demonstrated cu s interactions in the cu complexes of prp and prp fragments at ph 7.4. however, at this ph, the exafs spectra would have contributions from different protonation states of the complex (vide infra), obscuring the coordination mode assignments. in this study , we used different spectroscopic techniques (x - ray absorption spectroscopy and nmr) in combination with electronic structure calculations to elucidate the coordination modes involved in cu binding to his111 in the prp fragment. a detailed ph study of the cu binding properties of this fragment has allowed us to identify species that would be physiologically relevant. we have also evaluated the oxygen reactivity of these cu species, identifying specific sites for copper - catalyzed oxidation. the roles of met109 and met112 in cu and cu coordination, as well as oxygen activation have been evaluated. peptides ktnmkhmaga , ktnakhmaga , ktnmkhaaga , and ktnakhaaga were prepared by solid - phase fluorenylmethoxycarbonyl (fmoc) methods, using a fmoc - rink amide mbha resin, as previously described. all peptides were acetylated at the amino terminus and amidated in the carboxyl terminal. peptides were purified by semipreparative reversed - phase high - performance liquid chromatography (hplc). the final purity was determined by analytical hplc and was found to be > 95%. copper(ii) peptide complexes were prepared either at a 2 mm peptide concentration with 0.5 equiv of cu (at ph 6.5) or 1 mm peptide with 0.8 equiv of cu at ph 8.5, in a mixture of 20 mm 2-(n - morpholino)ethanesulfonic acid (mes) buffer with 20 mm n - ethylmorpholine (nem) and 50% glycerol; the ph was adjusted by adding small volumes of naoh or hcl. the nature of the copper(ii) peptide complexes was probed by electron paramagnetic resonance (epr) spectroscopy, yielding g and a values that are consistent with those previously reported. the copper(i) peptide complexes were obtained upon reduction of the copper(ii) peptide complexes with 100 equiv of ascorbic acid (adjusted to the corresponding ph), under anaerobic conditions. the reduced complexes were characterized by epr, and in all cases, they were found to contain less than 3% of the residual cu species (figure s1 in the supporting information, si). the cu k - edge xas data were collected at the ssrl on the unfocused 20-pole, 2.0-t wiggler beamline 7 - 3 under storage ring parameters of 3 gev and 300350 or 500 ma. a rhodium - coated premonochromator flat bent mirror was used for harmonic rejection and vertical collimation. the samples were loaded into 2 mm lucite xas cells with 38 m kapton windows and maintained at a constant temperature of 10 k during data collection using an oxford instruments cf 1208 continuous - flow liquid - helium cryostat. a canberra ge 30-element solid - state array detector was used to collect cu k fluorescence signals, using a soller slit and a zinc filter inserted between the sample and detector. internal energy calibration was accomplished by simultaneous measurement of the absorption of a copper foil placed between two ionization chambers located after the sample. the first inflection point of the foil spectrum was assigned to 8980.3 ev. extended x - ray absorption fine structure (exafs) data are reported to k = 12.8 in order to avoid interference from the zn k - edge. no photodamage was observed to cu samples, and thus all of the scans were used in the final average. each data set includes an average of 727 scans. data from cu samples were collected on four physically separate spots on the sample cells with 24 scans / spot to minimize the effect of photoreduction. the energy - calibrated and averaged data were processed by fitting a second - order polynomial to the preedge region and subtracting this from the entire spectrum as a . a three - region polynomial spline of orders 2, 3, and 3 was used to model the smoothly decaying postedge region. the data were normalized by scaling the spline function to an edge jump of 1.0 at 9000 ev. the least - squares fitting program opt in exafspak was used to fit the data. initial ab initio theoretical phase and amplitude functions were generated in feff 7.0 using calculated structures of cu models as the starting structures. atomic coordinates were adjusted as necessary as fits were improved. during the fitting process, the bond distance (r) and the mean - square deviation or bond variance in r arising from thermal and static disorder were varied for all components. the threshold energy (e0) was also allowed to vary for each fit but was constrained to the same value for all components in a given fit. coordination numbers (n) were systematically varied to provide the best chemically viable agreement to the exafs data and their fourier transform (ft) but were fixed within a given fit. the fits were evaluated based on a comparison of the normalized error (fn) of each fit along with inspection of individual fits to the data and the agreement of the fts and of individual wave components. cu - prp complexes were prepared as described above but at a lower peptide concentration (0.3 mm) and without glycerol, and the ph was varied from 3.4 to 9.2. h total correlated spectroscopy (tocsy), and h c heteronuclear single quantum coherence (hsqc) experiments were performed at ph 6.5 and 8.5. h tocsy and h c hsqc cross peaks affected by cu (0.8 equiv) were identified by comparing their chemical shift values in the absence and presence of the metal ion. all spectra were acquired at 288 k in nmr tubes sealed under a n2 atmosphere, using a bruker avance ii 600 mhz spectrometer with a triple - resonance probe equipped with z - axis self - shielded gradient coils. acquisition, processing, and visualization of the nmr spectra were performed using topspin 2.1 (bruker) and sparky. in all calculations, the structure of the prp peptide with sequence ktnmkhma was used, with an acetylated n - terminus and amidated c - terminus. the structures had a total of 141161 atoms, depending on the protonation state of the backbone amides and the number of explicit water molecules included in each model. each initial copper(i) peptide complex model was built in gaussview 4.1.2, starting from the previously reported cu models. using a restricted kohn sham (rks) approach, all copper(i) peptide structures with a spin multiplicity of 1 (singlet) were fully optimized without geometry constraints, and the stationary points of selected cu structures were characterized by a harmonic analysis (table s1 in the si). all geometry optimizations were performed using the demon2k code with the functional opbe (which combines handy and cohen s optx exchange functional with the pbe correlation functional). it should be noted that calculations at the lda level and with the nonempirical pbe functional were also performed for all models; however, only the obtained with opbe are presented here because this functional provides the best description and distances for cu s bonds. the orbital and auxiliary basis sets used were tzvp and gen - a2, respectively. solvent effects on selected optimized structures were evaluated in the demon2k code, including six explicit water molecules in the gas phase and reoptimizing with opbe and tzvp; in these cases, after reoptimization, the water molecules remained outside the coordination sphere (figure s2 in the si). implicit solvation of selected models was also calculated using the model cosmo, as implemented in the orca program. because explicit solvation with six water molecules provides a geometric description very similar to that of implicit solvation (table s2 in the si), herewith we report the corresponding to explicit solvation with six water molecules for all of the structures. the inner - sphere reorganization energy for the electron - transfer step was computed using the following expression, which depends on the energies of the oxidized (ox) and reduced (red) geometries at the selected structures that best reproduce the copper complex at ph 6.5 and 8.5:where eox(redgeom) is the energy of the oxidized state in the reduced structure and ered(oxgeom) is the energy of the reduced state in the oxidized structure. kinetic studies of the reduction of the copper(ii) peptide complexes by ascorbate were carried out on a sx20 stopped - flow system operated by the pro - data software (applied photophysics) with a 150 w xenon light source and equipped with a photodiode array for multiwavelength analysis. all experiments were performed in a single - mixing mode of the instrument, with a 1:1 (v / v) mixing ratio in a 1 cm optical path. the temperature was maintained at 25.0 0.1 c using a water bath and monitored via the internal sensor of the mixing unit. cu - prp complexes were degassed in a vacuum line and loaded into the driving syringes anaerobically. all flow lines of the instrument were extensively washed with degassed sodium dithionite and 20 mm nem / mes buffer, before charging the driving syringes with reactant solutions. the final concentration of the copper(ii) peptide complexes in the cell was 0.15 mm. the reduction was followed under pseudo - first - order conditions with a 20-fold excess of reductant, and the loss of absorbance was monitored at 570 nm (ph 8.5) or 600 nm (ph 6.5) as a function of time. the reported rate constants represent the average values and standard deviations of three independent runs. kinetic studies of the reoxidation of the copper(i) peptide complexes by oxygen were carried out on an agilent 8453 diode - array spectrometer. all experiments were performed in a screw - cap quartz cuvette with a 1 cm optical path. the cu - prp complexes at ph 6.5 were prepared by adding 1 equiv of ascorbate to a degassed solution of the copper(ii) peptide complex (0.8 mm peptide with 0.5 equiv of copper) in 20 mm nem / mes; full reduction under these conditions was confirmed by uv vis absorption. reoxidation of the cu complexes was followed by the appearance of a characteristic d d band at 600 nm after the addition of an air - saturated buffer (0.5 equiv of dioxygen). the reported rate constants represent average values and standard deviations of three independent runs. the cu - prp complexes have been spectroscopically characterized previously. the coordination mode of the cu - prp complex is ph - dependent with a pka of 7.5. at low ph, the equatorial coordination is the 3n1o mode, while at high ph, a 4n coordination mode is stabilized with a third deprotonated amide of the backbone (scheme 1). here, we use xas to evaluate these models and the role of met109 and met112 in copper coordination. the cu k - edge spectra of the cu - prp complexes at ph 6.5 and 8.5 are presented in figure 1. the extremely weak signal around 8979 ev is the 1s 3d transition, associated with the cu d ion. figure 1 shows that the spectra are not affected by the replacement of met109 and met112 by ala. thus, at both ph values, the thioether groups of the met residues are not involved in the coordination of cu. exafs fits for the data shown in figure 1 are consistent with 4n / o tetracoordinated complexes with cu ligand distances between 1.96 and 1.98 (table 1 and figure s3 in the si). these are in a good agreement with the previously proposed coordination models (scheme 1). the effect of the ph on the copper coordination is not reflected in xas because of the similar light - atom (n / o) ligands at both ph values. however, the difference between the two coordination modes (3n1o and 4n) is clearly observed by cd and epr. the reoptimized structures of the 3n1o and 4n models reported in ref were used to simulate the exafs spectra at ph 6.5 and 8.5, respectively, without any fits against the experimental data. the simulated exafs spectra show reasonable frequency agreement with the experimental data (figure s4 in the si), thus validating the proposed models for the cu - prp complexes in scheme 1. xanes (a and d), exafs (b and e), and ft (c and f) spectra of cu - prp (black line), cu - prpm09a (green line), cu - prpm112a (red line), and cu - prpm109&m112a (blue line, at ph 6.5 ( a c) and 8.5 (d f). the parameters obtained for the best fits for these data (figure s3 in the si) are listed in table 1. the goodness - of - fit fn ranges between 0.19 and 0.31 depending on the signal - to - noise ratio of the data set. the cu k - edge x - ray absorption near - edge structure (xanes) spectra of the cu - prp complexes show an intense signal assigned to the electric dipole - allowed cu 1s 4p transition at 8984 ev, characteristic of cu complexes (figures 2a, d). both xanes and exafs spectra (figure 2) of the cu - prp complexes change with the ph (figure s5 in the si), while the most drastic changes are observed upon substitution of the met residues by ala. regardless of the ph, the xanes spectra of the cu - prp complex (black line) and the m109a and m112a complexes (red and green lines) show a characteristic signal of four - coordinate complexes (figures 2a, d), while the cu complex without met residues (cu - prpm109&m112a, blue line ) displays a spectrum indicative of a two - coordinate complex. xanes (a and d), exafs (b and e), and ft (c and f) spectra of cu - prp (black line), cu - prpm09a (green line), cu - prpm112a (red line), and cu - prpm109&m112a (blue line) at ph 6.5 (a c) and 8.5 (d f). at ph 6.5, the exafs spectra (figure 2b) of the cu - prp complex change significantly upon each met - to - ala substitution, suggesting that both met109 and met112 participate in cu coordination. consistently, the fts of the exafs spectra (figure 2c) show a drastic loss in intensity in the met - to - ala variants compared to those of the cu - prp complex; the low intensity of these signals is best modeled by a lower number of sulfur atoms in the coordination sphere (see below). these changes are most dramatic for the cu - prpm109&m112a complex, consistent with the xanes . in contrast, at ph 8.5, the exafs spectra of the cu - prp complex (black line) and the single met - to - ala variants (red and green lines) are practically identical (figure 2e), while only the spectrum for the cu - prpm109&m112a (blue line), where there are no sulfur atoms available for coordination, differs from the original complex. consistently, the fts of the exafs spectra of cu - prp (black line) and the m109a (green line) and m112a (red line) variants (figure 2f) are also very similar in intensity, indicating that they have the same sulfur content in the first coordination sphere, while the spectrum of the cu - prpm109&m112a complex (blue line) shows essentially no intensity at the r value of a cu overall, the exafs indicate that at ph 8.5 only one met residue participates in cu coordination, while at ph 6.5, both met residues play an important role. consistently, a comparison of the spectra of the cu - prp complexes at ph 6.5 and 8.5 in figure s5 in the si shows differences that can be attributed to a larger contribution of sulfur atoms in the first coordination sphere at low ph. the best fits of the cu - prp complexes at ph 6.5 and 8.5 are shown in figure 3, and the corresponding parameters are listed in table 2. at ph 6.5, the best fit is achieved with two sulfur atoms at 2.37 and two nitrogen or oxygen ligands at 2.17. the four - coordinate nature of the complex is supported by a bond valence sum (bvs) analysis: the bvs value is in better agreement with a four - coordinate structure (0.98) compared to a three - coordinate fit (bvss = 0.82). at ph 8.5, the best fit of the exafs data involves only one sulfur ligand at 2.35 and three nitrogen or oxygen ligands displaying one short cu n / o bond at 1.97 and two longer cu2n / o bonds at 2.15 (table 2). again, bvs analysis is in better agreement with a four - coordinate structure (bvss = 1.10) relative to a three - coordinate complex (bvss = 0.74). exafs spectra (a and c) and their fts (b and d) of cu - prp at ph 6.5 (a and b) and 8.5 (c and d). experimental data are shown as solid black lines, and their best fits, using the parameters listed in table 2, are shown as dashed red lines. the reported goodness - of - fit fn is the normalized error given bywhere the summation is over the fitted k range, k is the photoelectron wave vector, and is the experimental or calculated data point. in order to further explore the nature of the ligands in the cu - prp complexes, 1d (h) and 2d h h tocsy and h c hsqc nmr spectra of prpm109a, prpm112a, and prp in the presence and absence of 0.8 equiv of cu at ph 6.5 and 8.5 were used to assign the signals of the h of met109 and met112, the h and h of his111, and other residues (figures s6s8 in the si). h tocsy and h c hsqc nmr spectra shows that the most affected amino acids in the presence of cu are met109, met112, his111, and lys110 (figures s6s8 in the si). figure 4 shows the aromatic (6.98.1 h ppm) and aliphatic (1.92.4 h ppm) regions of the h nmr spectra of the free peptide prp (black line) and the cu complex (red line) at ph 6.5 (figure 4a) and ph 8.5 (figure 4b). at ph 6.5, the chemical shifts associated with the h of met109 and met112 are clearly affected (figure 4a, aliphatic region) having a broadening and downfield shift (0.15 ppm of h met112 and 0.23 ppm of h met109) in the presence of cu. in agreement with xas data, nmr data indicate that cu is coordinated by both met residues at low ph. the aromatic region of the spectrum displays a broadening and an upfield shift in the h (0.12 ppm) and h (0.04 ppm) signals of his111 in the presence of cu (figure 4a). this indicates that at ph 6.5 the his111 imidazole nitrogen participates in cu binding. h nmr spectra of prp (black line) and cu - prp (red line) at ph 6.5 (a) and 8.5 (b). black lines indicate the chemical shifts for the h protons of met residues and the h and h protons of histidine in the peptide without copper. gray arrows show the shifts of these signals in the presence of cu. at ph 8.5, in the aliphatic region of the h nmr spectrum, the chemical shifts of h of met109 and met112 undergo a broadening and a downfield shift (0.12 ppm of h m112 and 0.21 ppm of h met109) upon cu binding (figure 4b). in the aromatic region, signals of the h and h of his111 also show broadening and downfield shift. the effect of cu on the signals of the h and h of his111 suggests that at ph 8.5 the his111 imidazole nitrogen participates in cu binding. however, the line broadening observed at ph 8.5 in both regions is much more dramatic than that observed at ph 6.5. such large signal broadening may be related to the presence of two species at ph 8.5, one with the sulfur atom from met109 coordinating cu and another complex with the sulfur atom from met112. this interpretation is consistent with the behavior of the met - to - ala variants observed in xas, where the only sulfur available in each variant is found to coordinate cu. in summary, our nmr data corroborate the involvement of sulfur atoms from met109 and met112 of the prp fragment in the coordination of cu, while they clearly indicate that the his111 imidazole nitrogen also binds to the metal ion. to further explore the role that met109, met112, and his111 play on the coordination with cu, we tested both the prp peptide and the cu - prp complex by h nmr spectroscopy at several ph values ranging from 3.5 to 9.5. figure 5a shows the behavior of h (dashed black line) and h (solid black line) of his111 in the free peptide and in the cu - prp complex (dashed and solid red lines). the pka associated with the free histidine is 6.2, while in the presence of cu, the pka of histidine shifts to 5 because of the coordination of his111 to cu in the cu - prp complex. this shift is consistent with that observed in cu complexes in blue copper proteins. the signals associated with the met residues in the cu - prp complex also change drastically with the ph. figure 5b shows in green (h met109) and red (h met112) dots chemical shifts of h of met109 and met112 in the presence of cu at several ph values (3.59.5). the chemical shift of these protons is 2.10 ppm in the free peptide (figure 4), and they do not change with the ph (data not shown). however, in the presence of cu, even at a ph as low as 3.5, the signals associated with h of met109 and met112 are both shifted to 2.29 ppm, indicating coordination to cu. also, at higher ph values (above 8.5), both signals show a tendency to return to the chemical shift associated with the free peptide (2.10 ppm). this indicates that, at higher ph values, both met residues leave the coordination sphere of cu, leading to a species without thioether ligands; such species would not be physiologically relevant and were not further characterized. (a) chemical shifts of the h and h protons of his111 in the free peptide (dashed and solid black lines) and in the cu - prp complex (dashed and solid red lines) as a function of the ph. a shift in the pka of his111 is observed in the presence of cu. (b) chemical shifts of the h protons of met109 (green dots) and met112 (red dots) in the cu - prp complex as a function of the ph. the ph - independent chemical shift of these protons is 2.10 ppm in the free peptide. these nmr data provide further insight into the role of met109, met112, and his111 in the coordination of cu to the prp fragment as a function of the ph. at very low ph (3.5) , cu is coordinated by both met109 and met112. with a pka of 5 , the nitrogen imidazole from his111 enters the coordination sphere, while the two met residues remain coordinated. as the ph is further increased, the met residues play a less important role in cu coordination, and only one met residue (met109 or met112) coordinates cu at ph > 8 (scheme 2). at ph < 5, cu is anchored by both met residues, while at a ph > 5, the his111 imidazole also participates in cu coordination. at ph > 8, the box in scheme 2 shows the structures for the cu - prp complexes under extracellular conditions, derived from the exafs and nmr data. in order to gain further insight into the nature of the unidentified n / o ligands in these cu complexes, several models were built and rks calculations were performed, using the cu models previously described as starting points. the copper ion was reduced and three groups of ligands were evaluated (table s3 in the si). group 1 included four - coordinate cu-1n2s1x models for the complex formed at ph 6.5, with two sulfur atoms from both met residues (met109 and met112), a his111 imidazole nitrogen, and a fourth ligand x, where x could be an oxygen atom from a backbone carbonyl group, a solvent h2o molecule, or a nitrogen atom from a backbone amide in a protonated (n) or deprotonated form (n) (including those of the most affected amino acid residues in the 2d tocsy and hsqc experiments ; figures s6s8 in the si). because at ph 8.5 the exafs indicate that one met is coordinated while nmr data show that both met residues are affected, group 2 included four - coordinate cu-1n1s2x models, where the sulfur atom is provided by either met109 (group 2a) or met112 (group 2b), his111 imidazole provides a nitrogen ligand, and 2x could be any of the following combinations: 2n, 1n1n, 2n, 1n1o, 1n1o, or 2o (described above). it should be noted that, because the xanes data indicate that cu complexes are four - coordinate, only the structures that optimized as four - coordinate complexes were analyzed in each group. five structures optimized as four - coordinate complexes in group 1 (table s3 in the si); these are shown in figure 6, and relevant geometric parameters with and without solvent effects are listed in table s4 in the si. the group 1 models are divided into two subgroups, according to the total number of atoms (160 or 161), which depends on the protonation state of the backbone amides. in this group, when x is a deprotonated amide (2n2sa and 2n2sb), the average cu s bond distances (2.365 and 2.34) are in good agreement with the experimental values; however, the two cu n bonds are quite short (2.092.10). when x is a backbone carbonyl (1n1o2sa and 1n1o2sb) or a water molecule (1n1o2sc1), the average cu s bond lengths (2.35, 2.34, and 2.36) and cu n / o distances are in good agreement with the experimental data (2.17, 2.145, and 2.16 ; table s4 in the si). however, the 1n1o2sb model is discarded because it is the structure that has the highest energy in this subgroup, 13.32 kcal / mol above 1n1o2sa. energy diagram of optimized four - coordinate cu-1n2s1x models for the cu - prp complex at ph 6.5 (group 1), with explicit solvent. most side chains are not shown for clarity; however, the geometry optimizations were done with the complete cu - prp complex. thus, the best candidates for the ph 6.5 cu structures (group 1) are the 1n1o2sa and 1n1o2sc1 models (figure 6) because they display metal ligand distances that best reproduce the experimental data in trends and values. furthermore, using 1n1o2sc1 to simulate an exafs spectrum using the exafspak program yielded a spectrum that was in good intensity agreement (but indicating an overall longer distance) with the experimental data for cu - prp at ph 6.5 (figure s9 in the si). therefore, at ph 6.5 the best model to represent the cu - prp complex has two sulfur atoms from met109 and met112, a his111 imidazole nitrogen, and an oxygen atom as the fourth ligand, which could be the carbonyl oxygen atom from his111 (as in model 1n1o2sa) or a water molecule (as in model 1n1o2sc1). in these models, the sulfur atom can be provided by met109 (group 2a) or met112 (group 2b). the group 2 models are divided into two subgroups, according to the total number of atoms (159 or 160), which depends on the protonation state of the backbone amides. five structures optimized as four - coordinate complexes in group 2a and only four in group 2b (table s3 in the si). the structures are shown in figure 7, and their geometric parameters with and without solvent effects are listed in tables s5 and s6 in the si. energy diagram of optimized four - coordinate cu-1n1s2x models for the cu - prp complexes at ph 8.5, with explicit solvent and either met109 (group 2a) or met112 (group 2b) as the ligand. most side chains are not shown for clarity; however, the geometry optimizations were done with the complete cu - prp complex. for the models with met109, when 2x is a combination of two deprotonated amides (3n1sm109a and 3n1sm109c), only the 3n1sm109c model yields cu ligand distances that are in agreement with the experimental trend, one short cu n and two long cu n bonds (2.04 and 2.13 , respectively) and a cu s bond distance at 2.36 (table s5 in the si), although it is the highest - energy structure of the group (figure 7). in contrast, in the models where 2x is the combination of a deprotonated amide and a backbone carbonyl (2n1o1sm109a, 2n1o1sm109b, and 2n1o1sm109c), the cu n / o bonds are reproduced in the 2n1o1sm109a model (2.32, 2.02, and 2.15 , respectively ; table s5 in the si), which is the lowest - energy structure in this subgroup. thus, we propose that the best model for the cu - prp complex at ph 8.5 with a sulfur atom from met109 is 2n1o1sm109a, with a nitrogen atom from the deprotonated amide of lys110 and an oxygen atom from the carbonyl of his111. the cu-1n1s2x models with the met112 ligand (figure 7 and table s6 in the si), having 2x as a combination of two deprotonated amides (3n1sm112a and 3n1sm112c), yield a cu s bond at 2.282.30 and three similar cu n bonds (2.032.04), which is not consistent with the experimental trend. on the other hand, when 2x is a combination of a deprotonated amide and a backbone carbonyl (2n1o1sm112a and 2n1o1sm112d), the 2n1o1sm112d model leads to a three - coordinate structure, while the cu ligand distances in the 2n1o1sm112a model show excellent agreement with the experimental data: a short cu n / o bonds (2.17), and a cu s bond at 2.34 (table s6 in the si). therefore, the best model for the cu - prp complex with met112 at ph 8.5 is the 2n1o1sm112a structure. overall, the best models for the cu - prp complex at ph 8.5 are 2n1o1s structures with a nitrogen from the deprotonated backbone amide of lys110 and an oxygen from the backbone carbonyl of his111, regardless of which met participates in the coordination shell. figure 8 summarizes the from dft modeling, showing the best models for the cu and cu complexes with prp at ph 6.5 and 8.5. it is clear that the cu complexes (cu-3n1o and cu-4n) have very different geometries and coordination spheres than their reduced counterparts (cu-1n1o2s and cu-2n1o1s). we calculated the inner - sphere reorganization energies associated with the reduction of the cu species to cu, at ph 6.5 and 8.5. for the low - ph structures cu-3n1o and cu-1n1o2s, the calculated i is 1.79 ev when the oxygen ligand is the backbone carbonyl of his111 and 2.15 ev when it is a water molecule. at ph 8.5, the cu-4n and cu-2n1o1s structures yielded a reorganization energy of 1.60 ev when met109 is bound and 1.66 ev for the met112 structure (table s7 in the si). these are necessary for evaluating the reduction and o2 reactivity in the next section. dft - derived models for cu and cu complexes with the prp peptide at different ph values. for the cu complex at ph 6.5, the best model has a 1n1o2s coordination sphere, where both met residues are bound to copper and the oxygen atom can be the carbonyl oxygen from his111 (as shown here) or a water molecule. at ph 8.5, the best models are 2n1o1s structures with a deprotonated amide of lys110, an oxygen atom from the backbone carbonyl of his111, and either met109 or met112 as the sulfur ligand. once the geometries and electronic structures of the cu - prp complexes had been elucidated, we evaluated the role of met109 and met112 on the reactivity of these complexes. first, the kinetics of reduction of the cu - prp complexes with ascorbate was studied, followed by reactivity studies of the ant cu - prp complexes with dioxygen. the reduction of both complexes cu-3n1o at ph 6.5 and cu-4n at ph 8.5 by ascorbate was examined by stopped - flow absorption spectroscopy at 25 c. electronic absorption at the wavelength for the maximum intensity of the ligand - field transitions (i.e., 600 nm at ph 6.5 and 570 nm at ph 8.5) was followed over time after rapid mixing of the cu complexes with the reductant. the reduction was followed under pseudo - first - order conditions, using at least a 20-fold excess of the reductant over the copper complex concentration. plots of the absorption intensity at 600 and 570 nm as a function of the reaction time are shown in figure 9, while representative data for the absorption spectra are shown in the insets; fits to the kinetic traces are summarized in table 3. the reduction of the cu-3n1o complex at ph 6.5 can be fitted to a single - exponential equation to obtain kobs = 0.287 0.010 s (figure 9a). in contrast, the reduction of cu-4n complex at ph 8.5 exhibited a slow biphasic behavior (figure 9b). the kinetic trace for cu-4n can be fitted to a double - exponential equation, yielding rates of 0.134 0.023 and 0.010 0.004 s. thus, even the fast phase of reduction of the cu-4n complex at ph 8.5 is 2-fold slower than reduction of the cu-3n1o complex at ph 6.5 (table 3). considering the reorganization energies calculated for these two complexes, this could be due to a difference in the reduction potentials of the cu-3n1o and cu-4n species, which would in different driving forces for the two reactions. using anaerobic reductive titrations of the cu complexes at ph 6.5 and 8.5 with ascorbate, we estimate a reduction potential of 152 mv for the cu-3n1o complex and 75 mv for the cu-4n complex. thus, a significant difference in g for their reduction by ascorbate is expected {g = = 3.52 kcal / mol}. using the semiclassical marcus equation for the rate of intermolecular electron transfer and considering the differences in the reorganization energies and reduction potentials, the ratio of the reduction rates for cu-3n1o and cu-4n species is calculated to be k3n1o / k4n = 2.3 (see the si), which is consistent with the experimental values. thus, the difference in the reduction potentials for these two complexes in a faster reduction for the cu-3n1o complex compared to that for the cu-4n species. stopped - flow absorption data for the reduction of the cu - prp complex at ph 6.5 (3n1o complex) (a) and at ph 8.5 (4n complex) (b) with 20 equiv of ascorbate. d transitions as a function of time, are shown in each inset. in order to evaluate the effect of met109 and met112 on the rate of reduction of the cu-3n1o complex , the reduction of the cu - prpm109a, cu - prpm112a, and cu - prpm109&m112a complexes by ascorbate at ph 6.5 was also studied; representative kinetic traces are shown in figure s10 in the si, and the reduction rates are summarized in table 3. within experimental error, cu - prpm109a, cu - prpm112a, and cu - prpm10&m112a exhibit a reduction rate constant that is the same as that of the cu - prp complex. these suggest that the met residues are not involved in the rate - limiting step of the reduction of the cu - prp complex at ph 6.5. thus, it is likely that this involves electron transfer, followed by a slower rearrangement of the peptide, to provide the met ligands to form the ph 6.5 cu-1n1o2s complex. we next investigated the first step of dioxygen activation by the cu - prp complexes. reduced complexes of prp, prpm109a, prpm112a, and prpm10&m112a were mixed with an oxygenated buffer, and the reaction was followed by uv vis absorption spectroscopy. d transition, indicating cu oxidation, while the reoxidation rate for each copper peptide is listed in table 4. the end products of the reaction of cu - prp complexes with oxygen were spectroscopically characterized by cd and epr; in all cases, at least 50% of the original cu complexes were recovered (figure s11 in the si). also, analysis of the samples by matrix - assisted laser desorption / ionization time of flight / time of flight (maldi - tof / tof) showed metal - catalyzed oxidation for the prp, prpm109a, and prpm112a peptides (figure s12 in the si). in particular, for the prp peptide, two new signals with m / z peaks corresponding to the mass of the peptide, + 16 and + 32 da, were observed, while single - variant peptides only show one + 16 da peak. in contrast, the double - variant prpm109&m112a peptide, which contains no met residues, suffered no modification. these indicate that redox cycling of these copper peptide complexes in oxidation of the met residues into sulfoxides, while no evidence was found for their oxidation to sulfone. kinetic traces for the reoxidation by dioxygen of the cu - prp complex (black) and the variants cu - prpm109a (green), cu - prpm112a (red), and cu - prpm109&m112a (blue) at ph 6.5, followed by uv vis absorption spectroscopy at 600 nm. inset: representative absorption data showing the increase in the intensity of the ligand - field transitions for the cu complex as a function of time. the reoxidation of cu - prp by dioxygen may follow inner- or outer - sphere electron - transfer pathways. in an outer - sphere mechanism, the cu complex would be oxidized by dioxygen in a single step to regenerate the cu complex, while an inner - sphere reaction would involve binding of dioxygen to the cu complex and formation of a potential copper(ii) superoxide intermediate, followed by the release of superoxide to regenerate the cu complex. the possibility of outer - sphere reoxidation of the cu - prp complexes was evaluated using the marcus equation to analyze the rates in table 4. using an estimated reduction potential for the copper peptide complex of 150 mv, the potential of one - electron reduction of dioxygen to o2 (165 mv vs nhe), and the calculated reorganization energy of the site, the outer - sphere electron - transfer rate from the cu complex at ph 6.5 to the oxygen molecule is estimated to be at most 2.81 10 s (see the si), which is 1010 times slower than the experimental rate of cu reoxidation from the kinetic data (3.6 10). thus, single - electron transfer from cu - prp to dioxygen likely proceeds via an inner - sphere pathway that would involve formation of a copper(ii) superoxide species. this is consistent with a previous study that showed the formation of superoxide species upon redox cycling of the cu - prp complex and with our finding that the met residues are oxidized to sulfoxides upon redox cycling of the cu - prp complex. it is important to note that the reoxidation rate shows an inverse correlation to the number of met residues in the initial cu complex, following the trend cu - prp < cu - prpm109a cu - prpm112a < cu - prpm109&m112a (table 4). the reoxidation rate of the cu - prpm109&m112a complex is 6 times faster than those of the single variants and 10 times faster than that of the cu - prp complex containing both met residues. these suggest that the presence of met ligands at the cu complex affects the rate - limiting step in the reoxidation process, which likely involves formation of the copper(ii) superoxide species. xanes data clearly show that, in the absence of met residues, the cu - prpm109&m112a complex is two - coordinate, while the cu - prp complex is four - coordinate (vide supra). thus, in an associative mechanism for formation of the copper(ii) superoxide species, a faster reaction would be expected for the cu - prpm109&m112a complex because of its coordination unsaturation, as observed. the nature of the mechanism (associative vs dissociative) for the inner - sphere oxygen reaction of the four - coordinate cu - prp complex requires further computational evaluation. cu - prp complexes at ph 6.5 and 8.5 were studied by xas, and the role of met109 and met112 in cu coordination was evaluated using peptides with met - to - ala variants. our xas show that cu coordination to his111 occurs through nitrogen and oxygen atoms at low ph, and all nitrogen ligation at high ph (pka = 7.5), which is consistent with the previously proposed coordination models, as shown in scheme 1, while the possibility of met residues acting as ligands for cu can be discarded. xas and nmr spectroscopy, in combination with electronic structure calculations, show that cu coordination to prp is also ph - dependent (scheme 2). at ph < 5, the thioether groups of met109 and met112 bind cu; with a pka of 5, the nitrogen imidazole from his111 enters the coordination sphere, while both met residues remain coordinated, adopting a 1n1o2s coordination mode. at ph 8.5, two cu species with 2n1o1s (2nosm109 and 2nosm112) coordination modes are formed, with a single met residue bound to cu (scheme 2). although this study has used a short fragment of prp that only includes his111, the 2s and 1n1o2s species described here would form in longer prp protein chains. at low ph (below 5) , all his residues would be protonated and met109 and met112 would dominate cu coordination to form the 2s species. at higher ph, other n - terminal his residues, such as his96, could participate in cu binding; however, a recent study of cu and ag coordination to the 91127 fragments demonstrated that a helical structure at the hydrophobic region of the 91127 fragments causes his96 to fall out of the coordination sphere, leaving his111 as the main copper anchoring site; thus, the 1n1o2s complex described herein would be the dominant species. prp is a synaptic glycolipid - anchored membrane protein predominantly expressed on presynaptic membranes, where it is exposed to cu ions in a range of concentrations: from 15 m at the synaptic cleft during synaptic vesicle release up to 100300 m during neuronal depolarization. thus, prp can interact with cu ions in the extracellular space, and at ph 7.4, there is a mixture of two species 3n1o and 4n (scheme 1 and figure 11a). also, biological reductants, such as ascorbic acid, are found in concentrations of 150 m in the cerebrospinal fluid. thus, cu - prp complexes anchored at his111 in the synaptic cleft may be reduced to cu by available reducing agents. our indicate that, in the presence of ascorbate, the cu-3n1o complex would reduce faster to generate a cu-1n1o2s complex. therefore, at physiological ph, 3n1o and 4n complexes are the relevant cu species, while the 1n1o2s complex is the relevant cu coordination mode at the his111 site in prp (figure 11b). schematic representation of the main cu - prp complexes under different physiological conditions. (a) at low cu concentration (nm), cu would be anchored at the or (cu - or) and at his96 and his111 sites (cu-3n1o and cu-4n). (b) cu complexes in the presence of reducing agents would be reduced. at the his111 site, (c) high copper concentrations (100500 m) can stimulate endocytosis; in the endosomes, only the his111 site would coordinate cu through met109 and met112. (d) in the extracellular space, oxygen activation by copper bound to his111 could generate superoxide, causing the subsequent oxidation of met residues. footnotes: a, ref; b, ref; c, ref; d, this work; e, ref; f, ref. unlike all other copper binding sites in the human prp protein, the his111 binding site contains the mkhm motif, which provides it with unique coordination and redox properties. mx2mx2 m motifs have been identified in copper - transport proteins (e.g., yeast yctr1) that bind cu selectively via met residues with affinities in each motif on the order of 2.5 10 m at ph 4.5. on the other hand, several proteins involved in copper transport that utilize his in combination with met residues for cu binding have emerged, such as pcoc with a cu-(his)(met)2 coordination mode, copc , and cusf. in these cases, the presence of the his residue in the coordination sphere plays two roles: (i) to increase the affinity for cu and (ii) to enable the coordination of cu ions. furthermore, it has been demonstrated that thioether ligands can be particularly effective at chelating cu when they are separated by no more than two amino acids. the mkhm motif in the human prp sequence fulfills these conditions, such that at physiological ph cu can be anchored via the his residue, while upon reduction, the cu ion is bound by his and met residues. high cu concentrations stimulate endocytosis of the prion protein, taking the protein from the extracellular space at ph 7.4 to the endosomal space, where there is a high concentration of protons (ph 45) and biological reductants, such as glutathione or nicotinamide adenine dinucleotide (nadh), that are found in concentrations of 13 mm and 200 m, respectively. under these conditions , his111 would be protonated and cu would be anchored only by met residues, as illustrated in figure 11c. thus, this site is optimized to chelate both cu and cu ions and is likely to be involved in cu transport into the cell. cu and cu bound to prp form complexes with very different geometries and coordination spheres: cu-3n1o versus cu-1n1o2s at ph 6.5 and cu-4n versus cu-2n1o1s at ph 8.5, ing in large reorganization energies of the site upon reduction of 1.79 and 1.6 ev, respectively. in spite of its higher reorganization energy, reduction of the cu-3n1o complex is faster than that of the cu-4n species because of its higher reduction potential. considering that, at physiological ph, cu-3n1o and cu-4n complexes are present, our indicate that, under reducing conditions, the cu-3n1o complex would reduce faster, shifting the equilibrium between 3n1o and 4n toward the cu-3n1o complex and favoring production of the cu-1n1o2s complex. overall, the reorganization energies for the cu - prp complexes are large compared to those of copper sites that are optimized to support electron transfer (blue copper t1 and cua sites), which range from 0.5 to 0.82 ev. despite its large reorganization energy, the cu - prp complex still reacts with dioxygen. several studies regarding the redox activity and functional implications of cu - prp complexes have been put forward. some studies have reported the generation of reactive oxygen species (h2o2 or superoxide) by cu - prp complexes; however, little is known about the mechanism of dioxygen activation by the different cu - prp complexes. this study investigated the kinetics of the first step of dioxygen activation by the cu-1n1o2s complex at ph 6.5. the estimate for the rate of outer - sphere reoxidation is 10 times slower than the experimental rate of cu reoxidation from the kinetic data. thus, we conclude that the cu-1n1o2s complex at the his111 site reacts with dioxygen in an inner - sphere electron - transfer pathway that involves binding of dioxygen to the cu complex and formation of a copper(ii) superoxide intermediate, followed by the release of superoxide and partial regeneration of the cu complex. our study also shows that the met residues can be selectively oxidized to sulfoxide by the copper - catalyzed reaction (figure 11d). it has been proposed that copper - catalyzed oxidation of met residues may trigger a structural transition, leading to aggregation of prp protein, or it may interfere with conversion of the prion protein into the fibrillar proteinase k - resistant conformation. beyond that, met residues in prp may act as an innate antioxidant defense in the protein by their ability to scavenge the produced superoxide and undergo oxidation to form methionine sulfoxide. without the met residues, the enzymatic peptide methionine sulfoxide reductase (msra) reverses methionine sulfoxide back to met, which once again is able to scavenge oxidants. in fact, in the alzheimer s disease brain, a decrease in the activity of msra compared to control subjects was observed, while the induction of msra activity protects neuronal cells from amyloid toxicity. it is plausible that a reversible oxidation / reduction of met residues at the n - terminal region of the prp protein could be acting as an ros sink. this would be consistent with the notion that prp plays a role in cellular antioxidant defense. in summary, our study shows that cu binding to his111 is highly ph - dependent and that the presence of one his and two met residues in the mkhm motif of the human prp fragment confers this site with unique cu binding properties. even upon drastic changes in the chemical environment, in particular those occurring during endocytosis, the two met residues in the mkhm motif allow prp to keep cu ions anchored, consistent with a copper - transport function for this protein. on the other hand, in the extracellular space, in the presence of reducing agents, the most populated cu - his111 complex would be a cu-1n1o2s species, which is capable of activating dioxygen. our study provides further insight into the molecular mechanism of oxygen activation by this site. the cu-1n1o2s complex reacts with dioxygen through an inner - sphere mechanism, likely involving the formation of a copper(ii) superoxide intermediate, followed by the release of superoxide and partial regeneration of the cu complex. the met residues are oxidized to sulfoxide in this process, and their ability to scavenge superoxide may play a role in the proposed antioxidant properties of prp.

the ability of the cellular prion protein (prpc) to bind copper in vivo points to a physiological role for prpc in copper transport. six copper binding sites have been identified in the nonstructured n - terminal region of human prpc. among these sites, the his111 site is unique in that it contains a mkhm motif that would confer interesting cui and cuii binding properties. we have evaluated cui coordination to the prp fragment of the human prp protein, using nmr and x - ray absorption spectroscopies and electronic structure calculations. we find that met109 and met112 play an important role in anchoring this metal ion. cui coordination to his111 is ph - dependent: at ph > 8, 2n1o1s species are formed with one met ligand; in the range of ph 58, both methionine (met) residues bind to cui, forming a 1n1o2s species, where n is from his111 and o is from a backbone carbonyl or a water molecule; at ph < 5, only the two met residues remain coordinated. thus, even upon drastic changes in the chemical environment, such as those occurring during endocytosis of prpc (decreased ph and a reducing potential), the two met residues in the mkhm motif enable prpc to maintain the bound cui ions, consistent with a copper transport function for this protein. we also find that the physiologically relevant cui-1n1o2s species activates dioxygen via an inner - sphere mechanism, likely involving the formation of a copper(ii) superoxide complex. in this process, the met residues are partially oxidized to sulfoxide; this ability to scavenge superoxide may play a role in the proposed antioxidant properties of prpc. this study provides further insight into the cui coordination properties of his111 in human prpc and the molecular mechanism of oxygen activation by this site.

the majority of chromosomal rearrangements in acute myeloid leukemia (aml) in fusion genes or position effects. indeed, according to the world health organization classification of hematopoietic tumors, a consistent group of aberrations is associated with specific aml subtypes, with both diagnostic and prognostic significance. in this report , we describe a case of aml (fab m5 subtype) in which the karyotype displayed a novel t(2;10)(q31;p12) balanced translocation as the sole cytogenetic abnormality in a bone marrow cell clone at onset. in november 2007, a 42-year old man was admitted to the department of hematology and oncological sciences of the sergnoli institute, bologna, northern italy. peripheral blood count showed white blood cells 81.710/l with 9% neutrophils and 84% blast cells, hemoglobin 12 g / dl, and platelets 8910/l. bone marrow aspirate was hypercellular and showed a wide population of medium and large sized cells characterized by a high nuclear / cytoplasmatic ratio and basophil cytoplasm. immunophenotyping identified a cd13, cd15, cd33, cd117, mpo7 population with aberrant expression of cd19. bone marrow biopsy confirmed a widespread infiltrate with cd68 (pgm1) positive blast cells. mutational analysis was negative for flt3-itd, flt3-tkd and npm1. in december 2007, induction chemotherapy with fludarabine (50 mg / die for 5 days), cytosine arabinoside (ara - c) (4 g / die for 5 days), idarubicin (20 mg / die on days 1, 3 and 5) followed by gemtuzumab - ozogamicin infusion (5 mg on day 6) was started and a morphological and cytogenetic remission was achieved. the patient later received a first consolidation cycle with idarubicin and ara - c, and a second consolidation cycle with high - dose ara - c. in may 2008, bone marrow examination revealed an initial relapse, confirmed by the cytogenetic analysis showing karyotype 47,xy,+8/46, xy. reinduction chemotherapy was started but the patient showed resistance to conventional chemotherapy. in june 2008, he started therapy with tipifarnib and bortezomib, but he experienced disease progression with peripheral blastosis, anemia and thrombocytopenia. fluorescence in situ hybridization analysis mapped the chromosome 2 breakpoint within rp11 - 25l17 (chr2:177,938,840178,096,310), precisely within the non - overlapping region (approx . 70 kb) of this clone with rp11 - 28m17 (chr2:178,009,023178,198,838) (figure 1a). the breakpoint was then located upstream from the heterogeneous nuclear ribonucleoprotein a3 (hnrnpa3) gene, encoding a protein involved in alternative splicing, and the nuclear factor erythroid 2-like 2 (nfe2l2) gene, encoding a basic leucine zipper (bzip) transcription factor. interestingly, both genes had been previously described as deregulated in some tumor types. on chromosome 10, the breakpoint was identified by the splitting signals of rp11 - 49d12 (chr10:28,369,83728,539,930) on both der and der (figure 1a). it encompassed the palmitoylated membrane protein 7 (mpp7) gene, encoding a member of the p55 subfamily of maguk proteins. figure 1a ) left: fluorescence in situ hybridization (fish) obtained with bacterial artificial chromosome clones delimiting the breakpoint regions on der and der. right: maps of the breakpoint regions in chromosome bands 2q31.2 (top) and 10p11.23 (bottom), according to the latest release of the ucsc human genome browser (grch37/hg19) (february 2009). the reported clones have the same color code as the fish image on the left. b ) expression analyses of exons 6 and 14 of mpp7 (red bars), exon 5 of hnrnpa3 (green bar), and exon 4 of nfel2l2 (yellow bar) evaluated by real - time quantitative polymerase chain reaction, in the present case , 4 control akute myeloid leukemia (aml) m5 samples, the mean ct value of the controls (mean value), and normal bone marrow. the showed comparable genes transcriptional levels in the patient with t(2;10) compared with the mean ct value of the 4 m5 aml controls. a ) left: fluorescence in situ hybridization (fish) obtained with bacterial artificial chromosome clones delimiting the breakpoint regions on der and der. right: maps of the breakpoint regions in chromosome bands 2q31.2 (top) and 10p11.23 (bottom), according to the latest release of the ucsc human genome browser (grch37/hg19) (february 2009). genes are indicated by yellow bars. the reported clones have the same color code as the fish image on the left. b ) expression analyses of exons 6 and 14 of mpp7 (red bars), exon 5 of hnrnpa3 (green bar), and exon 4 of nfel2l2 (yellow bar) evaluated by real - time quantitative polymerase chain reaction, in the present case , 4 control akute myeloid leukemia (aml) m5 samples, the mean ct value of the controls (mean value), and normal bone marrow. the showed comparable genes transcriptional levels in the patient with t(2;10) compared with the mean ct value of the 4 m5 aml controls. neither of these two breakpoint regions has ever been described as being involved in tumor - associated chromosome rearrangements. to assess the possible impact of this translocation on the hnrnpa3, nfe2l2, and mpp7 genes, real - time quantitative polymerase chain reaction assays were performed on the patient's bone marrow (bm) rna, and compared to 4 aml m5 control cases (without the translocation), as well as to normal bm. we used three reference genes (hprt1, ywhaz, and sdha) and the mean expression value of the control amls as calibrator. the obtained showed that there was no statistically significant change in the expression of any of the genes investigated (figure 1b). to summarize, we describe for the first time a novel, non - recurrent t(2;10)(q31;p12) translocation in aml which did not lead to any gene fusion or position effects. remarkably, the molecular consequences of this translocation are to be found outside the breakpoint regions' gene domains. we might speculate that the chromatin relocation due to the t(2;10)(q31;p12) rearrangement might have influenced the expression pattern of additional genes, mapping along both derivative chromosomes 2 and 10. however, we were not able to evaluate this because of the limited amount of rna material available. it is not possible to draw clear about the possible clinical impact of this translocation, also because the rearrangement was not present at relapse. however, on the other hand, the t(2;10)(q31;p12) was the only chromosomal aberration observed in the karyotype of a cell clone in the patient's bone marrow at onset, suggesting it might have an impact on leukemogenesis. notably, the patient was negative for flt3 and npm1 mutations, excluding the possibility that this translocation might be a secondary event to this type of alteration. the study of further aml cases with t(2;10)(q31;p12) would allow us to gain a better understanding of the clinical and molecular impact of this translocation on patient outcome.

we describe a case of acute myeloid leukemia m5 showing a balanced t(2;10) (q31;p12) translocation. this has never been described before as the sole cytogenetic abnormality in a bone marrow cell clone at onset. using fluorescence in situ hybridization with properly designed bacterial artificial chromosome probes, we mapped the breakpoint regions on both derivative chromosomes 2 and 10: der and der, respectively. the mpp7 gene, disrupted by the breakpoint on chromosome 10, was juxtaposed upstream of both hnrna3 and nfe2l2 genes on chromosome 2, without the formation of any fusion gene. using real - time quantitative polymerase chain reaction , we tested the possible disregulation of any of the breakpoint - associated genes as a consequence of the translocation, but we found no statistically significant alteration. considering the potential role of this clonal cytogenetic abnormality in leukemogenesis, we speculate that this translocation could have an impact on additional genes mapping outside the breakpoint regions. however, the limited amount of rna material available prevented us from testing this hypothesis in this present case.

criss - cross heart (cch), or superoinferior ventricles, is a complex congenital rotational abnormality in which the systemic and pulmonary venous streams cross at the atrioventricular (av) level without mixing. its frequency is less than 8/1000000 and accounts for < 0.1% of congenital heart defects (chd). in the normal heart, av structures are parallel to each other when viewed from the front, whereas in cch the av structures are not parallel but angulated by as much as 90 degrees.14 the atrium connects with the contralateral ventricle and the ventricular chambers are arranged in a superoinferior fashion, with the right ventricle (rv) superiorly and the left ventricle (lv) inferiorly located, regardless of whether the av connection is concordant or discordant. the diagnosis is made by using 2-dimensional and color doppler echocardiography.35 here, we report a rare case of cch with concordant atrioventricular connections with dorv which was diagnosed by echocardiography. a 2 month old female infant, weighing 3.7 kg presented with history of breathlessness, feeding difficulty and cyanosis. she was a second product of non - consanguineous marriage and born by normal vaginal delivery. respectively. blood pressure was recorded at 75/35 mmhg and oxygen saturation 85% in room air. cardiac auscultation revealed normal first and second heart sound along with a grade 3/6 ejection systolic murmur audible best at the left upper parasternal area. the two - dimensional echocardiography, subcostal coronal views demonstrated the connection of the left - sided left atrium and the right - sided left ventricle through the mitral valve and the right - sided right atrium to be connected to the left - sided right ventricle through the tricuspid valve by anterior angulation of the transducer. it also shows the two great arteries arising from the right ventricle (figure 1). subcostal coronal views shows the connection of the right - sided ra to be connected to the left - sided rv through the tricuspid valve, inlet vsd and the two great arteries arising from the rv. short - axis views shows the right ventricle was superior and left ventricle was inferior with horizontal position of the ventricular septum (figure 2). short - axis views shows the superior (rv) and inferior (lv) ventricle with horizontal position of the ventricular septum. the standard 4-chamber view was not showing simultaneously all four chambers and both atrioventricular valves (figure 3). a large size inlet type of ventricular septal defect (bidirectional shunt) and a moderate size ostium secundum atrial septal defect (bidirectional shunt) were present. cch is a complex congenital anomaly produced by the rotation of ventricular mass along its long axis. this positional anomaly can coexist with a horizontal displacement of the ventricular mass along the horizontal plane of long axis, which produces superior - inferior ventricles. in 1961, lev and rowlatt described unusual arrangement of the cardiac inlets that is ventricular chambers arranged in a superoinferior fashion, with the rv superiorly and the lv inferiorly.2 in 1974, anderson et al used the term cch for cardiac anomaly producing the illusion of crossing of the systemic and pulmonary venous stream without mixing at the av level.3 in the later years, other cases was described with situs solitus or situs inversus or isomerism, mostly with av concordance.45 the physiology is determined by the concordant or discordant atrioventricular (av) and ventriculoarterial (va) connection and the associated cardiac defects.67 cch may be seen in three forms, that is complete transposition, corrected transposition, and normal hearts. in our case, there was av and va concordance with malposed great arteries. a review of the medical literature does not reveal any isolated presentation of cch cases. most patients with cch has other anomalies such as vsd, transposition of great vessels, right ventricular hypoplasia, subpulmonary stenosis, straddling av and others.78 the diagnosis should be suspected by echocardiography when the parallel arrangement of the av valves and ventricular inlets can not be achieved, and the two valves are not easily visualized simultaneously on apical 4 chamber view. color flow mapping can help in assessing the av connection, visualization of the direction of intracardiac blood flows and recognition of the crossover of the inflow streams.910 yang yl et al11 reported that the failure to obtain a characteristic 4-chamber view was diagnostic for recognition of the cch. the echocardiographic features suggested by yang yl et al11 were present in our case. surgical options vary according to the exact sequential segmental analysis and associated abnormalities. in , cch is a rare cardiac anomaly than can be diagnosed by a transthoracic echocardiography by an alert echocardiographer to determine the relationships of the cardiac chambers and associated cardiac anomalies.

crisscross heart (cch) is a rare cardiac malformation characterized by crossing of the inflow streams of the two ventricles due to an apparent twisting of the heart about its long axis. the developmental mechanisms and causes of cch are remaining unknown. neonates mainly presents with cyanosis and a systolic murmur. we herein present a case of cch with concordant atrioventriculo connections with double outlet right ventricle (dorv) which was diagnosed by echocardiography.

while abdominal pain is a common presentation in patients with sickle cell disease, its progression to an acute surgical abdomen is relatively rare. only a few case reports describing ischemic bowel secondary to sickle cell crisis have been published. ischemic bowel secondary to sickle cell disease has been recognized with increasing frequency. most patients with ischemic bowel are elderly and often have a past medical history significant for atherosclerotic disease manifesting with congestive heart failure or cardiac dysrhythmias. in contrast, patients with sickle cell disease are considerably younger, often with no history of cardiovascular disease. a 42-year - old african - american man with a past medical history of hypertension, end - stage renal disease, and mechanical heart valve was admitted to the hospital in sickle cell crisis with complaints of chest and bilateral upper extremity digit pain. cardiac work - up showed an ekg with st depression in the left lateral wall leads. cardiac catheterization was performed, but demonstrated no significant coronary artery lesions and required no further cardiac intervention. the patient had an elevated amylase and lipase of 456 and 334 u / l, respectively. electrolytes were within normal limits except for a total bilirubin of 2.1 mg / dl. as such, the patient was managed conservatively and eventually started on oral intake. once the patient was started on a regular diet, he developed acute generalized abdominal pain which was similar in his estimation to previously experienced pain associated with sickle cell crisis. upon examination, nevertheless, his examination revealed a tense, distended abdomen which revealed diffuse rebound tenderness and guarding. complete blood cell count demonstrated a white blood cell count of 16,200 with 80% neutrophils. an upright abdominal film demonstrated pneumoperitoneum. the patient was immediately given intravenous piperacillin / tazobactam, hydrated aggressively, and taken to the operating room for exploration. intraoperatively, evaluation of the small bowel demonstrated 3 lesions that were grossly infarcted and leaking bile. the ischemic regions and infarcted / perforated portions of duodenum and jejunum were resected with primary anastomosis. the patient's operative course was uneventful and he was transferred to the intensive care unit for recovery. histologic sections of the duodenal and jejunal specimens of infarcted bowel demonstrated transmural infarction with necrotizing acute inflammatory cell exudates along with submucosal edema and necrotizing acute serositis consistent with ischemic bowel ( fig. 1, fig. its etiology has been attributed to a myriad of sources including mesenteric and retroperitoneal adenopathy, infarction in vertebral bodies, hepatobiliary disease and splenic infarction. while the above may at some point in the natural history of the disease contribute to abdominal pain, it is more likely that intermittent ischemia, stemming from transient low - flow states due to red blood cell deformity, is the primary source of the abdominal pain. at the capillary level, red blood cell deformability is the major determinant of viscosity with the capillary diameter at which red blood cell deformation inhibits normal blood flow being < 10 m. increased membrane rigidity ing in decreased deformability may be the primary contributor in transient vaso- occlusion in capillaries that often range in diameter from 3060 m. one classic study by boley et al. reproduced ischemic colitis pathology in dogs after injection of microspheres of 30100 m into the mesenteric circulation. we believe that the bowel injury found in our particular patient likely represents an extreme manifestation of what is likely to be a spectrum of pathology that ranges from minor abdominal pain secondary to transient ischemia to full - blown transmural bowel infarction. most case reports and reviews emphasize that the majority of patients with abdominal pain will not have clinically significant bowel injury and thus can be managed conservatively with bowel rest, aggressive hydration, and intravenous analgesics. however, our case highlights the fact that the far end of the clinical spectrum, i.e. transmural bowel injury with pneumoperitoneum requiring operative intervention, does indeed occur and should be kept in mind when managing and following sickle cell patients with abdominal pain.

we report a case of small bowel ischemia secondary to sickle cell disease. acute bowel ischemia is an uncommon presentation of patients with sickle cell disease. historically, only a handful of cases have been reported. we also provide a summary of the literature relevant to sickle cell patients with acute bowel ischemia.

the red spotted grouper, epinephelus akaara is one of the most important grouper species for aquaculture in korea, china, japan and southeast asian countries. in particular, groupers are ideal candidate species for intensive aquaculture in asia pacific region because of high consumer demand, desirable taste, efficient feed conversion, and rapid growth (kohno et al ., 1993 ; commercial importance culture species is still in infancy with adult management, natural spawning and seed production because most groupers require culture periods of several years until first maturation and spawning is achieved ( song et al ., 2005). the problems of grouper aquaculture development has been hindered by the difficulty of mature male grouper security, which is due to the lack of a standardized method of sex change and the unavailability of mature male broodstock. most groupers including the red spotted grouper are protogynous hermaphrodites, first become sexually mature as females and later change sex to become males. natural sex change in grouper species occurs between 3 and 11 years of age depending on the species (tan & tan, 1974 ; chauvet, 1988). the steroid hormones play important role of sex change in the grouper species (li et al . many study have been reported on different grouper species to develop standardized methods for sex reversal by treatment with androgens or aromatase inhibitors ( bhandari et al ., 2005). but sex - changed male grouper had testis with ovarian cavity and sperm production is very low. thus, in order to have a stable supply of sperm, it is necessary for male groupers without ovarian cavity using hormone treatment prior to ovarian cavity formation. in this study , we examined that the effect of 17-methyltestosterone (mt) to induce the primary male of juvenile red spotted grouper e. akaara. the experiments were carried out in marine science institute, jeju national university. at each experimental group, 200 fish were reared in 300 l circle tank with flowthrough sea water, under natural water temperature and photoperiod. at 70 days after hatching (total length, 3.33 0.38 cm ; body weight, 0.470.14 g), fish were immersed in 17-mt at 1 and 5 fish were sampled at 12 months after end of the treatment period in order to histological analysis. the gonads were fixed in bouin's solution, embedded in paraffin, cross - sectioned, and stained with haematoxylin and eosin using the standard methods for light microscopy. at the initiation of an experiment (70 day after hatching), juvenile red spotted grouper have the paired primordial gonads with somatic cells bellow kidney in the posterior portion of the body cavity. each lobe of the primordial gonads began elongation to form an ovarian cavity (fig . gonad development during sexual differentiation is a little studied but similar patterns as early characteristic of female differentiation were reported ( murata et al ., 2009 ; liu & sadovy, 2009 ; tsai et al ., 2011 ; sao et al ., 2012). in e. malabaricus, the initial ovarian cavity was observed at 47 day after hatching by elongations of somatic cells in the gonads on the sides facing the gonad lateral walls, an early event of initial ovarian cavity formation (murata et al ., 2009). in e. coioides and e. bruneus, the initial ovarian phase was observed at 4 month after hatching and 60 day after hatching, respectively (tsai et al ., 2011 ; sao et al ., 2012). in this study, early morphological change to form the initial ovarian cavity in red spotted grouper were observed by around 70 day after hatching. sex pattern of protogynous hermaphroditic fish can be divided into two types, mondary and diandry, according to the male development pathway (reinboth, 1967). monandric species follow a single male developmental pathway; all males in a population are secondary males derived exclusively from functional females via sex change. diandric species follow two male developmental pathways, i.e., primary males develop from juveniles through sexual differentiation and secondary males develop via sex change. in halichoeres most grouper is mondary type with secondary male via sex change from functional female (lee et al ., 2006). at 12 months after end of the treatment period, control group, 17-mt 1 mg / l treatment group for 4 and 8 weeks, and 17-mt 5 mg / l treatment group for 4 weeks were all female, which is contained oogonia and perinucleolus oocytes in ovary. however, sex - changed males were observed in the 17-mt 5 mg / l treatment group for 8 weeks. the gonad of sex changing individuals was consisted of numerous testicular lobules and filled with sperm, but can not observed the ovarian cavity (fig . successful induction of artificial sex change has been reported mostly in small sized females ( tukashima & kitajima, 1983 : chao & chow, 1990 ; tsuchihashi et al ., 2003). androgen administration including 11-ketotestosterone (11-kt) and the synthetic 17-methyl - testosterone (mt) widely used for sex reversal induction in grouper, but various dose concentrations and methods of hormone administration (chao & lim, 1991 ; tsuchihashi et al ., 2003 ; however, these methods of hormone administration have problem such as incomplete sex inversion and reverted back to being females during the next reproductive period ( marino et al . therefore, the most effective treatment time for artificial sex change induction by steroid hormone is during gonadal sex differentiation . in this study, sex - changed males without ovarian cavity were observed when juvenile red spotted grouper ( 70 dah) were immersed in 17-mt 5 mg these suggested that the red spotted grouper be able to induce the primary males by hormone treatment prior to gonadal sex differentiation.

we investigated the androgenic effects of 17- methyltestosterone (mt) on gonadal sex reversal in juvenile red spotted grouper epinephelus akaara. the fish were immersed in 17-mt at 1 and 5 mg / l. treatment method of 17-mt was once weekly for 4 and 8 weeks. fish were sampled at 12 months after end of the treatment period in order to histological analysis. at the initiation of an experiment (70 day after hatching), juvenile red spotted grouper have the paired primordial gonads with somatic cells bellow kidney in the posterior portion of the body cavity. formation of ovarian cavity indicates that the ovarian differentiation beginning at 70 dah in red spotted grouper. at 12 months after end of the treatment period , control group, 17-mt 1 mg / l treatment group for 4 and 8 weeks, and 17-mt 5 mg / l treatment group for 4 weeks were all female. however, sex - changed males without ovarian cavity were observed in the 17-mt 5 mg / l treatment group for 8 weeks. in grouper , we firstly reported that the red spotted grouper be able to induce the primary males by hormone treatment prior to gonadal sex differentiation.

a 35-year - old man with an unremarkable medical history sought treatment for headaches, hearing loss, and night sweats. they came on as the day progressed and were neither positional nor associated with nausea, vomiting, or visual changes. three weeks before arriving at the hospital, he noted a sense of " fullness " in his ears; he said that spoken voices sounded muffled, and he had difficulty hearing telephone conversations. when someone at the restaurant where he worked dropped a dish, he heard that sound clearly and reported that it was almost painful, causing him to become dizzy. his appetite was good, and he had not lost weight. although he reported no fever, he did report night sweats for several weeks. he had no rash, diarrhea, abdominal pain, chest pain, shortness of breath, joint complaints, or dysuria. he also said that he was exposed to dust at his workplace because of remodeling. he lived in new hampshire, had no pets, and worked as part owner of a restaurant. exposures included multiple male and female sexual partners with inconsistent condom use, and he had acquired several tattoos 8 months earlier while traveling in spain and italy. he had no known tuberculosis exposure. temperature was 36.7c, blood pressure 128/84 mm hg, pulse 80, respirations 16/minute. sclerae were anicteric, and his pupils reacted to direct and consensual testing and responded normally to accommodation. bedside testing showed that his hearing was diminished to low volume sounds, but he was able to hear loud sounds, which he found painful. when the examiner clapped his hands loudly a few feet from the patient's ear, the patient exhibited nystagmus. the patient's neck was supple; a 1.5-cm, nontender lymph node was palpated in the left upper anterior cervical chain. the remainder of the examination was unremarkable, with negative romberg test; normal gait; and normal motor, sensory, and reflex performance. rapid plasma reagin (rpr) was positive at a titer of 1:128 and was confirmed by a fluorescent treponemal antibody test. subsequent studies showed a cd4 receptor positive t - cell (cd4) count of 899/ml and an hiv viral load of 878 copies / ml. a lumbar puncture showed protein 33 mg / dl, glucose 78 mg / dl, 9 erythrocytes/l, and 8 leukocytes/l (100% lymphocytes). cerebrospinal fluid venereal disease research laboratory test (csf - vdrl) were negative. csf - vdrl, cerebrospinal fluid venereal disease research laboratory test; elisa, enzyme - linked immunosorbent assay; wb, western blot; rt - pcr, reverse transcription polymerase chain reaction. the patient was started on 24 mu of iv penicillin per day for 14 days. at the end of this period, he reported notable improvement in his headaches, hearing loss, and vertiginous symptoms. one month after completing treatment, his rpr titer was 1:32; 3 months after he completed treatment, it was nonreactive. the course of syphilis in an hiv - positive patient may be altered from the natural history of the disease in hiv - negative patients. an increased frequency of ocular disease, multiple and slower resolving primary chancres, and a higher titer rpr have been reported. in addition, delay or failure of titer decline after treatment, predilection for developing the jarisch - herxheimer reaction, and clinical relapse have also been described in hiv infection. treponema pallidum is thought to invade the central nervous system in 25% of patients with syphilis, irrespective of hiv status. most of these persons successfully clear the infection, but other patients harbor the spirochete and remain at risk for sequelae of neurosyphilis. syphilitic meningitis, meningovascular disease (often occurring with stroke), labyrinthitis, or cranial nerve palsies, as seen in this case, can be early findings. loud sounds or even routine acoustic stimuli can in vertigo, nystagmus, or nausea and vomiting. the physiologic underpinnings of the tullio phenomenon were first described in 1929, when tullio noted that experimentally induced fenestrations in the bony capsule of the lateral semicircular canals of pigeons caused the canals to be sound - responsive, inducing vestibular activation. shortly thereafter, benjamins described a tullio reaction in a human patient with fistulizing cholesteatoma. today, the term has been generalized to include vestibular activation in response to stimulation by sound of any part of the vestibular apparatus. the tullio phenomenon is seen in a range of clinical contexts, including congenital deafness, meniere disease, suppurative middle ear disease, and spirochetal infections, such as syphilis or lyme disease. nields et al. describe a woman who had nystagmus and vertigo with routine sounds; running tap water caused her to fall to the floor or retch in pain. describe a series of patients with oscillopsia induced by pencil tapping, telephone ringing, or the sound of cutlery falling on the floor. patients often report a vague sense of ear blockage and an unpleasant awareness of their own voice vibrating in their ear. symptomatic cranial nerve viii involvement in this patient prompted treatment for neurosyphilis, despite equivocal (but typical) csf findings. interpreting csf findings in hiv - positive patients with syphilis is challenging because commonly encountered mild lymphocytic pleocytosis may be attributable to hiv. csf - vdrl is often negative, with a 20%70% false - negative rate. optimally managing patients with hiv and syphilis has been debated; to date routine recommendations are the same as for hiv - negative persons. although any patient in whom syphilis is diagnosed and who has neurologic symptoms should undergo lumbar puncture, csf evaluation should also be considered in asymptomatic patients in whom syphilis is diagnosed and who have an rpr titer > 1:32 or a cd4 + count < 350, as these markers have been associated with increased risk for neurosyphilis. because of the high rate of relapse that has been reported in some series, close clinical and serologic follow - up is essential. a study of 59 patients with neurosyphilis showed that hiv - positive study participants were 2.5 times less likely to normalize csf - vdrl reactivity than hiv - negative patients. this effect was even more pronounced in patients with cd4 + counts < 200. the findings suggest that more intensive therapy for neurosyphilis in immunocompromised patients merits further study. after an all - time low case - rate of syphilis in 2000, reports of rising trends, specifically in groups at risk, such as men who have sex with men, were reported in 2001. the outbreaks marked a 9.1% overall increased rate of primary and secondary syphilis and were characterized by a high rate of hiv infection. parallel increases in hiv infection rates were a concern because of the common mechanism of transmission and the increased efficiency of hiv transmission with coexistent genital ulcers. in 2001, increases in hiv rates were reported in several states with large urban populations; men who have sex with men accounted for the largest known subgroup at risk in incident adult or adolescent hiv cases (32%) and aids cases (44%). new hiv diagnoses increased in 29 states with mandatory reporting from 1999 to 2002, notably by 17% among gay and bisexual men. syphilis incidence also continued to rise in the united states in 2002, with a 12.4% increase since 2001, and the highest trends were estimated to be among men who have sex with men. in san francisco, primary and secondary syphilis rates increased by > 1,000% from 1998 to 2002 among men who have sex with men. high - risk behaviors have been documented in this group and factor prominently in syphilis reemergence in the early 21st century. this case report describes a patient who had tullio phenomenon as the index symptom of neurosyphilis with previously undiagnosed hiv infection. his rpr titer was 1:128, his cd4 count was preserved, and he responded well clinically and serologically to standard therapy. to our knowledge, this case is the first report of syphilis occurring as tullio phenomenon in an hiv - positive patient. we suggest that syphilis be considered in patients who have cranial nerve viii symptoms and an appropriate risk - factor profile. eradicating treponemes may be limited from sanctuary sites such as the cns. because control of syphilis likely depends not only on antimicrobial effect but also on host immune response, awareness of unusual symptoms of a relatively common disease will benefit not only the patient but also public health efforts in managing both syphilis and hiv infection. preventive and educational efforts focused on men who have sex with men may prove particularly important in modifying behaviors that foster the growth of both epidemics.

the incidence of syphilis has consistently increased from 2000 to 2002. we report a case of acquired syphilis with symptoms of tullio phenomenon in a patient concurrently diagnosed with hiv infection. the resurgence of syphilis in hiv - positive groups at high risk has public health implications for prevention of both diseases.

postural stability is deeply linked with sports injuries, and copious evidence exists indicating that postural stability is greatly reduced by sports injuries. back in 1965, freeman first described stabilometric alterations in patients suffering from ankle sprain and correlated lateral instability of the ankle with a lack of postural control1. after freeman, several authors analyzed the effects of sports injuries on postural stability of athletes and concluded that its deterioration may cause re - injury or even new injuries2, 3. in addition to previous injuries, postural stability may be affected by changes in the level of activity, as well as by its type, intensity, and volume4,5,6, which constitute a risk of injury for athletes7. some authors have carried out prospective assessments of athletes in order to determine how stabilometric changes can be a causal factor for injuries8. assessed the first two weeks of the season for basketball players, and correctly described balance as a predictor of injuries, showing that those with higher postural sway values at the start of the season were the ones most injured in the long term9. similar were found by wang et al., who established a correlation between poor mediolateral stability and suffering from ankle injuries later on10. likewise, trojian and mckeag showed in 2006 that the ability to maintain single - leg balance of athletes at the beginning of the preseason was a predictor of ankle sprains for the autumn season11. also, soderman et al. correlated all lower extremity injuries with increases in postural sway in female soccer players12. from a physiological point of view, murphy et al. affirmed in 2003 that the main cause of deteriorated postural stability as a risk factor is an alteration in the neuromuscular control strategy, which increases intersegmental joint forces and consequently increases the development of forces involving ligaments, tendons, and muscles13. in spite of the findings reported by these authors, whether postural stability is hopper et al. analyzed single - leg stability by assessing the duration for which female netball athletes could maintain a unilateral posture; however, they did not find any correlation between postural stability and an increase of injuries14. similar were reported by beynnon et al. in soccer, field hockey, and lacrosse athletes15. based on the controversy existing regarding postural stability as an injury predictor and the evidence of effects of training on postural stability and considering that to date , no studies have taken into account the type of training as a cause of postural stability deterioration in their prospective investigations, the aim of the present study was to analyze the stabilometry in athletes during their training stage in order to determine whether injured athletes scored differently in stabilometric tests than those who were injury - free in two different training periods. this was a five - month descriptive prospective study (september january) that was divided into two parts according to the different training periods. the first part consisted of bipodal and monopodal stabilometry tests at the end of preseason (in september, mainly general exercises) and a record of injuries suffered by the athletes during the subsequent period of training, which was volume period (in october and november, with training based on alternating general and specific high - volume, low - intensity exercises). the second part consisted of bipodal and monopodal stabilometry tests at the end of volume period and a record of injuries suffered by the athletes during the subsequent period of training, which was pre - competition period (in december and january, with an majority of specific low - volume, high intensity exercises). in order to avoid interference from previous injuries, a total of 51 track and field athletes who were from an athletic club in the city, were 17 to 35 years old and had at least three years of experience took part in the first part of the present study, after excluding those who did not train regularly or who had been injured during the preseason period. in the second part of the study, athletes who had been injured previously were excluded in order to avoid interferences in the . therefore, 39 athletes between the ages of 17 and 35 participed in this part of investigation. anthropometric and demographic data are shown in table 1table 1. statistical description of the samplefirst part of the study n=51second part of the study n=39injured in volume period n=12non - injured n=39injured in pre - competition period n=6non - injured n=33age (years)23.28.022.37.526.59.021.67.0height (m)1.740.071.740.081.750.051.740.09weight (kg)61.011.062.911.761.57.963.212.4bmi (kg / m)20.12.420.72.520.11.520.82.7gendermen866.7%2769.2%583.3%2266.7%women4 33.3%12 30.8%1 16.7%11 33.3%experience (years)7.84.77.24.610.04.86.74.4quantitative variables are shown as means and sd. p values are from the student s t - test and test, respectively. p values are from the student s t - test and test, respectively. bmi: body mass index before the start of the study, all athletes were briefed on how they would be tested, and written informed consent was obtained from each subject or from their legal guardians in the case of underage athletes according to the standards of the declaration of helsinki (2008 revision)16. weight and height measurements were performed with a tefal (france) digital precision scale (100 g300 kg) and an asimed t201-t4 (spain) measuring rod, respectively. for stabilometric measurements, a modular electronic baropodometer was used comprising a 120 160 cm sensormdica (spain) platform with 19200 active sensors. athletes were subject to a bipodal and a monopodal stabilometric measurements once at the end of each period. for the bipodal test, athletes were instructed to remain as still as possible on the baropodometric platform for 52 seconds, with a between - heels separation of 5 cm and their feet forming a 30 angle. for the monopodal test , athletes stood on each of their lower limbs for 15 seconds (left leg first) at the center of the platform (fig . the following parameters were recorded for the bipodal test as well as for the left - leg and right - leg monopodal tests : length ( length) and area (area) of the path described by the center of pressure, the speed of center of pressure movement (speed), and the position of the center of pressure in the mediolateral (xmean) and anteroposterior (ymean) planes. r to indicate whether they belong to the left or right leg, respectively. tests were carried out before training started, in order to avoid any interference. also, athletes were instructed not to engage in any sports activity on the day of testing. bipodal, left monopodal and right monopodal stabilometry tests in order to track injuries, athletes were instructed in advance and then interviewed weekly by a sports physical therapist during the volume and pre - competition periods in order to record any musculoskeletal injury on the lower limbs they might have sustained during these periods. injury was defined as physical damage that ed in missing or modifying one or more training sessions or competitions (kolt et al . a description of data included means and standard deviations for the categorical variables ( table 1). differences in social and demographic variables between the injured and non - injured athletes each period (volume and pre - competition) were analyzed by means of the student s t - test for independent samples in the case of the continuous variables and a test for the categorical variables. for comparison between stabilometric variables between groups (injured and non - injured), the student s t - test for independent samples was used. a significance level of p0.05 was set for all statistical procedures, and the spss v. 19 software was used. table 1 shows social and demographic statistics for the injured and non - injured subjects during the volume and pre - competition periods. there was a similar number of injured athletes in both periods. no differences were apparent in the social and demographic characteristics of the injured and non - injured athletes (p>0.05). mean values of stabilometric variables in bipodal and monopodal support taken at the end of the pre - season periodtotal injuredin volume period n=12non - injuredn=39preseason measuresmeansdmeansdlength (mm)*366.5183.9340.2104.0area (mm)36.527.147.549.9speed (mm / sec)*7.43.96.72.0xmean (mm)3.94.74.43.2ymean (mm)4.54.16.05.1lengthl (mm)272.0112.5267.865.5areal (mm)419.1493.7408.7350.8speedl (mm / sec)24.411.424.76.8xmeanl (mm)4.93.66.013.5ymeanl (mm)6.65.1410.28.2lengthr (mm)255.6119.9275.582.8arear (mm)340.2282.9378.2298.8speedr (mm / sec)23.112.024.77.9xmeanr (mm)3.42.93.02.8ymeanr (mm)7.06.58.65.2 are split for athletes injured during the volume period.length= length of the path described by the center of pressure. area = area of the path described by the center of pressure. speed = speed of the center of pressure. the first section is for bipodal support, the second section is for the left foot (indicated by addition of l to each variable), and the third section is for the right foot (indicated by addition of r to each variable). * p<0.05 shows mean values for the center - of - pressure spread and position variables in both bipodal and monopodal support at the end of the preseason period for the athletes who were injured and non - injured during the volume period. bipodal stabilometry tests showed that those injured during the volume period had scored significantly higher in length and speed than those who were not injured (p=0.009 and p=0.003, respectively). the first section is for bipodal support, the second section is for the left foot (indicated by addition of l to each variable), and the third section is for the right foot (indicated by addition of r to each variable). mean values of stabilometric variables in both bipodal and monopodal support at the end of the volume periodtotal injuredin pre - competition n=6non - injuredn=33volume measuresmean sdmean sdlength (mm)310.1131.8307.7143.6area (mm)128.5103.198.8109.6speed (mm / sec)6.22.813.542.0xmean (mm)4.51.85.75.0ymean (mm)8.72.712.49.4lengthl (mm)335.395.9316.3127.4areal (mm)615.0512.0489.6506.8speedl (mm / sec)31.19.635.340.2xmeanl (mm)3.52.55.812.3ymeanl (mm)10.87.016.311.6lengthr (mm)349.089.3322.7132.8arear (mm)631.1328.3608.1624.0speedr (mm / sec)31.99.436.740.2xmeanr (mm)*15.230.34.33.6ymeanr (mm)12.06.815.39.6 are split for athletes injured during the pre - competition period.length= length of the path described by the center of pressure. area = area of the path described by the center of pressure. speed = speed of the center of pressure. the first section is for bipodal support, the second section is for the left foot (indicated by addition of l to each variable), and the third section is for the right foot (indicated by addition of r to each variable). * p<0.05 displays the mean values for the center - of pressure spread and position variables in both bipodal and monopodal support at the end of the volume period for the athletes who were injured and non - injured during the pre - competition period. right - leg monopodal stabilometry showed that injured subjects scored significantly higher with respect to xmeanr (p=0.041). left - leg monopodal and bipodal stabilometry did not show any statistically significant difference (p>0.05). the first section is for bipodal support, the second section is for the left foot (indicated by addition of l to each variable), and the third section is for the right foot (indicated by addition of r to each variable). the aim of the present study was to analyze stabilometric values of athletes through their training in order to determine their value as a predictor for injuries in each of the training periods. to this end, athletes were subjected to a bipodal and monopodal stabilometry at the end of the preseason period and a tracking of their injuries in the subsequent volume period (first part). in addition, bipodal and monopodal stabilometry was carried out at the end of volume period, and tracking of their injuries was carried out in the subsequent pre - competition period (second part). the obtained from stabilometry at the end of the preseason period show that athletes with poorer values for center - of - pressure length and speed in bipodal support at the end of the preseason were the ones who were injured in the two subsequent months, which comprised the volume period. although these findings agree with previous studies in which high variation of postural sway correlated or predicted posterior sports injuries9, 10, 12, our did not show any significant difference in monopodal stability, which differ from most of the previous studies. mcguine et al. observed in 2000 that basketball players with poorer preseason unilateral balance values were seven times more prone to ankle sprains during the season9, and soderman et al. reported similar findings for female soccer players, with the ones with lower scores for unilateral postural balance being more prone to leg - related injuries12. similar were found for unilateral stability of athletes by watson et al., who assessed the monopodal postural sway by making soccer players maintain unilateral balance for 15 seconds, and those who were not able to perform it without touching down were classified as having abnormal postural sway. this group was the most affected by posterior ankle sprains19. also, our findings contrast with the study of trojian and mckeag in 2006, in which they found a positive association between monopodal support stability and ankle sprains11. in addition, the of stabilometric tests carried out in the volume period show that athletes with worse center - of - pressure values in the mediolateral plane while performing during right - leg monopodal support were more commonly injured during the two subsequent months, which comprised the pre - competition period. in spite of the fact that these were found in monopodal stabilometry instead of bipodal stabilometry, these findings show the same trend ilustrated by the previous values of center - of - pressure spread found during the first part of the present study, in which injured athletes exhibited stability deterioration. besides, these support the research by mcguine et al. and soderman et al., who reported in basketball players and soccer players, respectively, poorer unilateral stabilometric center - of - pressure values in subjects who were later found to be injured more frequently9, 12. these are also in accord ance with those of trojian and mckeag, who associated worse monopodal stabilometry with later ankle sprains in athletes11, and agree with those of wang et al., who established in 2006 a correlation between poorer center - of - pressure mediolateral position and later ankle injuries10. on the other hand, in 2001 and 1995, respectively, beynnon et al. and hopper et al. did not find data to support the value of postural balance as a predictor of ankle sprains14, 15, which differs from the rest of the studies and the present investigation. the same procedure was used by willems et al. in 2005, who did not find significant differences in postural stability variables; however, these authors discovered that subjects with weaker control over their center of gravity were more prone to later suffering ankle sprains20. similar trends were seen for the of both forms of stabilometric testing performed in the preseason period and the number of athletes injured in the volume period compared with the testing performed in the volume period and the number of athletes injured during the pre - competition period. both sets of data point to athletes with higher values (and therefore poorer scores) for stabilometric variables being more prone to injuries in the subsequent periods. however, higher stabilometric variables were found for the bipodal stance in the first part, whereas higher stabilometric variables were found for the monopodal stance in the second part. based on the difference between the of the parts of the present study, it is important to take into account that the injuries that occurred during the pre - competition period could have been influenced by other risk factors such as higher intensity of trainings or explosive actions, which are thought to be actions involving more damage21 and characteristic of this training period. also, it is important to indicate that the present investigation is the only study to date in which all lower extremity injuries were correlated with increases in postural sway. in addition, certain limits of our study may have played a part in determining the lack of more significant differences. the low number of injuries recorded as a consequence of the small size of the sample might conceivably have contributed to this discrepancy in . indeed, the difference in number of athletes participating in both parts of the study could have affected the due to the small size of the sample. further research with a larger sample size is recommended, as well as inclusion of other age groups. in , athletes who show higher center - of - pressure spread during bipodal stabilometry at the end of the preseason are more prone to injuries during the subsequent training period. also, athletes who exhibit poorer stability in the mediolateral plane when performing right - leg monopodal support at the end of the volume period are more prone to injuries in the subsequent training period. as a practical application, inclusion of specific proprioceptive training in the training routines of athletes is recommended in order to improve stabilometric parameters and decrease and/or eliminate their role as risk factors.

the aim of this study was to analyze stabilometry in athletes during an indoor season in order to determine whether injured athletes show different stabilometric values before injury than non - injured athletes in two different training periods (volume and pre - competition periods). the subjects were 51 athletes from unicaja athletic club who trained regularly. at the end of the preseason and volume periods, athletes were subjected to bipodal and monopodal stabilometry. in addition, all injuries happening in the periods after performing stabilometry (volume and pre - competition periods) were tracked. variance analysis of bipodal stabilometric measurements taken at the end of the preseason period showed that athletes with higher values for the center - of - pressure spread variables suffered injuries during the volume period. the right - leg monopodal stabilometric measurements taken at the end of the volume period showed that athletes with higher values in the center - of - pressure position variables suffered injuries during the pre - competition period. athletes showing the worst values for center - of - pressure spread variables are more prone to sports injuries in the subsequent training period. in monopodal measurements, athletes with poorer mediolateral stability were more prone to injuries in the subsequent training period.

noise is one of the most harmful agents for health, primarily for hearing, and it is often unavoidable at workplaces and entertainment venues. noise - induced hearing loss (nihl), which is hearing loss that is induced by high levels of sound pressure, is one of the most common occupational diseases. currently, noise is a constant part of people's daily activities, as it is present in traffic, leisure, and work; therefore, nihl may become one of the main chronic diseases in the future (fiorini, 2000). nihl is defined as sensorineural hearing loss that occurs due to systematic occupational exposure to high levels of sound pressure, thus causing damage to the hair cells of the organ of corti. generally, this hearing loss is bilateral and symmetrical, insidious and irreversible, and is directly related to the period of exposure and individual susceptibility (brazilian national noise and hearing preservation committee, 1999 ; rabinowitz, 2000). this hearing disturbance is initially manifested at the frequencies of 6,000 hz, 4,000 hz, and 3,000 hz, and broadens progressively to the frequencies of 8,000 hz, 2,000 hz, 1,000 hz, 500 hz, and 250 hz. noise rarely leads to profound hearing loss, which generally does not exceed 75 db for high frequencies and 40 db for low frequencies, and reaching its upper limit in the first 10 to 15 years of exposure (luxon, 1998 ; hanger, barbosa - branco, 2004 ; gatto et al ., generally, professionals only notice a hearing difficulty when the lesion is at an advanced stage, as the hearing symptoms are insidious and manifest late ( sava, 2005). continuous exposure to high levels of sounds may not only in hearing damage, but also in a few secondary alterations, such as tinnitus, stress, physiological alterations to the heart rhythm and to blood pressure, as well as difficulty in discriminating speech sounds, especially in noisy environments. noise causes physical exhaustion, chemical, metabolic, and mechanic disturbances to the sensory hearing organ, thus ing in partial or total hearing loss of the organ of corti, which is located in the internal part of the ear. (otoni a, boger me, barbosa - branco a, shimizu he, maftum ma, 2008). dentists are typically exposed to 2 types of noise: external noise from the work environment and noise from their own work equipment, such as the noise from high and low rotation motors, compressors, air conditioners, amalgamators, aspirators, and others (hinze, deleon, and mitchel, 1999). in addition to noise, they are exposed to other factors, including chemicals (manipulated substances, especially mercury), biological agents (the oral cavity is full of microorganisms and the risk of contracting diseases such as hepatitis and aquired immune deficiency syndrome ( aids) is high ), mechanical agents (body lesions caused by to the instruments that are used), social stresses (having occupation that involves tension and requires mastery of the situation to facilitate the patient / professional relationship), and ergonomic challenges (due to the body position while working, the professional is subjected to back and arm problems and varicose veins) (souza, 1997). according to presta et al. dentists have frequently presented with discomforts that are related to the nature of their profession, which may progress to repetitive strain injuries or work - related musculoskeletal disorders. in 1959, the american dental association (ada) had already recommended periodic hearing assessments for dentists, due to their prolonged exposure to high - frequency sounds, which were caused by instruments like high speed drills, ejection systems, ultrasound equipment, model clippers, and instruments with high suction and vibration speeds, which can lead to hearing loss. studies (altinoz et al ., 2001 ; fernandes et al ., 2004) that measured the noise levels in dental practices observed noise levels that were higher than 80 db sound pressure level (spl). in brazil, law number 6.514 of the brazilian labor code (consolidao da leis do trabalho ; clt), relates the parameters that provide acoustic comfort for practitioners with the norms of the brazilian association of technical norms (abnt). the brazilian employment legislation considers the maximum tolerable level of noise to be 85 db spl in an 8-hour shift (segurana e medicina do trabalho, 1991); the brazilian standard (nbr) 10.152 indicates that this level should be between 35 and 45 db (a) for a dental office. paraguay verified that dentists with 5 or more years of practice presented hearing disturbances by tonal threshold audiometry. leggat stated that the reason that more experienced dentists suffer from hearing alterations may be due to previous exposure to older equipment. however, this possibility was not considered in this study, as the study population was relatively young. previous studies (oliveira et al ., 2007 ; torrs et al ., 2007 ; melo et al ., 2008) have shown that dental professionals should be aware of occupational noise, as well as of the harmful consequences that it can have on their health. this understanding should begin early, during the undergraduate course, when the professionals are being educated, so that, aware of the risks that they may be exposed to, they will be able to prevent them, instead of attempting to lessen or treat the problems caused by them. in brazil, the prevention of occupational diseases and occupational accidents began with the clt in 1943, and ever since, the attention to hearing in noisy work environments has intensified. with the promulgation of ordinance number 3.214/78 there was an important improvement in the scope of hearing conservation efforts, by means of the control program occupational health medical - pcmso (nr-7), which made the tonal threshold audiometry mandatory. the interest in early diagnosis has increased, and considering the progress of the technology that is aimed at the diagnosis of hearing loss, as well as the prevalence of hearing alterations even in the absence of complaints, which are commonly found by tonal threshold audiometry, other methods have been used to identify hearing alterations at an early stage. according to previous studies, high - frequency (between 9,000 and 18,000 hz) tonal audiometry is an instrument that is effective for the early diagnosis of hearing problems due to exposure to noise (porto et al . that investigated hearing at conventional frequencies and high frequencies in dental practices have shown a tendency toward lower thresholds, suggesting that significant hearing problems accumulate with time. as the frequency, time of exposure, and age increases these studies also observed a greater incidence of hearing loss in the frequencies of 6,000 hz and 14,000 hz. lopes and godoy compiled a literature review regarding the importance and contribution of high - frequency audiometry for the early identification of nihl. they demonstrated that conventional tonal threshold audiometry alone may not be effective in the identification or prevention of nihl, and also described the methodological variables for its proper execution. the authors also suggested this method should be added to the routine that is indicated by the occupational hearing loss prevention program. therefore, the purpose of this study was to investigate the hearing thresholds at conventional and high frequencies, thus enabling early prevention of hearing loss and and improving the overall hearing health of the population as a whole. this study began after the approval of the ethics committee of the university of so paulo under process number 043/2007. this research study was financed by the so paulo research foundation (fapesp) funding agency under process number 2007/01074 - 7. this was a cross - sectional study with 108 volunteer participants the bauru community, which were divided into 3 experimental groups. group i (gi) consisted of 44 dentists (16 males and 28 females) aged between 23 and 57 years (average of 34 years of age). group ii (gii) consisted of 36 female dental nurses, aged between 21 and 59 years (average of 38 years of age). lastly, group iii (giii) consisted of 28 prosthodontists (17 males and 11 females) aged between 17 and 53 years (average of 35 years of age). professionals from private dentistry offices or laboratories, universities in bauru, so paulo, and hospitals that employ dentists on their staff were invited to participate. the participants initially received clarifications regarding the purpose of this study, which only began after the agreement to participate and the signature of the informed consent were obtained. for the inclusion and exclusion criteria, only professionals in the dentistry field with at least 2 years of experience, who did not present any pre - existing diseases such as mumps, high blood pressure, diabetes, meningitis, human immunodeficiency virus (hiv), syphilis, and other conditions that can compromise hearing and/or pre - existing hearing impairment were considered. all the participants were underwent: a specific interview and middle ear inspection: these procedures were performed to investigate the individual features such as age, time in the profession, noisy entertainment habits, exposure to chemical products, as well as health conditions and other diseases that can aggravate the effect of environmental risks. conventional tonal threshold audiometry (250 to 8,000 hz), high - frequency tonal threshold audiometry (9,000 to 16,000 hz), and speech reception threshold tests were performed using a siemens sd 50 audiometer. for the tests of the tonal thresholds the warble tone was used, which was presented using aural headphones (hda 200). the hearing threshold was established when there was a 50% positive answer for sound detection (lopes and godoy, 2006). acoustic impedance test: the acoustic immitance measures and the ipsilateral and contralateral acoustic reflex tests of the stapedius muscle were performed with gsi tymp star the data analysis from the specific interview revealed that 65 participants were bothered by noise at work, 50 participants reported difficulties in speech comprehension, 8 had served in the army, 11 had acoustic trauma, 32 were exposed to chemical products, and 35 referred to being exposed to noise during recreational activities. graph 1 shows the mean hearing thresholds for all of the frequencies that were tested in the 3 groups. hearing thresholds of the right ear in all of the tested groups. hearing thresholds of the left ear in all of the tested groups. the comparison between the mean hearing thresholds of each frequency that was tested in the 3 groups was performed using the kruskall - wallis test, and considered significant by the dunn test. we obtained a statistically significant difference in the right ear for the frequencies of 2,000 hz (p = .0446), 8,000 hz (p = .0492), and 16,000 hz (p = .0441) when the mean of group i (mean threshold at 2,000 hz = 5.91 db, at 8,000 hz = 11.59 db, and at 16,000 hz = 21.59 db) was compared to the mean of group ii (mean threshold at 2,000 hz = 10.69 db, at 8,000 hz = 18.61 db, and at 16,000 hz = 32.78 db); hence, we verified that gii presented worse thresholds at the frequencies of 2,000 hz, 8,000 hz, and 16,000 hz in the right ear compared to gi. it can observed that the right and left ears presented similar configurations in conventional and high - frequency audiometry, when the mean hearing thresholds for all of the groups is considered; however, the right ear presented worse hearing thresholds than the left ear. for the left ear, the frequencies of 4,000 hz (p = .0238) and 6,000 hz (p = .0310) presented statistically significant differences between gi (mean threshold at 4,000 hz = 8.41 db and at 6,000 hz = 14.32 db) and gii (threshold mean at 4,000 hz = 14.03 db and at 6,000 hz = 20.69 db) and between gi and giii (mean threshold at 4,000 hz = 15.36 db and at 6,000 hz = 22.32 db). at the frequency of 9,000 hz in the left ear, there was a statistically significant difference (p= .0397) between gi (mean theshold at 9,000 hz = 10.91) and gii (mean threshold at 9,000 hz = 20.28 db). the comparison of the mean hearing thresholds at the frequencies of 500 hz to 2,000 hz, 3,000 hz to 6,000 hz, 9,000 hz to 16,000 hz, and 12,000 hz to 16,000 hz was performed using the kruskall - wallis test, and the statistical significance was determined by the dunn test. for these comparisons, a statistically significant difference (p = .0147) was obtained only when the mean thresholds between the frequencies of 3,000 hz to 6,000 hz in the left ear between gi (10 db) and gii (15.93 db) and those between gi (10 db) and giii (16.25 db) were compared. the speech audiometry, which was performed using the speech recognition threshold, confirmed the of the conventional audiometry in 100% of the participants, as did the speech recognition index, which was compatible with the hearing thresholds found in 100% of the participants. for the acoustic immitance and evaluation of the ipsilateral and contralateral stapedius muscle reflexes, normal tympanograms were obtained bilaterally in 100% of the participants, thus indicating that the middle ear did not interfere with the that were obtained. this study focused on a sample of dentists, dental nurses, and prosthodontists with more than 2 years of experience in the dentistry field and with an average age of 35 years among the 3 groups. in this population , hearing loss can occur due to prolonged exposure to high - frequency sounds that are produced by the instruments that are used in their daily practice, which in subsequent handicaps to their communication and quality of life (hinze, deleon, and mitchel, 1999). this study showed that only 60% of the participants were bothered by noise at work (souza, 1997 ; trres et al . , 2007 ; melo et al ., 2008), that 43.3% of the participants reported difficulties in speech comprehension, and that 32.4% mentioned that they were exposed to noise during recreational activities. this study revealed that the mean hearing thresholds of all of the groups worsened according to increases in frequency (matthews et al . , 1997 ; beltrami, 1999 ; fernandes and mota 2001 ; mota, 2002 ; porto et al ., 2004 ; silva and feitosa, 2006 ; lopes et al ., 2006 ; lopes and godoy, 2006 ; lopes, almeida, zanconato, and mondelli, 2007 ; carvalho, koga, carvalho, and ishida, 2007). for all of the groups, both conventional and high - frequency audiometry of each ear showed similar configurations; however, by comparing both ears in the 3 groups, we observed that the right ear showed worse mean thresholds than the left ear. these are in agreement with zubick, tolentino, and boffa, and are in disagreement with gijbels et al. , who gathered data on the effects of occupational health among dentists and observed that hearing loss was greater for the left side for right - handed professionals, which can be explained by the short distance between the left ear and the circular motion / vibration equipment for right - handed professionals. the tritonal means of 500 to 2,000 hz and 3,000 to 6,000 hz revealed that prosthodontists (gii) had the worst hearing thresholds. for the high - frequency mean (9,000 to 16,000 hz) the dental nurses (gii) had the worst hearing thresholds. these highlight the importance of using complementary tests in the hearing evaluation (which in this case was high - frequency tonal threshold audiometry), and that if these tests are used as a routine procedure in the evaluation of these professionals it would assist in the early detection and prevention of hearing problems. in this study, we concluded that the conventional hearing assessment did not identify hearing problems in the 3 groups that were tested. however, the assessments of the high - frequency thresholds indicated disturbances to the peripheral auditory system, specifically on the external hair cells, thereby demonstrating greater sensitivity for the early detection of hearing problems and favoring the use of complementary tests as prevention tools.

summary introduction: in the dentistry practice, dentists are exposed to harmful effects caused by several factors, such as the noise produced by their work instruments. in 1959, the american dental association recommended periodical hearing assessments and the use of ear protectors. aquiring more information regarding dentists', dental nurses', and prosthodontists' hearing abilities is necessary to propose prevention measures and early treatment strategies. objective: to investigate the auditory thresholds of dentists, dental nurses, and prosthodontists. method: in this clinical and experimental study, 44 dentists (group i ; gi), 36 dental nurses (group ii ; gii), and 28 prosthodontists (group iii ; giii) were included,, with a total of 108 professionals. the procedures that were performed included a specific interview, ear canal inspection, conventional and high - frequency threshold audiometry, a speech reception threshold test, and an acoustic impedance test. : in the 3 groups that were tested, the comparison between the mean hearing thresholds provided evidence of worsened hearing ability relative to the increase in frequency. for the tritonal mean at 500 to 2,000 hz and 3,000 to 6,000 hz, giii presented the worst thresholds. for the mean of the high frequencies (9,000 and 16,000 hz), gii presented the worst thresholds. : the conventional hearing threshold evaluation did not demonstrate alterations in the 3 groups that were tested; however, the complementary tests such as high - frequency audiometry provided greater efficacy in the early detection of hearing problems, since this population's hearing loss impaired hearing ability at frequencies that are not tested by the conventional tests. therefore, we emphasize the need of utilizing high - frequency threshold audiometry in the hearing assessment routine in combination with other audiological tests.

visceral leishmaniasis or kala - azar, an infective disease encountered in tropical and subtropical regions of the world including indian subcontinent, is caused by protozoan parasite, leishmania donovani. l. donovani has two morphological forms in its life cycle: the motile flagellated slender promastigotes that stay inside the midgut of sandfly vector and the immotile nonflagellated oval amastigote present in the phagolysosomal complex of mammalian host macrophages. the survival and proliferation of the amastigotes followed by the reinfection of new macrophages by evading the host immune machinery ensure the progression of the disease in the mammalian host. overall annual prevalence of the disease is approximately 12 million people and the size of the population at risk is approximately 350 million. it is estimated that about 500,000 cases of visceral leishmaniasis occur worldwide each year. two closely related metalloids like arsenic and antimony are the drugs of choice to treat diseases caused by the kinetoplastid parasites. antimony and arsenic are members of the same group, xv, of the periodic table and are transported into cells by the same channels, carriers, and pumps. interestingly, arsenic - resistant leishmania species show cross resistance to antimony. previously, it has been shown that 30 g / ml of sodium arsenite has antileishmanial activity. however, this concentration of arsenic is reported to be toxic in several systems. the selectivity of these arsenical drugs towards trypanosomes rather than the host is apparently conferred by the aromatic ring structure. nowadays, nanomedicine is rapidly evolving as an extensively studied potent therapeutic tool. various exciting new works were done throughout the world in the interdisciplinary field of therapy for leishmaniasis using nanomedicine. so far, only a few reports are known describing the usage of metal nanoparticles as antileishmanial agents. despite usages of arsenic (iii) form as antileishmanial agent, use of arsenic nanoparticle (as - nps) is yet to be reported. in our laboratory, for the first time, stable yellowish - brown colored spherical as - nps have been synthesized. the aim of this study is to investigate whether the antileishmanial efficacy of as is enhanced in its nanoparticulate form. here , we have tested the effect of as - nps on the growth, oxygen consumption, infectivity, and intracellular proliferation of this parasite at a concentration which is manyfold less than that of as (iii). unless otherwise mentioned, all biochemicals such as fcs, antibiotics, and g418 were of highest quality and were purchased from sigma chemical co. (usa). promastigotes of l. donovani (mhom / in/83/ag83) were cultured at 23c in rpmi 1640 media containing 10% heat inactivated fcs (fetal calf serum), 25 mm hepes, 2 mm l - glutamine, 50 u / ml penicillin, and 50 g / ml streptomycin. parasites were inoculated at a density of 1 10 cells / ml and grown for 3 days to obtain exponentially growing log phase cells, which were used for subculture of the parasites. elemental grey arsenic, in the bulk stage (as4), is solid and insoluble in aqueous solution. under ordinary condition thus, the effect of elemental arsenic is difficult to evaluate in an aqueous media; that is, why arsenic can not be applied in the cell culture medium. arsenic is made water soluble only by making it a nanoparticle. as - nps used in this work were prepared using a simple wet - chemical procedure and characterized by sem and tem as published earlier. briefly, in a typical procedure, 1.0 ml of naaso2 (loba chemicals) having concentration 1.0 10 m was mixed with 12 ml of distilled water, and, to this solution, 0.6 ml of ice - cold nabh4 solution was added having concentration 1.0 10 m. the mixture was allowed to stand at room temperature for 2 hours when yellowish - brown colored as solution appeared. the solution was further heated to ~60c for 15 minutes and cooled to room temperature. sem, tem, and dls (malvern) measurements of the as solution indicated that the particles were of spherical shape, and the calculated average particle size was 76 nm (see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/187640). the dls measurement was carried out in a malvern nano zs instrument equipped with a 4 mw he - ne laser (= 632.8 nm) according to manufacturer's protocol. the zeta potential of the solution has been measured and found to be 18.1 mv, indicating that the particles are negatively charged. parasites were inoculated at a density of 0.5 10 cells / ml and incubated at 23c in 5 ml rpmi-1640 media for 24 hours. following incubation, 0.6 10 cells / ml were incubated further without and with different concentrations of as - nps. promastigote numbers were counted at an interval of 24 hours for 2 consecutive days in a neubauer improved counting chamber (haemocytometer) using a phase contrast microscope. ic50 value was determined by plotting log of cell numbers after 24 hours of incubation with as - nps versus concentrations of as - nps used. briefly, log phase leishmania cells (1 10 cells per each well) in 96-well plate were incubated without or with indicated concentrations of as - nps or as (iii) solution in growth media at 23c for defined time intervals. after the treatment, 10 l mtt solution (cck-8, sigma) was added to each well, following which, the readings were collected at an interval of one hour up to three hours with a microplate reader (biorad ; model no . 5000 raw 264.7 cells were seeded in each well of a 96-well plate and incubated for 24 hours at 37c . in the following day, these cells were treated for 24 and 48 hours with indicated concentrations of as - nps in fresh media at 37c . after the treatment, 100 l fresh media was added to each well followed by 10 l of mtt reagent ( cck-8, sigma), following which, the readings were collected at an interval of one hour up to four hours with a microplate reader (biorad ; model no . the quantitation of the viable cells in control and sets treated with different concentrations of as - nps was obtained from these average values ( o.d./o.d . a standard curve was generated to determine the equivalence of the cell numbers and o.d . p values were determined using paired t - test between untreated sets and sets treated with as - nps . transformations of empty egfp ( enhanced green fluorescence protein) plasmid into l. donovani promastigotes were performed by electroporation with a bio - rad gene pulsar apparatus using 450 v and 550 f capacitance. briefly, late log - phase promastigotes (0.51.0 10) were harvested at 1200 g (4c) for 10 min and washed twice in electroporation buffer (21 mm hepes, 137 mm nacl, 0.7 mm nah2po4, and 6 mm glucose, ph 7.4). cells were finally suspended at a density of 1 10/ml and 0.40 ml was taken into a 0.2 mm ice - chilled electroporation cuvette. thirty micrograms of plasmid dna dissolved in 40 l of electroporation buffer was then added to the cuvette and incubated on ice for 10 min. the cells were incubated for another 10 min on ice and added to 10 ml of drug - free growth medium (rpmi-1640). after 24 hours of revival, 20 g / ml g418 was added and the cells were kept at 26c for another 10 days with mild shaking. the presence of transfected cells was monitored visually by microscope (supplementary figure 2) and the drug concentration was increased gradually with each passage. finally, all the transfected cells were maintained in 300 g / ml of g418. consumption of oxygen by parasites was measured by gilson oxygraph (model : gilson oxygraph 56) according to the procedure published earlier. parasites inoculated at a density of 1 10 cells / ml were incubated at 37c for 24 hours. from there, 1 10 cells were further incubated without or with indicated concentrations of as - nps or as (iii) for defined time periods. following incubation, 1 10 parasite cells either treated or untreated were taken in pbs and transferred in the cell of the oxygraph to record the oxygen consumption. total oxygen dissolved in pbs inside the oxygraph cell was measured by the addition of sodium metabisulphite and is expressed as 100%. averages of three independent measurements of percent of oxygen consumed by the parasite cells were presented graphically. raw 264.7 cell line (atcc tib-71) that was used for the purpose of infection studies with leishmania donovani promastigotes is a transformed murine macrophage cell line routinely used by several researchers in this field. parasites stably expressing egfp (parasite / macrophage = 20 : 1). after 4 hours of incubation for internalization at 37c, the coverslips were washed twice with media to remove nonphagocytosed promastigotes. the plate containing coverslips were then incubated without or with indicated concentrations of as - nps or as (iii) at 37c for indicated period of time. infected cells and the parasite positive dots within the infected cells were visualized in gfp and uv filters (for dapi) in the nikon inverted fluorescence microscope (nikon eclipse ti - u). pictures were taken in tiff image format and were processed and merged in adobe photoshop cs5. from the images, total and infected macrophage cell numbers were counted. the numbers of gfp positive punctate dots representing internalized parasites (amastigotes) were also determined. the numbers of dots per 100 infected cells thus obtained were plotted against corresponding concentrations of as - nps and the ic50 value was determined accordingly from the graph. to investigate the attachment after addition of the parasites to the macrophages, subsequent processing was similar to that of the infected sets. to see the curing effect of these nps, as - nps (2 m) were added to the 24-hour postinfected macrophages and were further incubated for 24 and 48 hours. numbers of total cells and average numbers of parasite positive (+) dots (from three independent experiments) within each infected cell were computed and represented graphically. statistical significance of the obtained was determined using the following analysis: standard deviation, paired t - test, unpaired t - test, and fisher exact test. the effect of different concentrations of as - nps and as (iii) on growth of l. donovani promastigotes was quantitatively determined by haemocytometer under a phase contrast microscope. the ic50 value of the as - np that was found to be 2.37 m was determined using the parasite cell numbers obtained from the 24-hour sample (methods and figure 1(a) ). to further confirm our , the cytotoxicity of as - nps and as (iii) on the promastigotes was assayed by mtt. our clearly demonstrated that treatment with 2 m as - nps for 72 hours has affected the growth of promastigotes most prominently in comparison with other lower concentrations of as - nps (0.1 and 1.0 m) (figure 1(b)(i) ). whereas as (iii) at the same range of concentration imparted minor effect on the survival of the parasites (red, blue, and green lines in figure 1(b)(ii) ), similar killing effect was only observed by treatment with 1020 m of as (iii) which is much higher than that of as - nps (figure 1(b)(ii) ). the of the mtt assay correlated with that of the cell counting by haemocytometer. having shown growth inhibitory activity on the promastigotes, both as - nps and as (iii) were tested for their effect on the parasite respiration. the respiratory activity of l. donovani parasites was directly assessed by measuring the percent of oxygen consumed using an oxygraph as described in materials and methods. oxygen consumption by the parasites decreased gradually upon treatment with 2 m as - nps for 24, 48, and 72 hours as compared to that by the cells left untreated (figure 2(a) ). however, as expected, the parasites treated with 2 m as (iii) showed almost no effect in their oxygen consumption (figure 2(b) inset ). even 10-fold higher concentration of as (iii) was shown to have much less effect on the respiration of promastigotes (figure 2(b) ). the effects of as - nps and as (iii) on the oxygen consumption by the parasites were found to be statistically significant (tables 1(a) and 1(b) ). the inhibitory effects of as - nps on the growth and metabolic activity of the parasites led us to investigate their effect on the infectivity of leishmania parasites in vitro. we have also compared the effect of as - nps and as (iii) on the infectivity of promastigote. the macrophages that were infected with promastigotes in presence of as - nps had modest number of gfp positive puncta (figure 3(a); panels (f) and (h) ) as compared to the set without as - nps indicating reduction of parasite burden in the macrophages (figure 3(a); panels (b) and (d) and table 2(a) ). however, in presence of as (iii), the numbers of internalized parasites (amastigotes) were very similar to those found in control sets (figure 3(a); compare panels (j), (l) and (b), (d) with (f), (h) ). ic50 values that were determined using range of concentrations of as - nps in the infection studies (37c for 24 hours of incubation) were found to be 1.5 m (figure 3(b) ). moreover, the number of fluorescent parasites attached on macrophage surface was also reduced by the treatment with the nanoparticles indicating an inhibition in the attachment of the parasites to the macrophages prior to internalization (figure 3(c); compare panels (b) and (d) and table 2(b) ). these experiments were repeated several times and ed in very similar observation. in the preceding section, we have demonstrated the suppressive effect of as - nps on the attachment and infectivity of the parasite. furthermore, we asked whether the nanoparticles can exert effect on the postinfection proliferation of the parasites inside the macrophages. to address that, 24-hour postinfected macrophages were treated with as - nps for another 24 and 48 hours (figure 4(a); panels (g), (h) and (i), (j), resp. ). our clearly showed that as - nps can also block parasite proliferation inside the macrophage significantly (figure 4(a): compare panels (d), (f) with (h), (j) and figure 4(b) ). while the parasite positive fluorescent puncta in as - np untreated cells increased significantly over time (figure 4(a); panels (b), (d), and (f) ), the parasite positive dots in the infected cells that were treated remained static (figure 4(a): compare panels (b), (d), and (f) with (h) and (j) and figure 4(b): in treated samples: parasite positive dots ). repeated addition of fresh as - nps after 24 hours of the first addition did not yield much effect (data not shown). the of this experiment is satisfactorily reproducible and the p values for this experiment that were determined by fisher exact test are statistically significant (figure 4(b) ). here, to explore the possibility whether the as - nps treatment had any effect on the macrophage itself, several microscopic images of nanoparticle treated macrophages were captured. figure 5(a) shows representative of such images which clearly indicate that there is no change in the morphology (phase and dapi) of macrophages treated with as - nps. additionally, we have checked the viability of the macrophages treated with the nanoparticles by mtt assay. figure 5(b) shows that as - nps treatment has ed in 25% loss in viable cells as compared to the untreated control. it is to be noted that the viable cell number in the control set (~4000) showed a reduction from the initially seeded cell number due to loss during the processing required for this experiment. it exists in nature mostly as two biologically relevant oxidation states: arsenate and arsenite. in proteins, as (iii) binds to both cysteine and histidine residues and causes depletion of intracellular glutathione. in the pentavalent form , arsenic is known to compete with phosphate, particularly in the citric acid cycle to alter the production of atp. several studies on the antibacterial, antiviral, and antifungal activities of arsenic have been carried out. according to a previous report , 30 g / ml of as (iii) (equivalent to about 230 m) is shown to be antileishmanial. this concentration of as is very toxic in mammalian system. the present study is the first to demonstrate that the novel as - nps possess better antileishmanial efficacy than that of as (iii). it is noteworthy that the concentration at which the as - nps imparted significant inhibitory effect on the viability, oxygen consumption, and infectivity of the parasite is too low for as (iii) to show any significant inhibitory effect on the parasites (figures 14). moreover, the antileishmanial effect comparable to that of as - nps is only achieved by at least 10-fold concentrated as (iii). ic50 value determination shows that the amastigotes are more sensitive to the as - nps than the promastigotes. this data indicates that the as - nps will be equally effective as an antileishmanial agent in vivo. furthermore, the negatively charged nanoparticles as indicated by the zeta potential value would possibly face no problem to reach the phagolysosomes of macrophages in the infected internal organs, namely, liver and spleen. while the cellular and nuclear morphologies of the host macrophage cells treated with as - nps remain unchanged (figure 5(a) ), the viability of the treated macrophages was reduced only by 25% (figure 5(b) ). it is noteworthy that 1 m as (iii) was shown to have severe toxicity in vitro and in vivo. moreover, it is not intuitive to predict the extent of its toxicity in vivo from the in vitro observation. there are reports where it was shown that the levels of toxicity between these two systems vary significantly. taken together , our findings suggest that the as - nps have very good potential as compared to the as (iii) to be developed as an effective antileishmanial agent with much less effect on the host macrophages. it is evident that the pathogenesis depends on the proliferative capacity of the parasite inside the macrophages. if, somehow, this proliferation can be controlled, the pathogen can be rendered ineffective. the main problem of the conventional antileishmanial drugs for leishmaniasis is their toxicity and high prices. so, the development of new drug(s) which would be cheaper with lesser side effects is a long lasting requirement. arsenic (iii) at a relatively higher concentration (30 g / ml) has been shown to have antileishmanial activity; but this concentration is reported to be toxic in several experimental mammalian systems. we have shown that the as - nps, developed for the first time in our laboratory, significantly inhibited the oxygen consumption and intracellular proliferation of the parasitic pathogen. the concentration of as - nps is about several fold lower than that of as (iii). moreover, this antileishmanial activity has comparatively less cytotoxic effect on the mouse macrophage cell line. thus, it is evident from our findings that as - nps have more potential than as (iii) to be developed as a cheap and less toxic antileishmanial agent.

visceral leishmaniasis, a vector - borne tropical disease that is threatening about 350 million people worldwide, is caused by the protozoan parasite leishmania donovani. metalloids like arsenic and antimony have been used to treat diseases like leishmaniasis caused by the kinetoplastid parasites. arsenic (iii) at a relatively higher concentration (30 g / ml) has been shown to have antileishmanial activity, but this concentration is reported to be toxic in several experimental mammalian systems. nanosized metal particles have been shown to be more effective than their higher oxidation state forms. there is no information so far regarding arsenic nanoparticles (as - nps) as an antileishmanial agent. we have tested the antileishmanial properties of the as - nps, developed for the first time in our laboratory. as - nps inhibited the in vitro growth, oxygen consumption, infectivity, and intramacrophage proliferation of l. donovani parasites at a concentration which is about several fold lower than that of as (iii). moreover, this antileishmanial activity has comparatively less cytotoxic effect on the mouse macrophage cell line. it is evident from our findings that as - nps have more potential than as (iii) to be used as an antileishmanial agent.

low back pain is a disease that frequently occurs in daily life. apart from headache, it is the most common pain experienced by humans. approximately 80% of people in the entire population experience low back pain at least once during their lives. this pain is one of the factors that greatly affect the quality of life of patients because it leads to socioeconomic problems associated with increases in the costs of treatment, together with problems in daily living activities1. simple low back pain can often be prevented by forming the habit of sitting with a proper posture and avoiding maintaining a sitting posture for prolonged periods. however, many types of low back pain are treated with therapeutic methods that are as diverse as the causes of low back pain. in most cases, therapeutic methods such as bed rest, assistive devices, traction treatment, hyperthermia, electric stimulation, and manual therapy are the first choice treatments2. when these fail, invasive therapeutic methods such as selective nerve root blocks and epidural injections are used, and surgery is performed if no response to the other treatments is seen, or if the condition of the disease is serious3. recently, new conservative treatments have been adopted, including extracorporeal shock wave therapy (eswt)4. eswt is a therapeutic method that applies shock waves to lesions from the outside of the body to promote revascularization and to stimulate or reactivate the curing process of connective tissues including tendons and bones, thereby relieving pain and improving function5. eswt can be used for pain relief as well as for muscle strength improvement through appropriate motor stimulation of muscles and tendons by the shock waves6. currently, eswt is administered for musculoskeletal system diseases, but studies of the effects of eswt on chronic low back pain are rare, and few studies have examined its effects on pain, disability, and depression. the purpose of this study was to examine the effects of extracorporeal shock wave therapy on pain, disability, and depression of chronic low back pain patients. this study was conducted with 30 patients (male : 9, female : 21) who had been diagnosed as having chronic low back pain by specialists in orthopedics, who had low back pain that had persisted for at least three months, and who had visited s orthopedics hospital located in daegu, korea. the subjects were divided into a conservative physical therapy group (cptg ; n=15 ; mean age 46.08.9 years, mean height 163.79.1 cm, and mean weight 61.914.3 kg) and an extracorporeal shock wave therapy group (eswtg ; n=15 ; mean age 49.78.3 years, mean height 161.35.9 cm, and mean weight 62.29.3 kg). ethical approval for the study was granted by the youngdong university institutional review board. the study subjects had not received any surgery to the lumbar vertebrae region; had no vertebral compression fractures, spinal tumors, or intervertebral disc infections; were free of inflammatory diseases such as rheumatism; and had no heart disease or structural abnormalities. the intention of this study and the experimental procedures were sufficiently explained to the study subjects and their voluntary agreement to participation was obtained before conducting the experiment. a vitera (comed, korea) was used to conduct extracorporeal shock wave therapy for eswtg. each patient assumed a prone position, and1,000 shock waves (7 times per sec) were applied at 2.5 hz at low energy flux densities of 0.010.16 mj / mm using a 17 mm head. the treatment was conducted at the quadratus lumborum muscle and the sacroiliac joint, where the patients complained of pain. cptg was treated with hyperthermia using hot packs (20 minutes), ultrasound (5 minutes), and electrotherapy using tens (15 minutes). this scale has 10 items which are scored on a scale of 05 points based on functional performance, with higher scores indicating more severe disabilities. in this study, nine items were assessed, excepting the item for sex life considering subjects without spouses. the scores measured for individual items were summed and the were divided by the maximum score of 45 points. depression was assessed using the beck depression index (bdi) developed by beck et al7. scores from 09 indicate conditions free of depression, from 1015 indicate slightly depressed conditions, from 1623 indicate quite depressed conditions, and from 2463 indicate seriously depressed conditions. for statistical processing , the degrees of the chronic low back pain patients pain, disability, and depression were examined using the paired sample t - test to analyze intra - group differences and the independent sample t - test for analysis of intergroup differences. spss 12.0 for windows was used for statistical processing and the significance level, , was chosen as 0.05. intra - group comparisons revealed significant decreases in vas, odi, and bdi scores of eswtg and the cptg at the end of the experimental period. intergroup comparisons showed that the decreases in vas, odi, and bdi scores were larger in eswtg than in cptg (table 1table 1.intra- and inter - group comparisons of vas, odi, and bdi scoresgroupprepostvas ( point)cptg**6.61.54.91.3eswtg**7.00.763.61.1odi (%) cptg**30.411.725.011.4eswtg**30.112.417.58.1bdi (point)cptg**18.76.416.34.5eswtg**19.66.712.55.7vas: visual analog scale, odi: oswestry disability index, bdi: beck depression index, cptg: conservative physical therapy group, eswtg: extracorporeal shock wave therapy group, *: paired t - test,: independent sample t - tests: p<0.05,: p<0.01 ). vas: visual analog scale, odi: oswestry disability index, bdi: beck depression index, cptg: conservative physical therapy group, eswtg: extracorporeal shock wave therapy group, *: paired t - test,: independent sample t - tests: p<0.05,: p<0.01 chronic low back pain that brings about discomfort and inconvenience in daily life tends not to respond to conservative therapy in many cases, which has prompted a search for other therapeutic methods. rompe et al.8 reported that when one - off wave therapy was implemented for patients with calcific tendinitis of the shoulder, 60% of the patients recovered to their normal states and 72% of the patients recovered to the extent that they felt only intermittent discomfort as assessed by the constant and murley scale. lee sb5 reported that when eswt was applied to patients with lateral epicondylitis of the elbow joint, the patients pain was relieved, their simple elbow test scores improved significantly, and 83% of the patients showed satisfactory outcomes. cho et al.9 reported that when patients with lateral epicondylitis of the elbow joint were treated with eswt, the patients pain significantly decreased and their muscle strength increased significantly. na jy3 found that when eswt was administered to chronic low back pain patients, the patients pain significantly decreased. in addition, some researchers have suggested that eswt is an effective and non - invasive new therapeutic method for the treatment of lateral epicondylitis of the elbow joint that does not respond to conservative therapy. lee et al.10 reported that when eswt was implemented for chronic low back pain patients, exercise programs used together with extracorporeal shock wave therapy relieved pain and improved dynamic balance ability to a greater extent than was seen with a combination of exercise programs and conservative physical therapy. the intra - group comparisons in the present study revealed that there were significant decreases in the vas, odi, and vas scores of eswtg and cptg at the end of the intervention. the intergroup comparison revealed that these decreases were larger in eswtg than in the cptg. rompe et al.8 advised that minute and repetitive stimuli applied through shock wave therapy were effective at relieving pain. hammer et al.11 recognized that breaking calculi and gallstones with extracorporeal shock waves increases the blood flow rate and rate of revascularization, and used this effect to stimulate and activate the curing processes of tendons, surrounding tissues, and bones to remove and stabilize inflammations in the tendons and ligaments, which ed in pain relief. the of the present study also indicate that decreases in vas scores were occurring through mechanisms described by presented in previous studies. in addition, eswt appeared to relieve chronic low back pain, thereby improving the patients physical functions and consequently their ability to perform daily living activities, leading to significant decreases in low back pain disability indexes. holmes12 argued that the limitations or disability in patients daily living would cause psychosocial problems that would further impair the quality of their lives. depression is closely related to physical disability and chronic pain in chronic disease patients, and it is an important element that interferes with pain relief by delaying recovery or aggravating diseases13. in the present study, eswt relieved pain and reduced disability indexes, thereby improving the patients physical functions and ability to perform daily living activities, which in turn had positive effects on the patients psychological, emotional, and cognitive states, decreasing the degree of their depression. decreased pain in chronic low back pain patients is a more influential factor than disability and depression. first, the number of chronic low back pain patient subjects was limited, so the can not be generalized. second, the lumbar lesion sites were limited and the subjects daily lives could not be completely controlled. third, no previous studies have measured the effects of eswt on depression, so our present could not be compared with those of other studies.

the purpose of this study was to examine the effects of extracorporeal shock wave therapy on pain, disability, and depression of chronic low back pain patients. in this study, 30 chronic low back pain patients were divided into an extracorporeal shock wave therapy group (eswtg, n=15) and a conservative physical therapy group (cptg, n=15). the eswtg received extracorporeal shock wave therapy and the cptg received general conservative physical therapy two times per week for six weeks. pain was measured using a visual analog scale (vas), the degree of disability of the patients was assessed using the oswestry disability index (odi), and their degree of depression was measured using the beck depression index (bdi). in intra - group comparisons, eswtg and cptg showed significant decreases in vas, odi, and bdi scores. intergroup comparisons revealed that these decreases in vas, odi, and bdi scores were significantly larger in eswtg than in cptg. extracorporeal shock wave therapy is an effective intervention for the treatment of pain, disability, and depression in chronic low back pain patients.

percutaneous renal biopsy has become an essential tool in the diagnosis and treatment of patients with renal disease, and although less invasive, it is fraught with potential complications. introduction of realtime ultrasound and automated biopsy needles increased quality of specimens and dramatically reduced the incidence of biopsy - related accidents. nonetheless, the potential for serious complications still remains. on average, clinically significant complications occur in 7.4% of cases, but also complications rates as high as 19.5% have been reported. many variability in complication incidence attains certainly to operator experience, but it is our opinion that a part of this heterogeneity is linked to the attention paid in planning correctly the optimal site of puncture, in order to avoid undesired puncture of anomalous vessels. while preprocedural ultrasonography is in fact widely used to assess the anatomical landmarks of the kidney and to exclude anatomical conditions considered as absolute contraindications, to date, power and colour doppler (cd) examination are used less frequently in this setting, if not only to discover postprocedural complications. in our experience, anomalous perirenal vessels were present in many biopsies complicated by renal bleeding. in our department, until august 2000, we performed a total of 332 biopsies, using b - mode ultrasonography, only occasionally integrated by cd study, in planning procedures. from september 2000 if this systematic approach reduced or not complication incidence after procedure, we designed a retrospective analysis of observed complications before and after the systematic introduction of cd study in the preparation of renal biopsy. present analysis includes 561 consecutive ultrasound - guided percutaneous renal biopsies performed in our department from august 1995 to december 2009 from the same operator. until august 2000, a total of 332 biopsies were performed, after a b - mode ultrasonographic study of kidneys. from september 2000 all patients (total 229) underwent a preliminary systematic cd study prior to biopsy. in these patients, vessels in the region of the biopsy low pulse repetition frequency were used, between 750 and 1000 hz. 332 patients biopsied before september 2000 were named group a. the group of 229 patients systematically cd - studied was named group b. demographical data are listed in table 1. postbiopsy complications were categorized as either minor (gross hematuria and/or subcapsular perinephric hematoma < 4 cmq of greater diameter) or major (> 4 cmq of greater diameter ; requiring transfusion of blood products or invasive procedures ; leading to acute renal failure, urine tract obstruction, septicaemia, or death). to minimize the risk of bleeding, prothrombin time, partial thromboplastin time, bleeding time (ivy), and salicilate, ticlopidine, and nsaids were withdrawn for at least 7 days before biopsy. use of heparin was prohibited two days before biopsy. in case of arterial hypertension under treatment, biopsy was performed only when normotensive range was achieved (less then 140/90 mm / hg). patients with renal cortex width < 10 mm and/or < 90 mm of kidney longitudinal diameter were not considered suitable for renal biopsy. all biopsies were performed using realtime ultrasound - fixed guidance (3.5 mhz convex probe, technos mp ; esaote, italy), and 16-gauge automated spring - loaded gun. in all cases the lower pole of kidney was used for biopsy, mainly at left side. only in cases in which cysts, small cortex, peri - renal vessels were detected in this location, the right kidney was biopsied. in group b, the presence of medium - size arteries / veins at lower pole of left kidney was an indication to perform biopsy on right kidney. biopsy was performed in prone position with patients lying with the abdomen on a firm pillow. the most appropriate needle - skin angle was selected by adjusting the puncturing adaptor (usually 20 ; see figure 1). after disinfection of the skin, lidocaine (1%) was applied locally under ultrasound control along the needle insertion tract. a small incision of the skin was made to facilitate the introduction of the biopsy needle. under us imaging guidance the needle was advanced at an angle of 20until the capsule of the lower kidney pole had been reached. patients were asked to hold their breath, the biopsy device was unlocked and tissue obtained. the sampling time was < 1 s. length of the obtained biopsy specimens was usually 1820 mm and at least two samples were taken. following the biopsy, patients lay in bed on their backs for a 24-hour observation time. during this time, both clinical (gross hematuria, flank pain, and hypotension) and imaging test (2-d realtime sonography, cd sonography, and power doppler sonography) were performed in order to evaluate the presence and sizes of potential bleedings. statistical analysis was performed through statistica 5.0 (statsoft inc .), using fisher's exact test. histological analysis, including conventional light and immunofluorescence microscopy, could be performed by the pathologist in 100% of cases. all complications registered during the study are reported in table 2. in group a an arteriovenous fistula was observed in one patient, and spontaneously resolved 18 months after procedure. in group one of this two requested an angiographic procedure to stop bleeding, through gianturco method. three major hematomas (> 4 cmq) were observed in group a, while only one in group b. in none of these cases surgical procedures were indicated after urological consulting. minor clinically significant complications occur in 7.8% of cases in group a and in 3.4% of cases of group b. microhematuria was highly frequent but it was not intended as a complication. prebioptic cd evaluation showed anomalous vessels at the lower pole of the right kidney in 15 patients and at the lower pole of left kidney in 19 patients, respectively, as shown in table 3. in 15 of 19 patients with anomalous vessels at the left kidney , it was impossible to identify an alternative trajectory for the needle (figures 1 and 2) and biopsy was carried out on the right kidney. in the other 4 cases was sufficient to accurately study the trajectory. only one patient in group a underwent right kidney biopsy, showing a large inferior pole simple cyst. anomalous interlobar vessels were observed in 9/229 pts while inferior pole arteries in 6 patients. cumulatively 23 pts (about 10% of whole group b) showed vascular abnormalities potentially at risk of postprocedural bleeding. several authors reported a drastic decrease in the incidence of complications after renal biopsy when procedure is guided by ultrasonography. systematic use of colour doppler ultrasonography in monitoring patients after a kidney biopsy, is crucial for an early diagnosis of postbiopsy av fistula. perhaps if the importance of colour doppler ultrasonography in postprocedural evaluation is clear and commonly accepted, its usefulness in preparing a biopsy is yet to be appreciated. this study, for the first time, showed a systematic, preparatory use of cd reduced postbiopsy complications incidence, in the same unit and from the same operator. it is to be underlined that during the time course of this study, the operator experience in biopsy practice grew, probably influencing in some part the of this study. perhaps it is obvious that this study has all limitations of a retrospective analysis. on the other hand it appears acceptable that preliminary cd study helped operators to identify the safest needle trajectory to the kidney, in some cases indicating to prefer right organ for biopsy, even in absence of other ultrasonographic indications to avoid left kidney puncture. data registered in our clinic before the default use of cd study showed an incidence of major and minor complications absolutely in line with those reported in the literature. cd module is largely available in most of ultrasonographers in use in biopsy - providing units. it does not require the exposition to ionizing radiations and does not expose patient to any kind of risk. perhaps it is an inexpensive tool, in term of money, and its systematic use requests little effort to the nephrologist in term of time. its systematic use can reduce complications, and then costs, frequently as high as dramatic from an ethical point of view. in other words it is an easy, useful hand and sure tool in percutaneous renal biopsy. on the other hand cd technique is highly operator - dependent, requesting experience and skill to warrant affordable reliefs. it is then to be underlined that cd module has to be used when the nephrologist plans a biopsy, to design ideal needle trajectory, and it is turned off when needle starts its descent to the kidney. in this phase, obviously, cd could be only a confounding factor for the operator, often used to vibrate needle to make it visible on ultrasonographic screen. this study opens a window on a new aspect in planning kidney biopsies: the presence of anomalous vessels potentially on the trajectory of biopsy needle. the systematic study of kidneys showed in fact anomalous vessels in about 10% of patients, giving a potential explanation to the observed reduction in complications rate in cd - studied patients. it is our opinion that this aspect could have more consideration in planning the better strategy for kidney biopsy. our data show clearly as the same operator, in the same setting, could obtain better and drastically reduce complications only enriching preliminary study with cd. it is obvious to remind to the reader as vascular structures, particularly in case of small ones, can slip from the attention even of a well - skilled operator using only b - mode ultrasonography. cd can help him to better clarify their nature and to identify potential sources of risk in his strategy to biopsy. on the basis of our data, clinically significant postbiopsy haematomas experienced nephrologist, planning a site of puncture for biopsy, has to take care of them and design a strategy to reach renal cortex without undesired experiences.

while ultrasonography is widely performed prior to biopsy, colour doppler examination is often used only to discover post - biopsy complications. aim of this paper was to evaluate the usefulness of colour doppler examination in planning the optimal site of puncture for renal biopsy. present analysis includes 561 consecutive percutaneous renal biopsies performed from the same operator. until august 2000 332 biopsies were performed after a preliminary ultrasonography (group a). from september 2000, 229 patients underwent even a preliminary colour doppler study (group b). postbioptic bleeding were categorized as minor (gross hematuria or subcapsular perinephric hematoma < 4 cmq of greater diameter) or major (hematoma > 4 cmq of greater diameter ; requiring blood transfusion or invasive procedures ; leading to acute renal failure, urine tract obstruction, septicaemia, or death). major complications were seen in 2.1% in group a while in group b only one case was reported (0.43%). minor clinically significant complications occur in 7.8% in group a and in 3.4% of cases of group b. colour doppler reduced drastically the incidence of complications observed before the introduction of routine colour doppler examination prior to biopsy. in our opinion, these data support the use of preliminary colour doppler study when a biopsy is planned.

in drosophila, widely - used mitotic recombination - based strategies generate mosaic flies with positive readout for only one daughter cell after division. to differentially label both daughter cells , we developed the twin spot generator technique (tsg) and demonstrate that through mitotic recombination, tsg generates green and red twin spots in internal fly tissues, visible even as single cells. we discuss the wide applications of tsg to lineage and genetic mosaic studies.

subacute granulomatous thyroiditis (sgt), also known as de quervain's thyroiditis, is a self - limiting, inflammatory disease of the thyroid that is believed to be caused by a systemic viral infection. it was first diagnosed in 1825, and 18 cases were reported as thyroiditis acuta simplex until 1895. it typically occurs in the area of the gland in mid - aged hyperthyroid women complaining of pain, tenderness, fatigue and mild fever. it is usually clinically diagnosed without the need for fine - needle aspiration (fna). therefore, there are few studies regarding the cytological features of sgt in the literature. however, it is important to exclude other diseases of the thyroid because sgt may be confused with them, particularly with malignancies. the objective of this study is to define the ultrasonographic (usg) and cytopathologic characteristics of sgt and highlight its differential diagnosis from the other lesions of the thyroid. a total of 21 cases diagnosed with sgt between 2001 and 2014 were included in our study. clinical data, such as the patients ages, clinical findings and clinical preliminary diagnoses and the usg findings, were obtained retrospectively from the pathology reports. cytological preparations were re - evaluated in terms of cellularity, multinuclear giant cells (mngcs), granulomatous structures, histiocytes, colloids, follicular epithelial cells, fibrous fragments and inflammatory cells. fine - needle aspirations were performed by a cytopathologist (np), who was accompanied by a radiologist (ky), and with ultrasonography guidance, by using a 22-gauge needle, 10 cc syringe and aspirator. on average, the procedure was concluded upon the detection of a sufficient number of air - dried preparations stained with the diff - quik dye at the bedside. the clinical and cytological characteristics of the 21 cases included into the study have been summarized in table 1. clinical and cytopathological features of subacute granulomatous thyroiditis all, except two cases, were women (90%) and the mean age was 38.19 years (1850 years). seven cases presented with a pain complaint in the area of the thyroid lasting a few weeks, whereas 2 cases had pain and previous upper respiratory tract infection and 1 case had a history of sgt that had occurred 3 months previously. the preliminary clinical diagnosis was sgt in 2 cases, thyroiditis in 6 cases, multinodular goiter (mng) in 4 cases, thyroid carcinoma in 4 cases and lymphoma in 1 case. the usg findings of the other 20 cases have been summarized in table 2. hypoechoic thyroid nodules with irregular margins were observed in 12 cases at usg, whereas heterogeneous, hypoechoic areas (lava flow) with indistinct margins were observed in 7 cases. in 1 case (case 16), one of the bilateral and multiple lesions, which located on the right side, appeared with a hypoechoic area, and the others presented as nodular lesions. in the 13 cases that presented as nodular lesions, the nodules were generally unilateral and single, with the largest mean size being 22.19 mm (range : 740 mm), whereas the largest mean size of the hypoechoic - heterogeneous areas was 27.52 mm (range : 1075 mm). ultrasonographic findings of subacute granulomatous thyroiditis a subacute granulomatous thyroiditis case sonographically characterized by an ill - defined hypoechoic nodule (case no . the cytologic features of the case included the following : ( b) multi - nucleated giant cell (papanicolau stain, 400), (c) an epithelioid histiocytic granuloma (papanicolau stain, 400), (d) a group of epithelioid histiocytes adjacent to a giant cell (papanicolau stain, 400) and (e) a collection of proliferated follicular cells (papanicolau stain, 200) a subacute granulomatous thyroiditis case with a sonographic image of a poorly defined hypoechoic area. the cytologic features of the case included the following: (b) multinucleated giant cell (diff quik, 400), (c) an aggregate of epitheliod histiocytes consistent with granuloma (diff quik, 400), (d) a cluster of epithelioid histiocytes (papanicolau stain 400) and (e) a group of proliferated follicular cells (diff quik, 400) cytologically, the diffusions were generally normocellular or moderately cellular, and there was no highly dense (tumoral) cellularity. all cases presented with follicular epithelial cells, although this was rare in some such cases. the cells were generally shaped as honeycomb fragments, with a partially degenerated and proliferated appearance, and it was noted that reactive atypia accompanied 1 case (case 15). however, no papillary or microfollicular structuring, plasmacytoid appearance or intranuclear inclusion or groove was observed in any sample. the mngcs were of variable sizes and forms, contained a cytoplasmic content and were generally large. phagocyted colloid and neutrophils appeared in the cytoplasm of some mngcs. in rare cases, there were also some smaller, round, vacuole - cytoplasm mngcs that contained less nucleus. though there were epitheloid histiocytes and clusters of epitheloid histiocytes, which were believed to be granulomas, in 10 cases, the epitheloid cells were isolated or formed loose cohesive clusters in 3 cases. the granulomas that were assessed appeared similar to those in other granulomatous diseases, specifically, to be derived from bare - foot or carrot - shaped nucleus cells constituting tight syncytial groups of variable sizes. this colloid generally appeared in thick, small and large masses inside epithelioid histiocytes, some of which occurred in a phagocytosed manner. all cases had a dirty that was composed of cellular debris, blood elements and pink amorphous acellular material, and this was accompanied by neutrophil leukocytes in 2 cases and mixed types of inflammatory cells with dominant lymphocytes in the remaining cases. the inflammatory diseases of the thyroid gland are classified into three groups, that is, acute, subacute and chronic. de quervain's thyroiditis, also named giant - cell or granulomatous thyroiditis, is the subacute inflammation of the thyroid and constitutes nearly 36% of all thyroid diseases. it is a nonspecific inflammation that generally appears 2 weeks after a viral upper respiratory tract infection and regresses spontaneously within 23 months. although its etiology remains unknown, it is believed have a viral origin, similar to the mumps virus, hepatitis b and c viruses, cytomegalovirus enterovirus, and type a and b coxsackie viruses. thyroiditis predominantly affects adult females, and the ratio of females to males is 2:16:1. clinically, patients present with localized anterior neck pain associated with glandular tenderness and diffuse pain in the ears and the jaw, which is accompanied by fatigue, weight loss, low - grade fever, elevated erythrocyte sedimentation rate, suppression in the thyroid stimulating hormone level and occasionally dysphagia. the thyroid is sensitive at palpation, and there is a mild - moderate growth in the thyroid. depending on the stage of the disease, hyperthyroidism, hypothyroidism or euthyroidism may be seen in patients. the patients recover spontaneously after weeks or months, and recovery may be supported by symptomatic treatment with nonsteroid anti - inflammatory drugs or, occasionally, cortisone. generally, an increase in the size of the thyroid and heterogeneous, diffuse, hypoechoic and confluent areas with negative margins, characteristically defined as lava flow, are observed on usg. however, these findings are nonspecific because they may also be observed in lymphocytic thyroiditis, mng and graves diseases, and the clinical findings may assist in the differential diagnosis. sgt may also present with nonsensitive solid nodules. unlike the literature findings related to this topic, the number of cases appearing as nodules in our study (13 cases) was relatively higher than the number of cases appearing as hypoechoic areas (7 cases). additionally, most of our cases were unilateral. due to the risk of infiltrative malignancies, such as generalized parenchymal abnormalities, which can be interpreted as diffuse nonneoplastic thyroid disease, including sgt, repeated usg and even fna should be performed to exclude other malignancies in sensitive or nonsensitive hypoechoic and solid nodules. due to the rare however, the frequency of cytopathological use has increased with the development of usg approaches. fine - needle aspiration is beneficial in the diagnosis of thyroiditis and may provide very accurate . fna plays an important role for accurate diagnosis, particularly when clinicians do not suspect sgt or in atypical cases. the fact that among all of our cases, there is no distinct clinical finding that may help us, except for pain in the area of the thyroid in 7 cases, pain and previous upper respiratory tract infection in 2 cases, and the presence of a history of sgt in 1 case as well as the absence of malignancies among clinical differential diagnoses in 5 cases; this reveals the importance of fna. the key cytological characteristics for sgt are as follows: (i) a high number of mngcs, (ii) epithelioid cells with a tendency for clustering, (iii) epithelioid cell granulomas, (iv) lymphocytes, macrophages and neutrophils, (v) frequently degenerated follicular epithelial cells displaying mild - moderate cellularity, and (vi) a dirty that is composed of cellular debris, naked, degenerated nuclei and thick colloids. the absence of one or a few of these does not exclude sgt but instead increases the number of thyroid diseases that are included in the scope of the differential diagnosis. the characteristic cytological feature of sgt is the presence of mngcs, which may comprise hundreds of large nuclei and contain colloidal residues in their cytoplasm. mngcs may be seen inflammatory conditions of the thyroid (granulomatous diseases, hashimoto 's thyroiditis, graves disease and palpation thyroiditis), hyperplastic conditions (mng displaying degenerative changes) and neoplastic conditions (papillary carcinoma, anaplastic carcinoma). (a) multinucleated giant cell secondary to a benign cystic nodule (our archival case) (diff quik, 400). (b) bizarre giant cells seen in a case papillary thyroid carcinoma (our archival case) (papanicolau stain, 400). (c) (d) multinucleated giant cell with an intracytoplasmic colloid like material (arrow) seen in one of our subacute granulomatous thyroiditis cases (case no . 21) (papanicolau stain, 400) two morphological types mngcs can be observed in fna samples: those with foamlike cytoplasm and those with dense cytoplasm. mngcs with foamlike cytoplasm are round cells with less diagnostic importance and are generally seen in cystic degeneration. they are usually composed of multinuclear forms of histiocytes, flat cytoplasmic contours, foamlike, vacuole and hemosiderin cytoplasms. further, mngcs with dense cytoplasm have angular cytoplasm and irregular shapes, are larger and contain a higher number of nuclei. primarily, the three following conditions should come to mind in diffusions containing mngcs: (i) sgt, (ii) granulomatous diseases, such as sarcoidosis, tuberculosis and fungal infections, and (iii) papillary carcinoma of the thyroid (pc). the presence of tumor cellularity and pc nuclear characteristics (powdery chromatin, nuclear groove and intranuclear inclusions), overlapping syncytial tissue fragments, real papillary fragments, if any, and psammoma bodies are characteristics in favor of pc. the mngcs that they contain are smaller in number and size compared with sgt and granulomatous diseases. these cells are called bizarre giant cells. however, in some pc cases, mngcs of sgt can be seen in addition to the bizarre giant cells. dirty with clinical, well - formed or poor - formed epithelioid granulomas and acute - chronic inflammation is included in the differential diagnosis for sgt. the mngcs that are present have a tendency to be more frequent and larger in size and to contain more nuclei. the main cells in the granulomatous diseases of the thyroid are epithelioid histiocytes, and the granulomas observed are generally well formed, comprise fewer lymphocytes and acute inflammatory components and do not contain a dirty . however, it should also be highlighted that mngcs may not always be seen in sgt aspirates, may sometimes be too few and scattered, or may very rarely be seen in sgt as intensely as they appear in other thyroid diseases. granulomas, which appear as epithelioid histiocyte clusters, are another key characteristic for sgt but are rare or may not be seen. different rates have been reported with regard to the presence of granulomas in sgt in different studies in literature, and it was suggested that these different rates were due to the association with the stage of the disease or a sampling error. epithelioid granulomas were defined in many cases, such as tuberculosis, sarcoidosis and fungal infections, postsurgically in the neck area, auto - palpation habit, hemorrhage in nodular goiter, proximity to histiocytic reaction, hashimoto's thyroiditis and graves diseases. the presence of a high number of mngcs accompanied by acute onset pain, which is also a characteristic clinical symptom for sgt, will be helpful for differential diagnosis. garca solano et al. reported the presence of follicular cells with intravacuolar granules and/or plump transformed follicular cells, though this was not a marked characteristic in our cases. in contrast, the follicular epithelial cells in our cases were generally degenerated / proliferated, and regenerative atypia findings were observed from an aspirate taken with the suspicion of malignancy. however, the patient was diagnosed as sgt due to the absence of cellular and structural malignant characteristics, despite the presence of a high number of mngcs and epithelioid cells accompanied by a dirty . if the patient has the cytological characteristics specified above when he / she comes to the clinic with a clinical diagnosis of sgt, the cytological diagnosis of sgt may not be too difficult. however, the following factors may rise to challenges in the diagnosis: (i) lack of clinical data, (ii) failure of the needle to reach the target, (iii) absence of the typical cytological characteristics (i.e. mngcs, granulomas) as in acute or advanced stage of the disease, and (iv) presence of atypical follicular epithelium cells without other diagnostic elements. according to ofner et al. although pain and tenderness in the thyroid gland is a very important finding for the diagnosis of sgt, it is not specific and may be seen in suppurative thyroiditis, trauma, radiation, nodular goiter, hemorrhagic and infarct areas, metastatic diseases and rarely, in hashimoto's thyroiditis. generally, the correct diagnosis can be made by combining detailed clinical and physical examination and using serological and cytological characteristics in combination. however, clinical follow - up and fna may be very beneficial in such cases. subacute granulomatous thyroiditis is a dynamic process, and usg and cytological findings may vary according to the stage of the disease. although there is no special radiological finding of thyroiditis, hypoechoic and heterogeneous areas with irregular margins in the thyroid are strongly associated with thyroiditis. however, sgt may also appear as a painful or painless hypoechoic and solid nodule, as occurs in most of our cases. they may be confused with malignancies, particularly in such cases, and give rise to difficulties in differential diagnosis. the characteristic cytological features in our series in fna were a high number of mngcs with dense cytoplasm, mild - moderate cellularity, degenerated - proliferated follicular epithelial cells and dirty accompanied with epithelioid granulomas and rare, mixed type inflammatory cells. although the presence of neck pain in the clinical history, the detection of mngcs at fna, their morphological characteristics and their number play a key role in the diagnosis. further, utilizing tbc - fungal cultures and assessing the accompanying cell population in the may help in the differential diagnosis. all authors of this article declare that we qualify for authorship as defined by icmje. each author has participated sufficiently in the work and takes public responsibility for appropriate portions of the content of this article.cv conceived of the study and drafted the manuscript. np participated in its design and coordination and helped to scientific revision of the manuscript. this study was conducted with the approval from all the institutions associated with this study as applicable. sgt = subacute granulomatous thyroiditis fna = fine - needle aspiration usg = ultrasonographic mngcs = multinuclear giant cells mng = multinodular goiter pc = papillary carcinoma to ensure the integrity and highest quality of cytojournal publications, the review process of this manuscript was conducted under a double blind model (authors are blinded for reviewers and vice versa) through automatic online system.

: subacute granulomatous thyroiditis (sgt) is an inflammatory disease that presents with different clinical and cytological characteristics. although the diagnosis is generally made clinically, imaging methods and fine - needle aspiration (fna) may provide assistance, particularly in atypical cases. the objective of this study is to reveal the ultrasonographic (usg) and cytological characteristics of sgt.materials and methods: the clinical, usg and cytological findings of 21 cases diagnosed with sgt were reviewed.:ultrasonographic data was available in 20 cases. a hypoechoic thyroid nodule with irregular margins was detected in 12 of the 20 total cases. of these, 9 cases complained about pain in the thyroid lodge and generally had unilateral lesions, heterogeneous and hypoechoic areas with indistinct margins, rather than nodular lesions, which were seen in 7 cases. cytologically, the multinuclear giant cells (mngcs) found in all cases were accompanied by a dirty containing varying numbers of granulomatous structures, including isolated epithelioid histiocytes, proliferated / regenerated follicle epithelium cells and inflammatory cells and colloid.:though hypoechoic and heterogeneous areas with irregular margins are strongly associated with thyroiditis, sgt may also appear as painful or painless hypoechoic, solid nodules and generate challenges in differential diagnosis. although the most remarkable characteristic observed in fna cytology was the presence of multiple mngcs with cytoplasm, a dirty accompanied by mild - moderate cellularity, degenerated - proliferated follicular epithelium cells, rare epithelioid granulomas and mixed type inflammatory cells are characteristic for sgt. the assessment of these radiological and cytological findings in conjunction with clinical findings will assist in the achievement of an accurate diagnosis.

. a single indel may alter gene length by disrupting the reading frame, increase substitution rate in flanking regions and catalyze adaptive protein evolution (tian et al . indels complicate multiple sequence alignments and may skew subsequent evolutionary analyses such as tests of positive selection ( fletcher and yang 2010 ; jordan and goldman 2012 ; westesson et al . 2012). integrating our knowledge of nucleotide substitutions with more detailed consideration of indel mutations is necessary to fully understand processes of molecular evolution. to this end, advances in high - throughput sequencing enable genome - wide analyses of indels at different divergence levels, thereby elucidating the context and effect of indels. indels impact ongoing genome reduction in obligate bacterial endosymbionts of insects, which have the smallest genomes reported for cellular life (moran et al . 2008). genome degradation in established endosymbionts, including buchnera of aphids and blochmannia of carpenter ants, is hypothesized to occur primarily through small deletions punctuated by occasional large deletions in nonfunctional sequence (gomez - valero et al . the spectrum of indel mutations in bacterial endosymbionts is affected by a combination of factors, including loss of dna repair mechanisms and relaxed purifying selection ing from population bottlenecks during vertical transmission of bacteria ( moran et al . 2008). the high at content of most bacterial endosymbionts in increased density of homopolymers, providing hotspots for indels as an important first step in gene degradation (gomez - valero et al . other repetitive regions, such as multimeric simple sequence repeats ( mssrs), may also serve as hotspots for indels (williams and wernegreen 2012). to investigate the roles of indels in genome evolution of bacterial endosymbionts, we assembled and annotated the genome of blochmannia chromaiodes (nc_020075), an endosymbiont of the carpenter ant camponotus chromaiodes. blochmannia chromaiodes is closely related to b. pennsylvanicus (degnan et al . 2004 ; wernegreen et al . 2009), which was previously sequenced (degnan et al . 2005). the genetic distance between these two species is close enough for accurate whole genome alignment but divergent enough that a meaningful number of indels have accumulated, thereby affording valuable opportunities to clarify the context and effect of indel mutations in endosymbiont genome evolution. camponotus chromaiodes ants were collected from two colonies in the same location less than 1 year apart. genomic dna from the two colonies was prepped separately and sequenced with either 454 or illumina technology. we used 454 reads for de novo assembly of a draft sequence, which we then corrected with illumina reads to ensure homopolymer accuracy in the final genome sequence. for 454 sequencing, we isolated endosymbiont cells from 12.9 g of ants using a percoll density gradient centrifugation protocol described previously (wernegreen et al . chloroform extraction as described previously ( williams and wernegreen 2010). for illumina sequencing , we prepared genomic dna from three gasters using the qiagen dneasy blood and tissue kit. 454 sequencing generated 295,496 single - end reads, which we assembled into a single draft contig using the gs assembler and gs mapper in newbler version 2.3. we closed the single gap with sanger sequencing. because 454 sequencing is known for homopolymer inaccuracy, we used illumina reads to correct the draft and obtain the final genome sequence. illumina sequencing generated 28,516,837 single - end 76 bp reads, which we aligned to the 454 draft genome using mosaik (http://bioinformatics.bc.edu/marthlab/mosaik, last accessed march 15, 2013). we corrected the draft genome and then confirmed the final genome sequence by aligning illumina reads with bwa (li and durbin 2009) and the genome analysis toolkit indelrealigner tool (mckenna et al . , we also assembled a large contig of the mitochondrial genome ( jx966368), which aligns with 100% identity to a c. chromaiodes mitochondrial sequence in genbank. we used an annotation engine hosted by the institute for genome sciences at the university of maryland school of medicine to obtain an automated annotation of b. chromaiodes, which we then manually curated. protein - coding genes predicted by the annotation engine were removed from the annotation if they lacked a blast extend repraze (ber) alignment score less than 10 to a non - blochmannia protein. we used the locus and gene names suggested by swissprot for the homologous gene in e. coli to provide consistency with other proteobacterial genome annotations. conserved hypothetical proteins or proteins with similarity to a protein family but not a specific family member were given a locus name reflecting the gene number (for example, bchro_042). we curated the start site for each protein - coding gene using the ber alignments to non - blochmannia species. we identified two uncalled protein - coding genes (cyod and a hypothetical protein homologous to bpen_539), three rna - coding genes (ffs, rnpb, and tmrna), and three pseudogenes (rpmd, uvrd, and yqic) by searching intergenic regions with blastx, blastn, and rfam (altschul et al . 2000) and dnadiff from mummer (kurtz et al . 2004), both with default parameters, to obtain whole genome alignments of b. chromaiodes and b. pennsylvanicus. when the two alignments disagreed, we applied a set of criteria to decide the final alignment. in priority order, the criteria are as follows: 1 ) preserve reading frame in protein - coding genes, 2 ) select the most parsimonious proposal (i.e., the proposal with the fewest substitutions and indels), 3 ) when both proposals have the same total number of events, select the proposal with fewer indels, and 4 ) when both proposals have the same number of substitutions, select the proposal with fewer transversions. for 12 cases, these criteria could not differentiate between the two proposals, and we selected the emboss stretcher proposal by default. we classified substitutions and indels according to the b. chromaiodes and b. pennsylvanicus annotations using variantclassifier (li and stockwell 2010). to estimate the neutral substitution rate, we generated amino acid - based alignments of protein - coding genes with translatorx (abascal et al . 2010) and mafft (katoh et al . 2005), omitting 23 genes with nontriplet indels. using codeml in paml with runmode 2 for pairwise comparisons (yang 1997), we estimated the number of synonymous changes and synonymous sites for each gene and then calculated the average number of synonymous changes per 1,000 synonymous sites across the genome. we did separate searches for homopolymers and mssrs in b. chromaiodes and b. pennsylvanicus using phobos (http://www.ruhr-uni-bochum.de/ecoevo/cm/cm_phobos.htm, last accessed march 15, 2013). we then identified overlap between indels and repeats using intersectbed from bedtools (quinlan and hall 2010). to determine whether compensatory indels are associated with protein divergence, we used the reciprocal blastp algorithm to identify orthologs of b. pennsylvanicus genes in e. coli (nc_000913). ortholog detection and calculation of amino acid distances followed methods described previously (wernegreen 2011). this strategy invokes paml to calculate amino acid distances based on an empirical amino acid substitution rate matrix and accounts for variation in evolutionary rates among sites. the gene content of b. chromaiodes is identical to that of b. pennsylvanicus (table 1). both genomes have evidence of three pseudogenes (rpmd, uvrd, and yqic), which differ slightly in length between the two species (supplementary table s1, supplementary material online). s1, supplementary material online ), the two nucleotide sequences are 98.0% identical. we detected 13,389 substitutions and 1,051 indels between the two sequences (table 2). substitution rates in pseudogenes and intergenic regions are very similar (26.8 and 29.3 substitutions / kb, respectively). these rates are higher than those of protein - coding and rna - coding genes (13.5 and 5.2 substitutions / kb, respectively), which is likely the of purifying selection acting on coding regions. however, substitution rates in pseudogenes and intergenic regions are significantly lower than our estimate of the neutral substitution rate using synonymous substitutions across the genome (49.2 synonymous changes/1,000 synonymous sites) (binomial test, p < 0.001). table 1gene content in blochmannia chromaiodes and b. pennsylvanicusspeciessize (bp)gc content (%) total genesprotein - coding genestrnarrnaother rnapseudogenesframeshifted genesb. pennsylvanicus791,65429.6658609403334 table 2substitutions and indels between blochmannia chromaiodes and b. pennsylvanicussubstitutionssubstitutions / kbindelsindels / kbindel bpindel bp / kbprotein - coding8,16613.5630.11750.3rna - coding455.260.7212.4pseudogene6326.8187.711247.6intergenic5,09429.39605.52,00411.5ambiguous21na4na9natotal13,38916.91,0511.32,3212.9note. na, not applicable.to account for differences in the amount of each sequence type in the two genomes, rates were calculated using the b. chromaiodes and b. pennsylvanicus annotations and then averaged.ambiguous refers to positions annotated differently in the two genomes (i.e., protein - coding in b. chromaiodes and intergenic in b. pennsylvanicus). gene content in blochmannia chromaiodes and b. pennsylvanicus substitutions and indels between blochmannia chromaiodes and b. pennsylvanicus note.na, not applicable. to account for differences in the amount of each sequence type in the two genomes, rates were calculated using the b. chromaiodes and b. pennsylvanicus annotations and then averaged. ambiguous refers to positions annotated differently in the two genomes (i.e., protein - coding in b. chromaiodes and intergenic in b. pennsylvanicus). rates of indel events in pseudogenes and intergenic regions are similar (7.7 and 5.5 indels / kb, respectively), and these are higher than those of protein - coding and rna - coding genes (0.1 and 0.7 indels / kb, respectively), further demonstrating the role of purifying selection. when we consider the number of nucleotides involved in indels, protein - coding and rna - coding genes still have relatively low rates (0.3 and 2.4 indel bp / kb, respectively) compared with pseudogenes and intergenic regions. however, we observed a higher rate for pseudogenes (47.6 indel bp / kb) compared with intergenic regions (11.5 indel bp / kb). conservation of intergenic spacer length was observed in previous comparisons of blochmannia and buchnera endosymbionts (degnan et al . 2005, 2011). blochmannia intergenic regions may contain regulatory sequences or other elements under constraint, as recently noted for buchnera (degnan et al . it is also possible that truncated products from the transcription and translation of pseudogenes are detrimental to the cell and increase selective pressure for deletions . we did not detect any large ( > 60 bp) indels between b. chromaiodes and b. pennsylvanicus. almost all of the 1,051 indels are small; 671 (63.8%) involve a single base, and 1,010 (96.1%) are 6 bp. previous intraspecific analyses of blochmannia and buchnera emphasized the importance of repeat regions as indel hotspots in the at - rich genomes of these endosymbionts (gomez - valero et al . 2008 ; moran et al . 2009 ; williams and wernegreen 2012). to explore this in our interspecific comparison , we determined the repeat context of indels, focusing on two repeat types: homopolymers and mssrs. we define homopolymers as tracts of 2 bp of a single nucleotide and mssrs as 2 contiguous occurrences of a 26 bp repeat unit. for our purposes , we did not consider these repeat types mutually exclusive when identifying repeat regions. in other words, a particular region may contain an mssr, which itself includes a homopolymer, such as caaatcaaat. to ensure that we are not overlooking the contribution of either repeat type of 1,051 indels, 1,034 (98.4%) occur in a repeat region (either homopolymer or mssr). regarding repeat types, 932 indels (88.7%) occur in homopolymers, and 611 indels (58.1%) occur in mssrs. to further assess the impact of repeat type on indels, we determined whether indels comprise 1 complete repeat unit. we found that 737 of 1,034 indels occurring in repeat regions (71.3%) comprise 1 repeat unit, with 579 of these comprising 1 homopolymer unit (78.6%) and 158 of these comprising 1 mssr unit (21.4%). the remaining 297 indels occurring in repeat regions (28.7%) either are not part of the repeat unit (e.g., an indel of g where the b. pennsylvanicus sequence is tattat - tat, and the b. chromaiodes sequence is tattatgtat) or include partial repeat units (e.g., an indel of ttcat where the b. pennsylvanicus sequence is cattcatt, and the b. chromaiodes sequence is ca-----t). these data provide strong support for the importance of repeat regions as indel hotspots in at - rich endosymbionts. our analysis not only reflects the recognized and significant role of homopolymers in indel mutations but also emphasizes the role of mssrs. we initially noted the possible contribution of mssrs in an intraspecific comparison of b. vafer (williams and wernegreen 2012). the larger pool of indels afforded by our interspecific comparison here reinforces the contribution of mssrs to indel mutation in this bacterial endosymbiont. the low rate of indels in protein - coding genes (0.1 indels / kb) reflects strong effects of purifying selection. of 63 indels in protein - coding genes (table 2) , 41 have lengths equal to a multiple of three and do not disrupt the reading frame. of the remaining 22 nontriplet, frameshift - inducing indels, 21 fall into one of two categories: compensatory indels or indels near the 3-end of genes. the deleterious effect of these indels may be limited, thereby enabling them to escape purging by purifying selection and possibly act as drivers of protein divergence. compensatory indels involve two or more indels that combine to preserve the reading frame. for our purposes, we define compensatory indels with three criteria: 1 ) indels occur within 20 bp, 2 ) length of each individual indel is not divisible by three, and 3 ) combined length of indels is either three or zero. using these criteria , we identified three instances of compensatory indels between b. chromaiodes and b. pennsylvanicus, each involving two single - base indels (fig . 1). these six indels comprise 27.2% of the 22 frameshift - inducing indels. studies of mammalian genomes show increased frequency of compensatory indels in exons compared with introns, suggesting selection for compensation (hu and ng 2012). our data indicate that this mechanism may also operate in bacterial genomes. in at - rich endosymbiont genomes, high densities of homopolymers prone to polymerase slippage may afford more opportunities for compensatory indels to occur and correct frameshifts. fig. 1.compensatory indels detected in a whole genome alignment of blochmannia chromaiodes and b. pennsylvanicus. for each, the alternative alignment hypothesis with only nucleotide substitutions is also shown. compensatory indels detected in a whole genome alignment of blochmannia chromaiodes and b. pennsylvanicus. for each, the alternative alignment hypothesis with only nucleotide substitutions is also shown. we explored whether compensatory indels are associated with levels of protein divergence by calculating pairwise amino acid distances between b. pennsylvanicus genes and their orthologs in escherichia coli, a free - living relative. pennsylvanicus comparison are among the most divergent proteins in the comparison with e. coli (supplementary fig . this pattern is consistent with the expected effects of purifying selection on frameshift - inducing indels . at highly conserved genes, a frameshift - inducing indel is likely to be removed by strong purifying selection . by contrast , a frameshift - inducing indel might persist longer in more divergent genes under relaxed selection, and compensation via a subsequent indel may act as a mechanism to prevent or reduce the accumulation of deleterious mutations in these genes . comparisons of yeast species suggest that compensatory indels may be a driver of rapid protein evolution ( kellis et al . even though out - of - frame sequence between compensatory indels is short ( < 6 bp) in our b. chromaiodes b. pennsylvanicus comparison, these indels in one or two amino acid substitutions (fig . 1), suggesting that compensatory indels may also impact protein divergence in endosymbiont genomes. interestingly, the nature of compensatory indels may hamper analyses of protein evolution, such as tests of positive selection, in more divergent species. others have explored the impact of indel - associated alignment error on tests of positive selection (fletcher and yang 2010 ; jordan and goldman 2012); however, these analyses used codon - based alignments constraining indels to frame - preserving triplets, thereby excluding the possibility of compensatory indels. when we similarly constrain alignments of the regions with compensatory indels in b. chromaiodes and b. pennsylvanicus, this in three or five nucleotide substitutions over < 6 bp (fig . sixteen frameshift - inducing indels do not have compensatory indels nearby to restore the reading frame . all but one of these occur less than 60 bp from the 3'-end of the gene ( table 3). although these indels disrupt the reading frame, their location minimizes the ing impact on protein sequence. in fact, two indels involve a complete mssr repeat unit at the exact 3'-end of the gene and therefore have no effect on gene sequence or length. thirteen indels in differences in protein length ranging from 1 to 13 aa. although we can not distinguish insertions from deletions in this comparison, previous analyses of buchnera endosymbionts of aphids showed higher prevalence of deletions in the 3'-end of genes (charles et al . downstream of these indels, the nucleotide sequence is highly conserved, whereas the amino acid sequence shows more changes ( on average, 3.5 aa substitutions per nt substitution), suggesting that these indels are drivers of rapid protein divergence. previous work showed that transcription of genes with frameshifts in homopolymers generates a mixed transcript pool, including full - length transcripts in which the frameshift was corrected by polymerase slippage (tamas et al . this may enable some frameshift - inducing indels to persist despite purifying selection, thereby catalyzing adaptive protein evolution or initiating the process of gene degradation . table 3indels occurring in the 3'-end of blochmannia chromaiodes and b. pennsylvanicus genesgeneindel size ( bp)repeat context of indeleffect of indeldifference in protein length (aa)amino acid substitutionsnucleotide substitutionsyral / rsmi12 g hp510psd18 a hp312ftsq12 a hp130def25 at mssr000ahpc42 taag mssr000ribe14 t hp131ubig17 a hp410nuoa18 a hp1330aroc16 a hp510ureg2none333ispg14 c hp161rplj17 a hp210rplk12 t hp100gpsa16 a hp230meta14 a hp421considering the region from the indel to the end of the longest gene sequence.hp refers to homopolymers, and mssr refers to multimeric simple sequence repeats.because this indel comprises a complete repeat unit at the exact end of the gene, there is no detectable effect on the gene or protein sequence. indels occurring in the 3'-end of blochmannia chromaiodes and b. pennsylvanicus genes considering the region from the indel to the end of the longest gene sequence. because this indel comprises a complete repeat unit at the exact end of the gene, there is no detectable effect on the gene or protein sequence. the genome - wide comparison of b. chromaiodes and b. pennsylvanicus presented here provides an important view of the spectrum of indel mutations impacting endosymbiont genome evolution. the role of homopolymers and mssrs as indel hotspots, which was previously observed in intraspecific comparisons, is strongly evident in this interspecific analysis. although purifying selection limits indel mutations in coding and intergenic regions, we detected frameshift - inducing indels in protein - coding genes that may act as important drivers of protein divergence and contribute to ongoing genome reduction. supplementary figures s1 and s2 and table s1 are available at genome biology and evolution online (http://www.gbe.oxfordjournals.org/).

indel mutations play key roles in genome and protein evolution, yet we lack a comprehensive understanding of how indels impact evolutionary processes. genome - wide analyses enabled by next - generation sequencing can clarify the context and effect of indels, thereby integrating a more detailed consideration of indels with our knowledge of nucleotide substitutions. to this end, we sequenced blochmannia chromaiodes, an obligate bacterial endosymbiont of carpenter ants, and compared it with the close relative, b. pennsylvanicus. the genetic distance between these species is small enough for accurate whole genome alignment but large enough to provide a meaningful spectrum of indel mutations. we found that indels are subjected to purifying selection in coding regions and even intergenic regions, which show a reduced rate of indel base pairs per kilobase compared with nonfunctional pseudogenes. indels occur almost exclusively in repeat regions composed of homopolymers and multimeric simple sequence repeats, demonstrating the importance of sequence context for indel mutations. despite purifying selection, some indels occur in protein - coding genes. most are multiples of three, indicating selective pressure to maintain the reading frame. the deleterious effect of frameshift - inducing indels is minimized by either compensation from a nearby indel to restore reading frame or the indel s location near the 3'-end of the gene. we observed amino acid divergence exceeding nucleotide divergence in regions affected by frameshift - inducing indels, suggesting that these indels may either drive adaptive protein evolution or initiate gene degradation. our shed light on how indel mutations impact processes of molecular evolution underlying endosymbiont genome evolution.

olanzapine is an atypical antipsychotic with efficacy in schizophrenia slightly higher than others.1 ) the extrapyramidal adverse effects are much lower than typical antipsychotics, though it has a higher metabolic adverse effect profile.2 ) orthostatic hypotension is common with first - generation antipsychotics with rates up to 77% compared to 15% with placebo.3 ) among atypical antipsychotics, clozapine, risperidone and quetiapine have higher rates of orthostatic hypotension.3 ) we report a case where olanzapine, in a dose dependent fashion, caused transient postural hypotension. a 22-year - old female was diagnosed with paranoid schizophrenia 16 months back and was started on olanzapine, which was increased up to 15 mg / day over two weeks. she responded well to it, resuming her higher studies as well as a part - time job of a school teacher. however, after two months, she had sudden falls on five occasions over one week, seriously injuring herself with bruises and lacerations. vitals were checked on all the occasions which showed pulse rate ranging from 7090 beats per minute (bpm) and blood pressure ranging from 110130/ 7080 mmhg. however, in all the occasions the examinations could be done after the patient had recovered and gained consciousness. investigations including complete blood count, fasting and post prandial plasma glucose, lipid profile and liver function test were normal. her body weight has been maintained at 52 kg over the past one year, her menstrual cycles were regular and abdominal ultrasonography revealed no abnormalities. however, on current assessment both cortisol (9.31 g / dl) and adrenocorticotropic hormone (14.5 pg / ml) levels were within normal range with respect to age, sex and time (morning / evening) of sampling. she was advised rest at home, dose of olanzapine was reduced to 10 mg / day and a round - the - clock nurse was appointed for observation and monitoring. thereafter, the frequency of fall reduced, and there was only one such event when she was dizzy and about to fall but prevented by the attending nurse. her pulse rate was 140 bpm and blood pressure was 76/40 mmhg in supine position, and three minutes later pulse rate was 80 bpm and blood pressure was 110/70 mmhg in standing position, which was suggestive of orthostatic hypotension. she recovered from her dizziness and was fully oriented within five minutes of the event. subsequently, olanzapine was cross - tapered with aripiprazole up to 15 mg / day, on which she is maintaining well without further recurrence of adverse effects during follow up at two months. our patient developed orthostatic hypotension with olanzapine 15 mg per day that subsided following discontinuation of medication. there are few reports of hypotension associated with olanzapine,4,5 ) one in an elderly individual, and the other with intramuscular administration. the summary of product characteristics for olanzapine reports 5% subjects develop orthostatic hypotension as compared to 2% in placebo,6 ) which is much less as compared to the rates observed with first generation antipsychotics.3 ) nevertheless, hypotension and bradycardia has been reported with olanzapine as low as 5 mg per day.7 ) most commonly orthostatic hypotension is an incidental finding during routine examinations, and symptomatic hypotension is not usually observed except in the elderly patients. in contrast, our patient, a young female, developed symptomatic orthostatic hypotension with olanzapine that ed in several episodes of falls, which required treatment discontinuation. interestingly, nourian et al.8 ) had shown through animal experiments that olanzapine, along with ziprasidone and aripiprazole has the least propensity to cause postural hypotension. several other putative mechanisms such as calcium blockade, inhibition of centrally mediated pressor reflexes, and negative inotropic effects, have also been proposed for this adverse effect.9 ) sedative effect of olanzapine may additionally contribute to the falls, though such mechanism appeared unlikely in our case. although the rates of orthostatic hypotension appear lower with olanzapine, an exercise of caution is definitely warranted in the light of the current case.

olanzapine is an atypical antipsychotic which is efficacious in the treatment of schizophrenia. the adverse effect profile for olanzapine is benign except for higher rates of metabolic events. orthostatic hypotension is less commonly reported with olanzapine as compared to first - generation and few atypical antipsychotics. we report a case where olanzapine, in a dose dependent fashion, caused transient postural hypotension.

the population included patients at hospital virgen de las nieves (granada, spain) who had suspected ame. routine virologic investigation included reverse transcription pcr (rt - pcr) of cerebrospinal fluid (csf) samples for detection of hev (xpert ev system, cepheid, sunnyvale, ca, usa), tosv, and mumps virus, and pcr of herpes simplex virus (hsv) and varicella - zoster virus (lc vhs 1/2 qual and lc vzv systems, respectively ; roche diagnostics, mannheim, germany). negative samples were subsequently tested for epstein - barr virus, cytomegalovirus, human herpes 6 virus, flavivirus, arenavirus, adenovirus, and phlebovirus. specific lcmv rt - pcr was conducted by using a previously described protocol. finally, csf specimens from pcr - negative case - patients were inoculated in vero and mrc-5 cell lines for viral culture. csf and acute - phase serum samples were also tested for igg and/or igm against tosv (eia enzywell toscana virus igg / igm, diesse, siena, italy), west nile virus (elisa igg and igm - capture elisa ; focus diagnostics, cypress, ca, usa), and lcmv by indirect fluorescent assay (ifa) with further confirmation by western blot. we studied 159 csf samples by using pcrs for the presence of hev, hsv, varicella - zoster virus, tosv, and mumps virus, yielding 68 positive cases. a viral agent was detected in 70 (44%) cases: hev accounted for 44 (63%) of positive cases, followed by varicella - zoster virus in 11 (16%), hsv-1 in 8 (11%), tosv in 4 (6%), lcmv in 2 (3%), and hsv-2 in 1 (1%). case - patient 1, a 21-year - old woman, came to the hospital s emergency unit in april 2008 exhibiting headache, chills, fever (38.9c), confusion, nausea, vomiting, and slight nuchal rigidity. laboratory were 415 leukocytes / mm (100% mononuclear cells), 43 mg/100 dl glucose level (reference 3565 mg / dl), and 128 mg / dl protein level (reference 1545 mg / dl) in the csf. igg and igm titers of 640 and 128 against lcmv were detected in the serum sample by ifa and titers of 400 and 200 by western blot assay, respectively. viral culture of the csf in vero cells revealed no cytopathic effect after 1 month of incubation. viral isolate (eeb-7) was used for genetic characterization. to sequence the genome, degenerate and specific primers were designed on the basis of an alignment of all complete lcmv large (l), glycoprotein complex and nucleocapsid protein sequences (table).terminal sequences were generated by using a universal arenavirus primer, targeting the conserved viral termini (5-cgc acm gdg gat cct agg c-3), combined with 4 specific primers positioned near the ends of each segment. amplification products were size - fractionated by electrophoresis in 1% agarose gels, purified (minelute, qiagen, valencia, ca, usa), and sequenced in both directions on an abi prism 3700 dna analyzer (pe applied biosystems, foster city, ca, usa). to determine the evolutionary history of isolate eeb-7 , we performed bayesian phylogenetic analysis of all available complete genome sequences of lcmv using beast, beauti, and tracer analysis software packages. clinical diagnosis was acute meningitis and the patient was discharged from hospital on day 9. * lcmv, lymphocytic choriomeningitis virus; gpc, glycoprotein comlex; np, nucleocapsid protein. the name of the strain is followed by genbank accession number, country, and year of detection. phylogenetic tree showing genetic lymphocytic choriomeningitis virus sequences relationship within the small segment coding for the glycoprotein complex (a) and nucleocapsid proteins (b). the name of the strain is followed by genbank accession number, country, and year of detection. case - patient 2, a 39-year - old man, sought treatment in may 2010 with headache, nausea, vomiting, increased perspiration, and a temperature of 37.5c. csf analysis demonstrated 1,715 leukocytes / mm (95% mononuclear cells), normal glucose level (68 mg / dl), and elevated protein levels (240 mg / dl). further virologic investigation detected lcmv rna in the csf and an igg titer of 640 by ifa and igm antibodies against lcmv in the serum sample. the sequence of a 194-nt pcr product (nucleocapsid protein gene) obtained was most closely related to sequences of the lineage i. sequence homology among the lcmv amplicon from case - patient 2 and lineage i strains was > 87% versus 77%79% sequence homology among case - patient 2 and strains from lineages ii iv. pcr has become the reference standard for identifying common viruses involved in ame. however, no commercial tests are available for lcmv; and in - house pcrs have to be optimized according to the natural genetic diversity of the virus. neurologic lcmv infection in spain has been diagnosed primarily by serologic tests. the diagnosis in case - patients 1 and 2 in the current study was achieved by serologic testing as well. nonetheless, pcr was useful because it allowed genetic characterization of the lcmv strain from case - patient 2. furthermore, the viral isolate of case - patient 1 was evident only by detection of lcmv rna in the cell culture supernatant because no cytopathic effect was observed. isolates from both lcmv case - patients belonged to the classical lineage i, which has been detected elsewhere in europe. lineage i is usually associated with human disease (as are lineages ii and iii) and is linked to the common house mouse as its reservoir. lineage iv viruses were previously detected in spanish wood mice and have not been associated with human disease. although no further epidemiologic studies could be conducted to search for infected reservoirs, the common genetic lineage and the fact that both case - patients resided within the same community might suggest transmission of lcmv by a common vector. previous seroprevalence studies have associated human lcmv infection with high exposure to the common house mouse. human lcmv infections might be underdiagnosed because the clinical characteristics of lcmv meningitis are similar to those of other viral meningitis; no commercial tests are available for serologic or molecular diagnostic assays, and usually no clear epidemiologic clue is available at the moment of diagnosis. thus, epidemiologic and virologic surveillance might ascertain that the true incidence of lcmv ame is more frequent than reported.

lymphocytic choriomeningitis virus (lcmv) was detected in 2 patients with acute meningitis in southern spain within a 3-year period. although the prevalence of lcmv infection was low (2 of 159 meningitis patients), it represents 2.9% of all pathogens detected. lcmv is a noteworthy agent of neurologic illness in immunocompetent persons.

oxytocin (ot) is a maternal hormone with effect on uterine contraction and milk ejection. more recently, ot has been shown to influence a wide spectrum of behavioural, physiological and endocrine functions mediated through receptors within the brain. ot induces several anti - stress - like effects; decrease in heart rate (hr), blood pressure and levels of stress hormones. administration of low dose ot can induce anxiolytic - like effect and in higher doses sedative effect. however, very few studies have explored the role of ot on anaesthetic and analgesic consumption. this study was conducted to determine the effect of ot secreted during breastfeeding (bf) on consumption of propofol and sevoflurane and assessment of its effect on intra - operative haemodynamics as the secondary outcome. before commencing this single blind prospective study, approval was obtained from the institutional review board. about 120 women with american society of anesthesiologists physical status i and ii, aged 2030 years, who had uncomplicated pregnancy, delivered full - term, healthy infants by the vaginal route and scheduled for tubectomy were enrolled for the study between july 2014 and december 2014. parturients with a history of cerebrovascular disease, hypertension, diabetes mellitus and other endocrine disorders, treatment with psychoactive medication, anticipated difficult airway, parturients on sedatives or analgesics and weighing < 70% or more than 130% of ideal body weight were excluded from the study. thorough pre - anaesthetic check - up of all patients including routine investigations was carried out. the parturients were allocated to three groups of 40 each depending on their lactation status. lactating women were randomly allocated to either bf group or withhold feeding (wf) group based on computer - generated randomisation technique (www.random.org), whereas non - lactating women were considered for the control group (non - feeding). the bf group (n = 40) consisted of lactating women who breastfed just before induction of anaesthesia. wf group (n = 40) included lactating women who withheld bf for 4 h before induction of anaesthesia. nf group (control) (n = 40) included non - lactating women (coming for interval tubectomy) who had delivered more than a year back and stopped bf on their own for a minimum of 4 weeks before surgery. the details of randomisation were kept with the principal investigator and not revealed to other investigators until the end of the study. the anaesthesiologist monitoring the patient intra - operatively and post - operatively was not aware of the lactation status of the women or their group allocation; none of the cases were primigravida. on arrival in the operating room , patients were monitored for hr, blood pressure and oxygen saturation by continuous electrocardiogram, non - invasive blood pressure and pulse oximetry, respectively, and basal parameters were recorded. depth of anaesthesia was monitored by entropy analysis (entropy module, datex - ohmeda s/5 avance workstation, ge healthcare). patients were started on intravenous (iv) infusion of ringer's lactate solution solution premedicated with injection midazolam 1 mg iv, injection glycopyrrolate 0.2 mg iv, injection fentanyl 2 g / kg iv and preoxygenated with 100% oxygen for 3 min. anaesthesia was induced with iv propofol 10 mg increments injected over 5 s, at 10 s intervals until the state entropy (se) levels dropped to 45, total dose of propofol required to achieve se 45 was recorded, and airway secured with laryngeal mask airway - classic (lma) (as per manufacturer 's recommendations), and patients were maintained on assisted spontaneous ventilation. if more than 2 attempts were required for lma insertion, cases were excluded from the study and were managed as per standard institutional protocol. additional doses of propofol 10 mg were administered as necessary when se values increased to more than 60 during lma insertion, and the amount of supplemental propofol used was noted. anaesthesia was maintained with sevoflurane in 60% n2o and o2, with ventilation adjusted to maintain end - tidal co2 between 35 and 40 mmhg. sevoflurane concentration was set at 2% initially and adjusted subsequently to maintain se levels between 40 and 60, and the difference between response entropy (re) and se < 10. if the difference between re and se exceeded 10, additional doses of fentanyl 1 g / kg was administered. fresh gas flow rate was set at 4 l / min for initial 5 min and then reduced to 2 l / min. sevoflurane and n2o were discontinued at the end of closure of abdomen and lma removed after complete recovery. patients were monitored in the post - operative period for 6 h. hr, mean arterial pressure (map), etco2, se and re were recorded at baseline, at induction and every 5 min after lma insertion until 15 min into post - operative period. end - tidal concentration of sevoflurane was recorded at 5 min intervals from the time of lma insertion until discontinuation of anaesthetics. parameters recorded were total duration of surgery, duration of anaesthesia, duration of sevoflurane use, total dose of propofol consumed and total volume of sevoflurane consumed (obtained by the anaesthesia gas module of ge datex - ohmeda s/5 anaesthetic delivery unit system). sample size was calculated based on findings of a pilot study conducted in our institute involving ten lactating women. the average end - tidal sevoflurane concentration was 1.45 0.32. keeping the power of study at 80% and confidence limits at 95%, to detect a minimum of 15% difference in sevoflurane consumption between groups, assuming normal distribution of values in both groups, the minimum sample size required was thirty in each group, we included forty patients in each group for better validation of . analysis of variance has been used to find the significance of study parameters between three or more groups of patients, student's t - test (two - tailed, independent) has been used to find the significance of study parameters on continuous scale between two groups (inter - group analysis) on metric parameters. chi - square or fisher exact test has been used to find the significance of study parameters on a categorical scale between two or more groups. the statistical software, namely sas 9.2, spss 15.0, stata 10.1, medcalc 9.0.1, systat 12.0 and r environment version 2.11.1 (ibm, usa) were used for the analysis of the data and microsoft word and excel were used to generate graphs, tables, etc. a total of 120 patients were included in the study with 40 in each group. the mean duration of the surgery, mean duration of anaesthesia and mean duration of sevoflurane use were comparable between the three groups. post - partum day distribution in lactating women were comparable among the bf group and wf group (p = 1.0). post - partum week distribution in lactating women (breastfeeding, withhold feeding groups) baseline hr was comparable in all the three groups (p-0.4); intraoperatively, a significant increase in hr was observed in wf group compared to other groups at all - time points until post - operative period (p < 0.001), bf group had lower hr throughout the procedure compared to other groups (p < 0.001). changes in heart rate (mean standard deviation): baseline (pre - induction); at induction (state entropy 45); after intubation; at 5, 10, 25, 30 and 15 min post - extubation baseline map was comparable in all the three groups (p-0.1), map was persistently higher in wf group compared to other groups (p < 0.001) and bf group had lower and stable map throughout the procedure compared to other groups (p < 0.001). comparison of mean arterial pressure (mm hg) in three groups of patients studied changes in mean arterial pressure (mean standard deviation); baseline (pre - induction); at induction (state entropy 45); after intubation; at 5, 10, 25, 30 and 15 min post - extubation; (se 45 - state entropy 45, map - mean arterial pressure) se values were comparable at baseline and throughout the procedure among all the groups. state entropy: baseline (pre - induction); at induction (state entropy 45); after intubation; at 5, 10, 25 and 30; (se 45 - state entropy 45) the difference in requirement of propofol to reach se 45 was statistically significant in bf and wf groups when compared to control group, and requirement was highest (102.88 5.30 mg) in wf group and least (74.13 6.97 mg) in bf group. comparison of propofol and sevoflurane usage the difference in requirement of sevoflurane to maintain anaesthesia was statistically significant in bf and wf groups when compared to control group, requirement was highest (6.43 0.75 ml) in wf group and least (3.80 0.76 ml) in bf group. the average end - tidal concentration of sevoflurane was significantly lower in bf group and higher in wf group compared to group nf (p < 0.001). post - hoc analysis between groups with regards to propofol and sevoflurane consumption showed statistically significant difference between groups nf, bf and wf, respectively. in the current study, the effect of ot which is secreted during bf on haemodynamics and anaesthetic drug consumption was determined. ot is mainly produced and synthesised in magnocellular neurons of the hypothalamic paraventricular (pvn) and supraoptic nuclei and secreted into the periphery via the posterior neurohypophysis. ot is also produced in parvocellular neurons within the pvn, and these neurons project to many areas within the brain such as other hypothalamic nuclei, the median eminence, amygdala, hippocampus, locus coeruleus, striatum, raphe nuclei, the dorsal motor nucleus of the vagus nerve (dmx) and nucleus tractus solitarii (nts). in addition, oxytocinergic fibres project down to the spinal cord, where they terminate on the presynaptic neurons of the sympathetic chain in the intermediolateral cell column and also in the dorsal horn in the area where pain modulation takes place. the central actions of the ot are mediated via ot receptors (otr) which are distributed widely in the central nervous system (cns). until date , only one otr is identified which is a member of class 1 g protein - coupled receptor family. during physiological and psychological stress responses, oxytocinergic neurons are activated, particularly in pvn of the hypothalamus and secreted into the circulating blood. ot induces several anti - stress - like effects such as decrease in hr, blood pressure and the levels of stress hormones,. these effects of ot are probably mediated through the hypothalamus and the vagal nuclei (dmx and nts). increase in ot release from the hypothalamus inhibits hypothalamic - pituitary - adrenal (hpa) axis by acting at three different levels. legros et al. demonstrated that human males treated with exogenous ot followed by synthetic adrenocorticotropic hormone (acth) showed a blunted response in cortisol secretion, suggesting that exogenous ot inhibits corticosteroid synthesis in the adrenal gland. second, neumann et al. demonstrated that peripheral ot inhibits acth release from the pituitary gland. they injected exogenous corticotrophin - releasing factor (crf) into lactating rats and measured acth responses, finding a reduction in acth response. the earlier study conducted on humans showed that bf women had significantly lower hormonal stress responses (as evident by lower cortisol and acth) during exercise stress than non - bf mothers and women without children. several follow - up studies have detected lower cardiovascular markers of stress (as evidenced by lower basal systolic blood pressures, higher levels of cardiac parasympathetic control and modulation of hr reactivity) during the task in breastfeeders as compared with non - bf mothers and women without children. light et al. found similar cardiovascular patterns for bf mothers during the anticipation of the public - speaking stressor; it is possible that any stress - buffering effects of bf are more potent during the immediate bf period. have shown that intranasal administration of ot, which passed directly into the brain, suppressed cortisol response to psychological stress, as well as attenuated emotional functions after stress episodes, indicating in humans, the inhibitory effect of ot on hpa activity mediated through cns in addition to peripheral effect. however, short - term iv administration of ot to women to enhance uterine contractions or decrease blood loss during labour or caesarean delivery confirms its effect in decreasing blood pressure. this hypotensive response to ot is due to decreases in total vascular resistance despite compensatory increases in hr, stroke volume and cardiac output. grewen et al. tested 28 early post - partum mothers, obtaining multiple blood samples for ot, the sympathetic marker, norepinephrine (ne) and the lactation hormone, prolactin while monitoring their cardiovascular responses to two stressors. they observed that greater overall ot level was related to lower plasma ne and higher prolactin levels; in contrast, higher ne was linked to increases in hr and decreases in stroke volume. these data support a cardioprotective role for ot, which may influence the magnitude and haemodynamic determinants of cardiovascular stress responses. similar findings were observed in our study, with women receiving anaesthesia immediately after bf showing a better haemodynamic profile compared to those who withheld bf and nf women. the anxiolytic - like effect seems to be mediated within the amygdala, which is richly provided with otrs, during bf, there may also be increased cns gamma - amino butyric acid, a major inhibitory neurotransmitter, which may inhibit the affective state and behaviour of the lactating animal. it was observed that administration of ot can induce both anxiolytic - like effects and in higher doses, sedative effects. the suggested there was temporary impairment although there was no correlation between ot levels and cognitive performance. the evidence that ot impairs some memory - related tasks has led to suggestions that it has a role in the forgetting of delivery pain in mothers. analgesic effect has been linked to the periaqueductal grey and the dorsal horn of the spinal cord. ot increases nociceptive thresholds through an enhancement of endogenous opioids. classical studies have demonstrated that chronic ot treatment induced not only anti - stress effects but also analgesia; this analgesic effect was blocked by the opioid antagonist naloxone but not by an ot antagonist, indicating that ot increases endogenous opioid transmission. the above - described physiological properties of ot may have contributed to decreased requirement of induction dose of propofol and maintenance dose of sevoflurane in this study in women who breastfed just before induction of anaesthesia. plasma levels of ot normally vary between 10 and 100 pg / ml. ot secretion peaks during child birth and during bf, levels returning to normal in between bf. blunting of plasma stress hormone levels were seen up to 30 min after bf, and bf women did not have attenuated physiological or subjective anxiety responses to a laboratory psychosocial stress administered 1 h after their last episode of lactation. this may be the reason for the higher requirement of sevoflurane and propofol in women in whom bf was withheld. the possible rebound increase in noradrenaline levels following ot - induced temporary suppression may explain higher hr and map in these women, practice implication of this concept include the possibility that the withhold feeding mother might be physiologically more fragile and more susceptible to post - partum illness, further research is required to provide sufficient evidence for this effect. when ot levels are measured in the cerebrospinal fluid, levels 510 fold higher than in plasma are normally found, but the concentration of ot at its receptor sites is not known. ot does not readily cross into the brain and needless to say, blood levels often do not correlate well with brain levels, so measuring peripheral ot may not correlate levels at otr within the cns. in humans, minimum alveolar concentration (mac) is reduced by 30% during early gestation, and in post - partum women mac returns to that of the non - pregnant state within 2 days of delivery, possibly due to sedative effect of progesterone which returns to baseline within 2448 h after delivery. post - partum, gastrointestinal tract related and mechanical factors are relieved immediately after delivery, gastric emptying time returns to normal as early as 18 h post - partum and gastric volume and ph are comparable with non - pregnant women. all cases in our study were elective and were fasting overnight and received acid aspiration prophylaxis; no signs of acid aspiration (gastric contents / stain on lma at extubation time) were seen in our cases, but it is recommended to use lma - proseal in post - partum period. the present findings in lactating women were restricted to after 3 days and within 1 week after giving birth, and we could not compare with exclusive formula feeding mothers to confirm if the same associations occur in these groups. as these data are suggestive rather than conclusive, further research is required to provide sufficient evidence on effects of ot parse on the requirement of anaesthetic and non - anaesthetic drugs. the of this study indicate that bf before induction of anaesthesia attenuates the stress response to surgery and maintains haemodynamic stability, and reduces the consumption of both propofol and sevoflurane, and with - holding of bf increases the stress response with increased consumption of both propofol and sevoflurane.

and aims: unique post - partum endocrine hormone oxytocin secreted during breastfeeding (bf) has amnestic, sedative properties and down - regulates stress responses. this study was done to assess the effect of bf on consumption of propofol, sevoflurane and haemodynamic stability in women.methods:study was conducted on 120 women aged 2030 years of american society of anesthesiologists i and ii physical status scheduled for tubectomy under general anaesthesia who were randomly allocated to three groups 40 of each; bf, withhold feeding (wf), and non - feeding (nf) groups. all received standard premedication. heart rate (hr), mean arterial pressure (map) and state entropy (se) values were recorded at regular intervals. all patients were induced with intravenous propofol until the se levels dropped to 45, and dose of propofol recorded. airway was secured with laryngeal mask airway and anaesthesia was maintained with sevoflurane in 60% n2o and o2. sevoflurane concentration was adjusted to maintain se between 40 and 60. end tidal concentration of sevoflurane and consumption of sevoflurane (ml) was recorded by ge datex - ohmeda s/5 system. were analysed by analysis of variance and chi - square test.:demographic parameters were comparable. dose of propofol and sevoflurane consumption in group bf was significantly reduced by 20% and 35%, respectively (p < 0.05) compared to group nf. intra - operative hr and map were persistently low in group bf and elevated in group wf (p < 0.05).: bf before induction of anaesthesia decreases the consumption of propofol, sevoflurane and maintains the intra - operative haemodynamic stability, whereas withholding bf increases propofol and sevoflurane consumption with intra - operative higher hr and map, compared to control group.

leaf springs like all other springs serve to absorb, store, and release the energy. in heavy vehicles, leaves are stacked one upon another to ensure rigidity and strength. during its operation, the leaf spring requires that the load rate should vary within limit (7%) and the maximum stress induced should be lower than the maximum design stress. for a given stress range the leaf spring should have maximum fatigue life or as specified. the stress range and the maximum stress induced in the leaf spring play a vital role in deciding the load rate and fatigue life of the leaf spring. the maximum stress induced can be reduced by assembling the leaves with different radii of curvature and establishing a common curvature under no load. the proper distribution of the stress between the leaves can enhance the fatigue life of the leaf springs. evaluated the axial fatigue strength of en45a spring steel specimen experimentally as a function of shot peening in the conditions used. s / n curves of the specimens were correlated with leaf springs curve in vehicles. concluded that the influence of high contact pressure and temperatures ed in microweld between the two leaf surfaces. the fatigue strength of the leaf springs was studied as a function of shot peening parameters. an experimental leaf springs model was verified by using a leaf springs test rig that could measure vertical static deflection of leaf springs under static loading condition. the showed a nonlinear relationship between the applied load and the leaf springs deflection for both directions of loading, in form of a hysteresis loop.. studied the origin of premature failure analysis procedures, including examining the leaf spring history. the visual inspection of fractured specimens and simulation tests on real components were also performed. it was concluded that fracture occurred by a mechanism of mechanical fatigue initiated at the region of the central hole, which suffered the highest tensile stress levels. kumar and vijayarangan described static and fatigue analysis of steel leaf springs and composite multileaf springs made up of glass fibre reinforced polymer using life data analysis. the dimensions of an existing conventional steel leaf spring of a light commercial vehicle were taken and verified by design calculations. static analysis of 2d model of conventional leaf springs was also performed using ansys 7.1, and the obtained were compared with experimental . patunkar and dolas worked on nonlinear force displacement of each leaf spring as well as the spring characteristics of a pack consisting of two to four leaves using ansys. the from ansys were compared with those from the test, which showed a fairly good agreement with each other. the objective of the present work is to determine the effect of assembly stresses on fatigue life reliability of leaf springs. it is available in open literature that with proper combination of stepping and individual leaf camber the stress distribution can be uniform along the leaf. the 65si7 leaf springs design parameter of a light commercial vehicle is taken into consideration for this work. this paper is divided into two parts. in part one, a design procedure for determination of total moment of inertia, number of leaves, and stepping and individual leaf camber is established. the stepping and individual leaf camber is proposed with a view to lower stresses. using sae approach fatigue life of the leaf spring , three leaf springs specimens lots (four leaf spring assemblies in each lot) have been manufactured. all the specimens are tested on a full scale leaf spring testing machine for fatigue life of the leaf spring. the theoretical and experimental fatigue life (with and without assembly stresses) are compared for validation. the material is heat - treated at 880c and oil - quench - hardened and it is tempered at 410c for 90 minutes to get tempered martensite structure. a leaf spring is considered as a beam of uniform strength composed of leaves of equal thickness where the fiber stress is the same throughout the length of the beam. this approximation is justified for most of the springs within the accuracy necessary for layout work and with certain correction factors for estimate of required length, overhang, camber, width, thickness, and number of leaves. the calculation of the leaf spring parameters involves certain number of steps. as per sae spring design manual approach, the steps involved in leaf springs design are as follows. consider i = kl332esf=34351.7 mm4, where k = load rate, l = spring span, e = young's modulus, and sf = stiffening factor. consider tmax=8ismaxlpmax=8.20 mm. consider i1=n1i1, where n1 = number of leaves with thickness t1, i1 = moment of inertia for section t1 = 8 mm, and width b = 70 mm and i1=b1t1312 + 3.1428t1464=2846.5 mm4, where b1 = b t1 = 62 mm and n1 = 11. consider i1=112846.13=31311 mm4i2=n2i2, where n2 = number of leaves with thickness t2, i2 = moment of inertia for section t2 = 7 mm, and width b = 70 mm and i2=b2t2312 + 3.1428t2464=1918.6 mm4, where n2 = 1 and b2 = b t2 = 63 mm and i2=11918.6=1918.6 mm4itotal=i1+i2=33230 mm4. n / mm% age variation of k=3.271% (hence acceptable). the seat clamp is taken into consideration for stress calculation between the leaves. the inactive length in the seat for a spring without liners is estimated as distance between the outside edges of the clamp bolts which is 100 mm for this spring. the active length for both front and rear cantilever will be 50 mm less than the distance from centre of the eye to centre of an axle seat. the stress at 50 mm distance from the centre of axle seat can be calculated as the following: front cantilever stress, sfa = palat2itrear cantilever stress, srb = pblbt2it. front cantilever (a) length = l = l/2 = 575 mm and rear cantilever (b) length = l = l/2 = 575 mm. cantilever ratio y = a / b = 1 (symmetric spring) and seat length sl = 100 mm. active length of front cantilever la = (l sl/2) = 525 mm. active length of rear cantilever lb = (l sl/2) = 525 mm. design load on front cantilever pa = p/2 = 6479.5 n. design load on rear cantilever pb = p/2 = 6479.5 n. maximum load on front cantilever pamax = pmax/2 = 14005 n. maximum load on rear cantilever pbmax = pmax/2 = 14005 n. as the leaf spring is symmetric cantilever ratio = y = a / b = 1; therefore, la = lb, pa = pb (at design load) and pamax = pbmax (at maximum load). also sfa = srb = sd (at design load) and sfa = srb = sm (at maximum load). front cantilever (a) length = l = l/2 = 575 mm and rear cantilever (b) length = l = l/2 = 575 mm. cantilever ratio y = a / b = 1 (symmetric spring) and seat length sl = 100 mm. active length of front cantilever la = (l sl/2) = 525 mm. active length of rear cantilever lb = (l sl/2) = 525 mm. design load on front cantilever pa = p/2 = 6479.5 n. design load on rear cantilever pb = p/2 = 6479.5 n. maximum load on front cantilever pamax = pmax/2 = 14005 n. maximum load on rear cantilever pbmax = pmax/2 = 14005 n. as the leaf spring is symmetric cantilever ratio = y = a / b = 1; therefore, la = lb, pa = pb (at design load) and pamax = pbmax (at maximum load). also sfa = srb = sd (at design load) and sfa = srb = sm (at maximum load). the total stress induced in the leaf spring is the summation of load stress and assembly stress: s = sl+sa. if all the leaves are fitted with the common unassembled curvature, then the assembly stress is zero; but the leaves are fitted in such a manner that the radius of curvature goes on increasing from the master leaf to the last leaf. with the use of different individual leaf camber, the assembly stress can be added to or deducted from the load stress of the assembled spring to obtain desirable stress pattern in the leaf spring. the assembly stress is arbitrarily chosen except they must be selected in increasing order from the main leaf to shorter leaf such that sat = 0. a negative assembly stress in the main leaf is required to reduce the maximum stress induced in the master leaf to safe limits as the longitudinal and lateral forces are more in the master leaf. a negative assembly stress is provided in the main leaf so as to reduce the maximum stress to about 866 mpa. the longitudinal and lateral forces imposed on the main leaf and also its greater stress range are the reasons for reducing its bending stress. in view of the condition sa t = 0, several selections of deducted and added stresses for the individual leaves are analysed before deciding on the best arrangement as shown in table 4. in almost all leaf springs the unassembled curvatures qn are different in the unassembled leaves. in assembly, a common (unloaded) curvature qo is established which is variable along the spring even if the leaves are made of circular arcs. the individual leaf curvature is calculated from common curvature as the folowing: qn = qosaey. curvature is called positive in the direction of increasing load and camber is conventionally positive in the opposite direction. camber can be converted into curvature as qo = curvature=8camberlength2=0.000574669 mm1. the relationship between free curvature, assembly curvature, and loaded curvature is given by qfree+q = qloaded. for flat leaf, the curvature is zero and it increases in the direction of load application; and for most of the springs the free curvature is negative. the no load camber requirement for this leaf spring assembly is 95 4 mm. the deflection can be determined as force per unit load rate. for different values of the load the corresponding values of deflection the stresses induced at different loads can be determined by substituting the values of the load in the stress induced formula. the analytical for load, deflection, and bending stress are shown in table 6. fatigue life is expressed by the number of deflection cycles a spring will withstand without failure or permanent set. a leaf spring used in a suspension will undergo a large number of cycles of small amplitude near the design load position without failure. under the greater amplitude the number of cycles without failure will be reduced since the maximum stresses as well as the stress range are increased and both are determining factors in fatigue life of the spring. as per sae spring design manual, this criteria is frequently used for determination of approximate fatigue life of the spring; initial stress (horizontal scale) and maximum stress (vertical scale) are intersected to estimate the number of cycles the spring will withstand for given loading condition. the fatigue test stroke for leaf spring without considering assembly stress is determined as the following: deflection to design load = 12959/153.1 = 84.6 mm, maximum load = 28 kn, metal - to - metal clearance (compression stroke) = 94.6 mm, total deflection to maximum load = 182.9 mm, stress at metal - to - metal position = 969 mpa, stress rate = 969/182.9 = 5.29 mpa / mm, release stroke = 0.5 94.6 = 47.3 mm, fatigue test stoke = 47.3 + 94.6 = 141.9 mm, initial stress = 969 (141.9 5.29) = 218.3 mpa. deflection to design load = 12959/153.1 = 84.6 mm, maximum load = 28 kn, metal - to - metal clearance (compression stroke) = 94.6 mm, total deflection to maximum load = 182.9 mm, stress at metal - to - metal position = 969 mpa, stress rate = 969/182.9 = 5.29 mpa / mm, release stroke = 0.5 94.6 = 47.3 mm, fatigue test stoke = 47.3 + 94.6 = 141.9 mm, initial stress = 969 (141.9 5.29) = 218.3 mpa. the fatigue test stroke for leaf spring by considering assembly stress is determined as the following: deflection to design load = 12959/153.1 = 84.6 mm, maximum load = 28 kn, metal - to - metal clearance (compression stroke) = 94.6 mm, total deflection to maximum load = 182.9 mm, stress at metal - to - metal position = 885 mpa, stress rate = 885/182.9 = 4.83 mpa / mm, release stroke = 0.5 94.6 = 47.3 mm, fatigue test stoke = 47.3 + 94.6 = 141.9 mm, initial stress = 885 (141.9 4.83) = 199.6 mpa. deflection to design load = 12959/153.1 = 84.6 mm, maximum load = 28 kn, metal - to - metal clearance (compression stroke) = 94.6 mm, total deflection to maximum load = 182.9 mm, stress at metal - to - metal position = 885 mpa, stress rate = 885/182.9 = 4.83 mpa / mm, release stroke = 0.5 94.6 = 47.3 mm, fatigue test stoke = 47.3 + 94.6 = 141.9 mm, initial stress = 885 (141.9 4.83) = 199.6 mpa. for the production of high strength leaf springs, the process is comprised of shearing, punching, heat treatment, hot cambering, shot peening, scragging, and testing for load rate and durability. the processing of the raw material plays a vital role in achieving the required load rate and fatigue life. after punching and shearing, the raw material the structure of the raw material is partial austenite, and after quenching the structure is martensite, but after the tempering process the structure should be tempered martensite. the material is heated in the furnace in the temperature range of 880910c depending upon the cross section thickness and width. the spring steel is having the thickness of 8 mm and width of 70 mm which is heated at 880c to achieve full austenite structure. hot cambering of the spring is done in this state by passing through finger cambering tools followed by quenching in oil at temperature of 80c. tempering is done at a temperature of 410c for 90 min slow cooling till the tempered martensite structure is achieved. the final assembly is done by pulling all the leaf with a centre nut and bolt. the 65si7 leaf springs assembly consists of two full length leaves and ten graduated leaves, four rebound clips of mild steel, four shim pipes with four nut and bolts, four rivets, centre nut and bolt, and bush of bronze. the full scale testing of leaf springs was carried out in an electrohydraulic static component testing system. the laminated leaf springs were placed in a fixture simulating the conditions of a vehicle. the setup consists of a hydraulic power pack to give a hydraulic pressure of 20.6 mpa with a flow rate of 210 liters per minute (lpm), which was sent to a hydraulic actuator to operate at a frequency of 0.3 hz with the displacement specified by the alternating load. this involves applying the axial load on the leaf springs and measuring the deflection and bending stress. the conventional leaf spring was tested under static load condition by using hydraulic static load ram for load application. mounting of the leaf spring was done by keeping it in inverted manner on the test bed. two eye ends were held in the clamping devices and load was applied from the top, at the center of leaf springs. to measure the load dial indicator was used, which was located beside the full scale testing machine and deflection was measured by strain gauges located at the clamping of the test rig. the springs were loaded from unladen load (i.e., 7.6 kn) to maximum load (i.e., 28 kn). the vertical deflection of the springs at the unladen load, design load, flat load, rubber touching load, and metal - to - metal contact or maximum load was recorded, respectively, as per the standard operating procedure prescribed. the leaf springs were tested on a full scale testing machine under the unladen load, rated load, flat load, rubber touching load, and metal - to - metal load, and the corresponding deflection and stress values observed are shown in table 7. table 7 depicts the observed values of deflection and stress corresponding to the loads applied on the shorter leaf by a static hydraulic ram. as per the is1135 typically this can be between 0.5 times the rated load and twice the rated load unless otherwise specified by vehicle manufacturer. consider oa = ob(ocob)2, where oa = load / deflection corresponding to rated load. consider ob = load / deflection corresponding to maximum load experienced under actual vehicle conditions typically 2 g, where g is the load shared by springs under the laden condition of the vehicle. for determination of experimental fatigue life, four specimens (s-1, s-2, s-3, and s-4 per batch) for three stress ranges (i.e., 269896 mpa, 218969 mpa, and 200885 mpa) are manufactured with proposed parameters. the stress range was considered for first lot of the specimens to be 627 mpa, 1.3 0.7 g. all the four specimens were tested under same stress range and fatigue life was determined. the stress range for second lot of the specimen was 751 mpa based on sae spring design manual approach, without considering assembly stresses; that is, all the leaves with common curvature were assembled. the stress range for third lot of the specimen was 685 mpa based on sae spring design manual approach, by considering assembly stresses. the spring was clamped in the centre to simulate its installation in the vehicle as shown in figure 3. as per the requirement specified by the vehicle manufacturer, the leaf springs are to be tested on full scale testing machine as per 1.3 0.7 g. the maximum load will be 2 g and the minimum load will be 0.6 g. here g represents the design load. the material processing for all the twelve specimens is the same, that is, normal rolling, quenching at 880c, tempering at 410c for 90 mins, shot peening at 18 a intensity, bhn 380432, and scragging at 0.9% of yield stress. for the first lot of the specimens the maximum stress is 896 mpa and minimum stress is 269 mpa as specified by the vehicle manufacturer. the fatigue life for the four specimens is 84212, 81961, 82226, and 85656 number of cycles. the second lot of specimen is assembled by considering common curvature, that is, without assembly stresses. the fatigue life for the four specimens is 66796, 69320, 70119, and 69956 number of cycles. the third lot of specimens is assembled by considering the assembly stresses and leaves with proposed individual leaf camber assembled by pulling against each other to establish common curvature. the fatigue life for the four specimens is 74827, 76658, 79010, and 77229 number of cycles. it is observed that the stress range for first lot, second lot, and third lot is 627 mpa, 751 mpa, and 685 mpa, respectively. the maximum stress for first lot, second lot, and third lot is 896 mpa, 969 mpa, and 885 mpa, respectively. it is observed that the lower the stress range is the higher the fatigue life will be. but for almost same stress range, the fatigue life decreases by increasing the maximum stress. it is also observed that the experimental fatigue life increases by 11.41% by considering assembly stresses. other factors like shape, size, temperature, surface, and so forth also affect the fatigue life of the leaf springs which can be considered for estimation of fatigue life. it is observed from figure 4 that the individual leaf camber is 99 mm, which is decreasing from the main leaf. the individual leaf camber is 84 mm for the second leaf with military wrapper. hence, the individual leaf camber decreases from the main leaf to the last leaf (almost flat). from figure 5 it is observed that the maximum stress induced in the leaf springs without considering the assembly stresses is 969 mpa and the initial stress value is 218 mpa. the intersection of 969 mpa and 218 mpa lies in the zone of 30000 to 50000 cycles. as the point of intersection it is observed that the maximum stress induced in the leaf springs by considering the assembly stresses is 885 mpa and the initial stress value is 200 mpa. the intersection of 885 mpa and 200 mpa lies in the zone of 50000 to 75000 cycles. as the point of intersection therefore, fatigue life of leaf springs without and with considering assembly stresses would be approximately (460001.2) 55200 cycles and (580001.2) 69600 cycles, respectively. table 9 shows the fatigue life comparison between the sae spring design manual and experimental testing, by considering and not considering the assembly stresses. it is observed from table 9 that as per sae approach the fatigue life without considering the assembly stresses is 55200 cycles and by considering the assembly stress the fatigue life is 69600 cycles. as per sae the fatigue life is 69047 and 76931 cycles without and by considering the assembly stresses, respectively, in the experimental testing. in experimental testing it is observed that the fatigue life increases by 11.41% due to the reduction in stress range, and maximum stress has reduced from 969 mpa to 885 mpa by considering assembly stress. the theoretical and experimental fatigue life of a light commercial vehicle leaf spring is determined by considering assembly stresses and without considering assembly stresses, and following are made.the maximum stress induced in the leaf spring reduces and uniform stress distribution is achieved by considering the assembly stresses. the fatigue life increases due to negative assembly stresses, which reduces the maximum stress.it is also concluded that for the same stress range, higher maximum stress reduces the fatigue life of the leaf spring and higher initial stress improves the fatigue life of the leaf springs. the maximum stress induced in the leaf spring reduces and uniform stress distribution is achieved by considering the assembly stresses. the fatigue life increases due to negative assembly stresses, which reduces the maximum stress. it is also concluded that for the same stress range, higher maximum stress reduces the fatigue life of the leaf spring and higher initial stress improves the fatigue life of the leaf springs.

the maximum stress induced plays vital role in fatigue life improvement of leaf springs. to reduce this maximum stress, leaves with different unassembled cambers are assembled by pulling against each other and a common curvature is established. this causes stress concentration or sets assembly stress in the assembled leaf springs which is subtractive from load stress in master leaf while it is additive to load stress for short leaves. by suitable combination of assembly stresses and stepping , it is possible to distribute the stress and improve the fatigue life of the leaf spring. the effect of assembly stresses on fatigue life of the leaf spring of a light commercial vehicle (lcv) has been studied. a proper combination of stepping and camber has been proposed by taking the design parameters into consideration, so that the stress in the leaves does not exceed maximum design stress. the theoretical fatigue life of the leaf springs with and without considering the assembly stresses is determined and compared with experimental life. the numbers of specimens are manufactured with proposed parameters and tested for load rate, fatigue life on a full scale leaf springs testing machine. the effect of stress range, maximum stress, and initial stress is also discussed.

the past years have witnessed a growing interest in the therapeutic use of cannabis and its constituents to manage chronic pain.1 irrespective of the number of new trials examining the use of cannabinoids for chronic pain, the evidence of its effectiveness and its safety remains limited.2 the pharmacology of cannabis sativa (the strain generally used to treat chronic pain) is quite complex, as it contains ~100 distinct cannabinoids,3 the relative levels of which largely determine their therapeutic effect.4 of the numerous cannabinoids, 9-tetrahydrocannabinol (9-thc) is considered the most psychoactive.5,6 the current definition of cannabinoids includes all endogenous and exogenous compounds that act on cannabinoid receptors.7 thc is not the only cannabinoid with therapeutic effects8, as cannabidiol (cbd) is used in the treatment of several different conditions. cbd is important not only for its therapeutic effects but also for its ability to mitigate euphoria and other side effects caused by thc,9 thereby increasing the potential therapeutic applications of cannabis. a lack of standardized dosing and uncertainty regarding the ideal ratio between thc and cbd continue to limit the medicinal usage of marijuana.10 a review by koppel et al11 analyzed six different neurological disease states and symptoms and a variety of thc / cbd ratios and methods of administration (e.g., oral, oromucosal, and inhaled) without obtaining clear evidence for specific indications. as there are numerous products containing myriad thc / cbd ratios, it remains extremely challenging to draw reproducible data regarding their effectiveness in the treatment of pain.10 there are several methods of administration of cannabinoids including smoking, orally (by infusion or by extraction in oil, as well as through edible products), vaporizing, and transdermally. while smoking remains the most common form of administration, a recent study found that in jurisdictions in which medical marijuana is legal, smoking is not necessarily the preferred route of administration.12 however, variance in routes of administration makes comparison of of different studies questionable. as a number of different compounds and methods of administration exist, thorough education of the physician (as well as the patient) is imperative prior to prescribing or recommending these compounds.13 the most comprehensive meta - analysis of inhaled cannabis for chronic pain to date14 has recently supported its short - term effectiveness in treating neuropathic pain. the authors also concluded that more studies are needed in order to evaluate long - term effectiveness and safety. these are consistent with those from schatman s 2015 comprehensive review.10 regarding safety, aggarwal15 opined that although little data are available on the risks associated with long - term medical use in published clinical trials, it can still be used to treat complex chronic pain conditions. more recently, ware et al16 published a 1-year follow - up trial in order to better investigate the long - term efficacy and safety. the authors did not find differences in serious adverse events between chronic pain patients treated with or without cannabis. nevertheless, a higher incidence of mild adverse effects was registered among patients treated with cannabis. even though the evidence is still weak and more studies are needed, there is significantly increasing availability of cannabis to treat chronic pain in several different countries, especially in the us and canada.17 understanding of complex policy and public health issues is imperative in order to fully understand the distinction between medical vs. recreational utilization of cannabis. savage et al recently concluded that there is a need not only for additional empirical investigation but also for increased research funding to help us develop a better understanding of how to make cannabinoids more effective and safer.17 questions have also been raised regarding the legitimacy of dispensaries clientele.10 it has been demonstrated that most dispensary customers had initiated marijuana use in adolescence, with one - half presenting with indications of risky alcohol use and 20% presenting with recent histories of prescription medication or illicit drug abuse.15 hence, many concerns are arising regarding the distinct possibility that in the us and canada, some of the same problems that these nations have had with prescription opioid abuse will potentially develop in relationship to medical marijuana. currently, the us is aggressively fighting its opioid crisis while simultaneously liberalizing access to cannabis for both medical and recreational utilization.18 an italian law approved in 201519 authorizes the use of cannabis to treat chronic pain. the law allows for the utilization of cannabis not only for neuropathic pain but also for all chronic pain conditions, as well as for spasticity, cachexia, and anorexia among aids and cancer patients, ocular hypertension in glaucoma, the alleviation of spasms in tourette syndrome, and some types of epilepsy, reiterating that cannabis - based drugs should be prescribed only when other available medications have proven to be ineffective or inadequate to the therapeutic needs of the patient. in order to reduce the costs of cannabinoid products, the italian government in 2014 committed its military chemical - pharmaceutical factory to cultivate cannabis to be distributed to all pharmacies across the country (initial production is scheduled for early in 2017). in the meantime , physicians can legally prescribe different cannabis products (such as bedrocan, bediol, and bedrolite), with different thc and cbd concentrations; these drugs can be administered orally (e.g., through infusions in olive oil) or via inhalation. bedrocan s constituents are 22% thc and < 1% cbd, while bediol contains 6.5% thc and 8% cbd and bedrolite contains 9% cbd and 0.4% thc and is accordingly considered non - psychoactive.20 all patients treated with cannabinoids have to be registered through a ministry of health database. in recent months, the society that includes all second - level (hubs) pain centers (pinhub, www.pinhub.it) has received a directive to evaluate all of these data among its centers. this paper presents a retrospective analysis of a case series of all chronic pain patients treated in one pinhub center over the past year in order to provide a snapshot of the initial italian experience with legalized cannabis use for chronic pain. the end point of this study has been the evaluation and identification of clinical indications and dosages currently used in second - level center of pain therapy. although our intention was to include data from all six of the centers initially involved in the study, a paucity of data from five of the six centers precluded doing so. following the approval of the italian law sanctioning the medical use of cannabis, all patients who initiated the use of medical cannabis have had to be registered, with their consent, in a database to evaluate specific data (figure 1). a retrospective group analysis of a case series of all chronic pain patients treated in one of the second - level pain clinics affiliated with the pinhub group in the first year following the approval of the italian law (december 2015 to november 2016) was performed. the clinical centers intended to be involved in this study were the pain therapy services of the following hospitals: ss antonio e biagio hospital (alessandria), ss annunziata hospital (chieti), monaldi hospital (naples), verona university hospital (verona), siena university hospital (siena), and azienda ospedaliera universitaria pisana (pisa). patients gave their permission to use their data when the physician filled out the case report form (crf). this research did not require approval by the institutional review boards of the aforementioned clinical centers as we used only raw data that was completely de - identified and anonymous. the data were presented in accordance with the strengthening the reporting of observational studies in epidemiology (strobe) guidelines.21 this study evaluated all patients registered as they had initiated treatment with cannabinoids for chronic pain based upon the judgment of their pain therapists. in accordance with the italian law, all patients treated with cannabinoids have had to be 18 years of age and determined to suffer from refractory chronic pain. according to italian law, cannabis could be prescribed with only two routes of administration: orally (infusion or extraction in olive oil) or through inhalation. smoking the cannabis is not permitted, so vaporization was the technique used for inhalation. currently, there are no national guidelines to be followed by physicians. furthermore, comorbidities and their severity that might preclude the treatment are not commonly predefined among the centers that are using this treatment. the primary end point of this study was to provide insight into how the italian second - level pain centers are utilizing medical cannabinoids in terms of routes of administration. secondary end points included the determination of the types of cannabis products most commonly utilized, as well as dosing. participating physicians were able to choose among infusion or inhalation via vaporization of the following types of cannabinoid products: pure bediol, bediol combined with bedrocan, pure bedrocan and bedrolite, and oil infused with bedrocan. as ~92% of the patients in the study used the high - thc, low - cbd bedrocan, it was unable to assess differences in efficacy and adverse events between the strains utilized in this preliminary investigation (figure 1). furthermore, the study evaluated effectiveness and safety through the analysis of patients who had at least one follow - up subsequent to initial prescription based on the data provided by the crfs defined by the italian health ministry. through the data requested by the italian health ministry for follow - up purposes, the study evaluated the dosages utilized, whether the therapy has been continued, clinical efficacy (determined by patient - reported levels of pain severity), and the reason for discontinuing treatment (pain worsened, pain not clinically improved, or presence of intolerable side effects). although assessing other outcomes such as functionality would have been useful, the italian health ministry has requested the provision only of data pertaining to pain relief. data have been obtained by the crf requested by the italian health ministry that has to be filled whenever patients initiated the therapy or had subsequent contact with the pain center at which they were being treated. the sample size was not calculated a priori as this study was an explorative retrospective analysis of all patients treated with cannabinoids for a year in second - level centers in italy after the approval of the law that legalized their use for chronic pain. all data are presented with percentages or means, as well as standard deviations. as this initial study is a case series, it is not surprising that the data are quite heterogeneous. however, the authors chose not to formally evaluate any statistical differences between the different treatments, as there were insufficient number of subjects using any product besides bedrocan. the sample size was not calculated a priori as this study was an explorative retrospective analysis of all patients treated with cannabinoids for a year in second - level centers in italy after the approval of the law that legalized their use for chronic pain. all data are presented with percentages or means, as well as standard deviations. as this initial study is a case series, it is not surprising that the data are quite heterogeneous. however, the authors chose not to formally evaluate any statistical differences between the different treatments, as there were insufficient number of subjects using any product besides bedrocan. of the 659 patients who initiated treatment with oral cannabis at all of the facilities originally intending to participate in the study, the study included only the subjects from the azienda ospedaliero universitaria pisana (pisa) for the analysis of data. as only 6% of the patients came from the other five facilities, a comparison of inter - facility data would not have been possible. on a positive note, utilizing only the data from the azienda ospedaliero universitaria pisana also allowed for a more homogeneous evaluation of current treatment. of all the subjects, 181 were male and 422 were female, and data on gender were missing for 11 subjects. of 614 patients whose data were used, 341 (55.5%) had at least one follow - up at 98.42 (144.66) days. figure 3 indicates the specific symptoms for which physicians initiated cannabis treatment. in 89.2% of patients, follow - up, 76.2% patients continued the cannabinoid therapy (of whom 64.7% reported an improvement associated with the therapy, while 34.1% reported neither an improvement nor a worsening), and 23.8% discontinued treatment (3.7% due to a worsening of their pain, 61.7% due to side effects, 29.6% due to an unsatisfactory change of their clinical condition ; for 4.9% of patients, the data were missing). there were no complaints of severe side effects, even though this may not have been directly assessed at follow - up visits. figure 4 illustrates the dosages (mg / day) of bedrocan and bediol at the initiation of the study and at initial follow - up, respectively. over the past year, a dramatic increase in the use of cannabis to treat chronic intractable pain has been witnessed.11 nevertheless, specific guidelines are still missing, and there is considerable heterogeneity in the use of this drug throughout the world. in italy, cannabis was approved legally 18 months ago for its use for several indications, including treatment of intractable pain (not specifically neuropathic pain). all patients who initiated treatment with cannabis had been required to be registered in a specific national database in order to evaluate the clinical effectiveness and safety of cannabis in the 2 years immediately following its legalization. hence, a retrospective analysis of a case series of patients treated in a second - level pain clinic that is a part of the pinhub society was performed. this analysis attempted to provide an initial snapshot of how cannabinoids are used in italy in order to understand how cannabis is currently used for chronic pain treatments and its implications for clinical practices. the initial data that were obtained are those only from one of the six centers that have extensively used cannabinoids for intractable chronic pain. it was observed that cannabinoid treatment is not yet common, even though the new law has been approved. this situation could be compared to a similar situation observed in italy several years ago with regard to opioids; despite the passage of legislation intended to facilitate opioid prescription, several years passed prior to the initiation of an appropriate increase in opioid prescription. the population in this investigation that was treated was similar to that generally observed in pain clinics (average age of 61 years, and majority being female). regarding the type of cannabinoid and modality of administration, almost all patients received infusions of bedrocan, while administration of the extract of cannabinoids in olive oil or vaporization was quite uncommon. hence, as these data are quite discrepant from those from canada and the us, it is extremely difficult to compare the effectiveness and safety of the treatment to those data from studies performed in other nations. this heterogeneity currently limits the possibility of developing guidelines available and useful on an international basis. although the method of administration in this study was relatively homogeneous, a large variety (as demonstrated through a large standard deviation) of concentrations (especially for bediol) used both at the initiation of treatment and (even more so) at the first follow - up was observed. furthermore, in italy, a formidable variety of indications for the prescription of cannabinoids. several types of chronic pain syndromes have been treated. in order to obtain more reproducible data, in the near future, the authors intend to better focalize indications for specific pain syndromes, such as intractable neuropathic pain. that a low rate (22%) of discontinuation of cannabinoids (certainly compared to discontinuation rates of opioid analgesics) was observed is clearly encouraging, particularly given that the iatrogeneses of cannabinoids in pain treatment are likely less substantial than those of opioids. first, it is a retrospective analysis of only one center, and unfortunately, we are compelled to acknowledge that this somewhat limits our understanding of the efficacy of medical cannabinoids for chronic pain. accordingly, we will continue to collect data from the five hospitals other than the azienda ospedaliero universitaria pisana and intend to publish data that will address inter - facility variance in outcomes. second, future investigation will analyze the impact of concomitant treatments, which will require a sufficient number of subjects for the performance of analyses of covariance. thus, we expect to move toward a better understanding of whether cannabinoid products are more effective as a monotherapeutic approach to chronic pain treatment as opposed to a component of multimodal care. finally, as the vast majority of patients enrolled in the study used the high - thc bedrocan, we were unable to assess the difference between cannabinoid treatments with different ratios of thc and cbd. future investigation will certainly look at this issue, as questions regarding the medical benefits and safety of high - thc, low - cbd cannabis have arisen.10 an initial analysis of the italian clinical practice of the use of cannabinoids for chronic pain syndromes in a reasonably large population is presented. even with the heterogeneity of the sample size and the limited data available, it can be stated that the treatment seems to be effective and safe in the majority of patients, even though the safety and effectiveness data should be confirmed in a trial better designed to assess them. nevertheless, additional data from a variety of types of trials are needed in order to better understand the benefits of cannabinoids to chronic pain sufferers. it is important for the italian and other european pain societies to more thoroughly investigate this topic in order to provide clearer and more useful guidelines, which will more adequately guide physicians in the use of this drug in the treatment of chronic pain.

despite growing interest in the therapeutic use of cannabis to manage chronic pain, only limited data that address these issues are available. in recent years , a number of nations have introduced specific laws to allow patients to use cannabis preparations to treat a variety of medical conditions. in 2015, the italian government authorized the use of cannabis to treat several diseases, including chronic pain generally, spasticity in multiple sclerosis, cachexia and anorexia among aids and cancer patients, glaucoma, tourette syndrome, and certain types of epilepsy. we present the first snapshot of the italian experience with cannabis use for chronic pain over the initial year of its use.methodsthis is a retrospective case series analysis of all chronic pain patients treated with oral or vaporized cannabis in six hubs during the initial year following the approval of the new italian law (december 2015 to november 2016). we evaluated routes of administration, types of cannabis products utilized, dosing, and effectiveness and safety of the treatment.as only one of the six centers has extensively used cannabinoids for intractable chronic pain (614 patients of 659), only the population from azienda ospedaliero universitaria pisana (pisa) was considered. cannabis tea was the primary mode of delivery, and in almost all cases, it was used in association with all the other pain treatments. initial and follow - up cannabinoid concentrations were found to vary considerably. at initial follow - up, 76.2% of patients continued the treatment, and < 15% stopped the treatment due to side effects (none of which were severe).we present the first analysis of italian clinical practice of the use of cannabinoids for a large variety of chronic pain syndromes. from this initial snapshot, we determined that the treatment seems to be effective and safe, although more data and subsequent trials are needed to better investigate its ideal clinical indication.

percutaneous nephrolithotomy (pcnl) is an effective choice in the treatment of large kidney stones because of excellent outcomes and acceptably low morbidity, and it is usually performed with the patient in the prone position.1 this approach provides a larger surface area for the choice of puncture site and a wider space for instrument manipulation. however, the prone position has several disadvantages: respiratory and cardiovascular risks; ventilatory difficulties, especially in obese patients and in elderly patients with compromised cardiopulmonary status;2,3 and the need for position changes during the procedure. for these reasons, many urologists have tried modified positions for pcnl (flank, supine, and modified supine).4,5 supine pcnl was first reported by valdivia ura et al. in 19983 and is regarded as possessing several advantages. another advantage of the supine position is that there is no need for position changes to perform other endoscopic procedures such as cystoscopic or ureteroscopic operations.6 the supine position enables simultaneous retrograde ureteroscopic procedures during pcnl without any position change. a nephrostomy catheter has routinely been placed after pcnl, because it provides proper drainage of urine, prevents urinary extravasation, and enables tamponade of bleeding.7,8 the need for placement of a nephrostomy catheter has been questioned, however, owing to increases in postoperative discomfort and morbidity, and several studies in the past two decades have demonstrated the feasibility of tubeless pcnl. in the current study, we evaluated the feasibility and efficacy of intermediate - supine pcnl in our initial experiences with this approach. the study was approved by the institutional review board of our institution. between january 2012 and october 2012, a total of 15 patients with renal stones underwent pcnl in the intermediate - supine position performed by a single surgeon (k.s.c .). preoperative data, operative outcomes, and postoperative outcomes were retrospectively collected and evaluated. before surgery , the desired calyx was punctured under fluoroscopy guidance and a guidewire was inserted by an interventional radiologist. then a pcnl was performed with the patient under general anesthesia in the intermediate - supine position. for intermediate - supine pcnl , the patient was placed in the supine position with a 1-l saline bag below the ipsilateral flank. thus, the ipsilateral flank was elevated 20, causing the posterior calyx to project more laterally (fig . , a retrograde ureteral occlusion catheter was fixed through the ipsilateral ureteral orifice by use of cystoscopy . under the guidance of c - arm fluoroscopy, we accurately assessed the punctured calyx through a guidewire placed in the intervention port . after amplatz sheath insertion, the nephroscope was introduced, and the stones were fragmented by using a lithoclast lithotripter ( electro - medical systems, nyon, switzerland) and were extracted by use of stone forceps or a suction catheter. after completion of stone removal, antegrade placement of a double - j ureteral stent was performed in most cases. if there was no significant bleeding and no significant injury to the renal pelvis or ureter, a tubeless pcnl was performed. in all cases, the stone size was measured with noncontrast computed tomography (ct) and was calculated by use of the following formula: widthlength0.25.9 complete stone - free status was defined as no stone fragments visible on the postoperative image study, and the operation was considered successful if the patient had either no or clinically insignificant residual stone fragments (largest stone diameter less than 4 mm and asymptomatic, nonobstructive, and noninfectious).10,11 length of hospital stay was defined as the time interval between the day of surgery and the day of discharge from the hospital. perioperative complications were evaluated according to the dindo - modified clavien system validated in 2004.12 pain was evaluated every 8 hours by a trained nurse using the visual analogue scale (vas) for pain. patients with moderate to severe pain were given tramadol 50 mg intravenous (iv) or pethidine 25 mg intramuscular (i m) if postoperative pain was not controlled with nonsteroidal analgesics. all patients were monitored for levels of hemoglobin and serum creatinine preoperatively and by the seventh postoperative day. a plain x - ray or an abdomen - pelvis ct the patient was discharged when he or she was thought to be free of complications and when the patient's pain was controlled with oral analgesics (vas pain score lower than 3). the preoperative and postoperative vas pain scores and serum creatinine variations among the subjects were analyzed by use of the wilcoxon signed rank test. comparisons with a p - value less than 0.050 were considered to be statistically significant. the study was approved by the institutional review board of our institution. between january 2012 and october 2012, a total of 15 patients with renal stones underwent pcnl in the intermediate - supine position performed by a single surgeon (k.s.c .). before surgery, the desired calyx was punctured under fluoroscopy guidance and a guidewire was inserted by an interventional radiologist. then a pcnl was performed with the patient under general anesthesia in the intermediate - supine position. for intermediate - supine pcnl , the patient was placed in the supine position with a 1-l saline bag below the ipsilateral flank. thus, the ipsilateral flank was elevated 20, causing the posterior calyx to project more laterally (fig . , a retrograde ureteral occlusion catheter was fixed through the ipsilateral ureteral orifice by use of cystoscopy . under the guidance of c - arm fluoroscopy, we accurately assessed the punctured calyx through a guidewire placed in the intervention port . after amplatz sheath insertion, the nephroscope was introduced, and the stones were fragmented by using a lithoclast lithotripter ( electro - medical systems, nyon, switzerland) and were extracted by use of stone forceps or a suction catheter. after completion of stone removal, antegrade placement of a double - j ureteral stent was performed in most cases. if there was no significant bleeding and no significant injury to the renal pelvis or ureter, a tubeless pcnl was performed. in all cases, the stone size was measured with noncontrast computed tomography (ct) and was calculated by use of the following formula: widthlength0.25.9 complete stone - free status was defined as no stone fragments visible on the postoperative image study, and the operation was considered successful if the patient had either no or clinically insignificant residual stone fragments (largest stone diameter less than 4 mm and asymptomatic, nonobstructive, and noninfectious).10,11 length of hospital stay was defined as the time interval between the day of surgery and the day of discharge from the hospital. perioperative complications were evaluated according to the dindo - modified clavien system validated in 2004.12 pain was evaluated every 8 hours by a trained nurse using the visual analogue scale (vas) for pain. patients with moderate to severe pain were given tramadol 50 mg intravenous (iv) or pethidine 25 mg intramuscular (i m) if postoperative pain was not controlled with nonsteroidal analgesics. all patients were monitored for levels of hemoglobin and serum creatinine preoperatively and by the seventh postoperative day. a plain x - ray or an abdomen - pelvis ct the patient was discharged when he or she was thought to be free of complications and when the patient's pain was controlled with oral analgesics (vas pain score lower than 3). the preoperative and postoperative vas pain scores and serum creatinine variations among the subjects were analyzed by use of the wilcoxon signed rank test. comparisons with a p - value less than 0.050 were considered to be statistically significant. we performed a total of 15 pcnl procedures with patients in the intermediate - supine position (table 1). the mean patient age was 58.7717.33 years, and the patients' mean body mass index was 26.174.22 kg / m. a nephrostomy catheter was inserted in two cases (13.3%); significant venous bleeding was suspected in one case, and a second - look operation was considered in one case. in 13 cases (86.7%), an antegrade ureteral stent indwelling at the ipsilateral site was performed in 14 cases. in two patients, the retrograde procedures were simultaneously performed without a change in position (one ureteroscopic ureterolithotomy and one transurethral placement of an occlusion catheter). the success rate was 80.0%, and the complete stone - free rate was 73.3%. one patient underwent a second - look operation owing to a large stone burden (26.00 cm) and long operative time (191 min) at the first operation. in the other two patients, it was anatomically difficult to obtain proper access to the remnant stones, which were relatively small, and the stones were successfully treated with shock wave lithotripsy. only two patients had mild complications according to the clavien - dindo classification (grade i in one patient and grade ii in one patient). serum creatinine levels measured preoperatively, on the day of surgery, and on postoperative day one were 1.120.89 mg / dl, 1.100.91 mg / dl (vs. preoperatively ; p=0.851), and 1.090.87 mg / dl (vs. preoperatively ; p=0.490), respectively. generally, pain was tolerable within 2 to 3 days; the vas scores for pain on the day of surgery, on postoperative day one, and on the day of discharge were 4.261.79 (vs. operative day ; p=0.040), 2.801.20, and 1.730.79 (vs. operative day ; p=0.003), respectively. many urologists have attempted to overcome the disadvantages of conventional pcnl, and the technique of pcnl has largely evolved in terms of the placement of tubes and changes in position over the past two decades. many cases of both supine pcnl and tubeless pcnl have been accumulated, but data are lacking for supine pcnl in conjunction with the tubeless technique. our early experiences suggested that intermediate - supine and tubeless pcnl is a safe and effective choice that offers several advantages with excellent outcomes. these experiences are consistent with a previous report by rana et al.13 those authors maintained that pcnl with the patient in the supine position has several advantages, such as a lower rate of complications, a shorter operative time, and the simplicity and ease of performance of the procedure. therefore, the procedure can provide uniform comfort for the anesthesiologist, patient, and surgical team. when the pcnl procedure is performed with the patient in the prone position, a ureteral catheter is commonly fixed in the lithotomy position before the patient is turned; however, pcnl in the supine position does not require turning. furthermore, pcnl in the intermediate - supine position facilitates the completion of ureteroscopic processes at the same time. therefore, the operative time for pcnl in the supine position is shorter than that for pcnl in the prone position.14 pcnl with the patient in the supine position saves 30 to 40 minutes of operative time and avoids the risk associated with a position change under general endotracheal anesthesia. de sio et al.14 reported that the operative time was 68 minutes (range, 55 - 140 minutes) in the prone position but 43 minutes (range, 25 - 120 minutes) in the supine position (p<0.001), and karami et al.15 reported that the operative time was reduced in the lateral position (74.426.9 minutes), prone position (68.737.4 minutes), and supine position (54.225.1 minutes) (p<0.040). de sio et al.14 reported that in their randomized controlled study, the stone - free rate was excellent in both the supine (88.7%) and the prone (91.6%) position pcnl groups. shoma et al.16 reported similar success rates for the supine (89%) and prone (84%) positions in a prospective nonrandomized study, and karami et al.15 reported that the success rate was 92% in the prone position, 86% in the supine position, and 88% in the lateral position. the success rate of supine pcnl is also dependent on the stone size and location. hoznek et al.17 demonstrated that the success rate was 90% for single stones, 78% for multiple stones, and 43% for staghorn stones during supine pcnl. xu et al.18 also suggested similar stone - free rates of 85.7% overall, 92.2% for a single calculus, and 72.9% for staghorn calculi. in the supine and intermediate - supine positions, mobile fluoroscopy (c - arm) can be utilized without the interrupted artifacts (spine, ribs, etc .) that are present in the lateral position, which might be helpful in improving surgical outcomes such as the stone - free rate.16,19 on the contrary, there are some limitations in using fluoroscopic examination during pcnl in the lateral position because of the possibility of the aforementioned artifacts. for the pcnl procedure, several studies have reported that the rate of significant bleeding requiring transfusion was about 1.5% to 9%3,13,16,19 and was directly related to the stone size, procedure time, and creation of multiple tracts.20 basiri and mohammadi sichani21 suggested that the risk of bleeding might be less in the supine position than in the prone position because a backflow of blood to the renal vein is caused by obstruction of the inferior vena cava. also, there might be less risk of colonic injury during supine pcnl because of the more anterior displacement of the colon away from kidney in the supine position than in the prone position.3,22 valdivia ura et al.3 reported no damage to the peritoneum and colon in a ct study and confirmed that the colon was further away from the kidney in the supine position than in the prone position. in their report on 557 patients, the operation was successful in 93% of the cases, with a low complication rate and no colonic perforation.3 however, a different study reported similar colonic injury risk between the supine position and the prone position.18 meanwhile, severe anesthesia complications are rarely reported in the prone position, and it is generally accepted that the supine position is more satisfactory for the anesthetist, especially in obese or high - risk anesthesia patients.23 in the prone position, it is difficult for the anesthetist to observe the patient effectively, and the prone position may not be favorable for resuscitation when acute syndromes such as obstruction of respiratory passages or acute myocardial ischemia occur during the operation.24 nevertheless, pcnl in the supine position also has several disadvantages. because the angle between the surface of the operation table and the anterior calyxes is smaller than that in prone position, it is difficult to access stones in the anterior calyxes.14,16,21 approaching the upper calyx is more difficult in the supine position, especially if placed overly medially, and this problem is more obvious on the left side.13,21 another disadvantage in the supine position is the mobility of the kidneys, which is greater than in the prone position. therefore, the kidneys are easy to move anteromedially during tract formation in the supine position. finally, the pyelocaliceal system is constantly collapsed in this position; thus, nephroscopic procedures might be more difficult.19 although there have been some debates on the efficacy and safety of tubeless pcnl, successful experiences by many urologists have rekindled clinical interest in tubeless or totally tubeless pcnl. according to recent studies, there is no significant difference in the rate of complications between standard pcnl and tubeless pcnl, and tubeless pcnl has several advantages.25,26 tubeless pcnl is associated with a shorter length of hospital stay; thus, patients can return quickly to everyday life, experience less pain, and incur a lower cost. sofikerim et al.27 reported that tubeless pcnl is a safe and effective technique even after supracostal access and is associated with less postoperative pain and a shorter hospital stay. shen et al.28 suggested that the hospital stay of the tubeless group was less than that of the middle - tube and large - tube groups in a multiple center metaanalysis. they recommended that a nephrostomy catheter be placed in certain situations (multiple access, major damage to the collecting system, possibility of a second look operation, severe intraoperative bleeding, complicated cases, and intrathoracic trauma).29 the disadvantages of the tubeless procedure, including the need to place a ureteral stent, should be considered; patients may have bladder irritation symptoms such as flank pain, gross hematuria, urinary frequency, and urgency because of the placement of a ureteral catheter, and patients must undergo cystoscopy to remove the ureteral stent.30 there were several limitations to our study. in addition, we did not attempt multiple accesses or an upper pole approach, and we selected relatively simple cases, although there were also some difficult cases with large staghorn stones among our early experiences. nevertheless, intermediate - supine and tubeless pcnl might be a safe and effective option that offers several advantages with excellent outcomes in the management of renal calculi. thus, a prospective randomized study with a larger population is needed to confirm our observations. in , intermediate - supine pcnl is believed to be feasible and effective and is associated with several advantages. it may shorten the preparation period of the operation and the operative time, and it can in a high stone - free rate with low complications. in our series, there was no increase in the bleeding risk with the intermediate - supine position; therefore, the tubeless technique could be applied in most cases. the tubeless technique also seems to have contributed to the favorable surgical outcomes in our early experiences.

we evaluated the feasibility and efficacy of intermediate - supine percutaneous nephrolithotomy (pcnl) in patients with renal calculi. fifteen patients were included in this study. the intermediate - supine operative position was modified by using a 1-l saline bag below the ipsilateral upper flank. a nephrostomy and stone extraction were performed as usual. after completion of the stone removal, a nephrostomy tube was used when necessary according to the surgeon's decision. if there was no significant bleeding or renal pelvic injury, tubeless pcnl was performed. the mean stone size was 5.485.69 cm2, the mean operative time was 78.9338.72 minutes, and the mean hospital stay was 2.601.29 days. tubeless pcnl was performed in 13 cases (86.7%), and retrograde procedures were simultaneously performed without a change of position in 2 patients (ureteroscopic ureterolithotomy in one patient and transurethral placement of an occlusion catheter in one patient). there were two complications according to the clavien - dindo classification (grade i in one patient and grade ii in one patient). the success rate was 80.0% and the complete stone - free rate was 73.3%. three patients with a significant remnant stone were also successfully managed with additional procedures (one patient underwent a second - look operation, and the remaining two patients were treated with shock wave lithotripsy). in the treatment of renal calculi, intermediate - supine pcnl may be a safe and effective choice that offers several advantages with excellent outcomes. thus, a prospective study with a larger population is needed to verify our outcomes.

this was a historical cohort study using data from the health improvement network (thin) database. thin is a population - based database of electronic health records of approximately 6 million patients from more than 300 general practices in the united kingdom. thin has been validated for a wide range of medical conditions, including stroke, and individuals contributing data to thin are representative of the uk population. data available include prescribed medications, medical diagnoses, lifestyle conditions, demographic / personal information, and feedback from specialist appointments and hospital admissions. the present study population was a subset of a larger thin study, in which patients with an autoimmune disease enrolled in thin between 1987 and 2007 had each been matched by age, sex, and general practice to up to 6 individuals without any autoimmune disease. ait was the exposure of interest; unexposed individuals comprised patients without ait (or any other autoimmune disease) who had been matched on age, sex, and general practice to the patients with ait in the original thin dataset. the outcome of interest was first - ever stroke (including ischemic and hemorrhagic) or tia during follow - up. exposure and outcome were defined using prespecified read code lists (appendix e-1 on the neurology web site at neurology.org). start of follow - up for individuals with ait and their unexposed counterparts was the date of the first thyroxine prescription in the patients with ait (the index date). end of follow - up was defined as the day of the outcome (stroke or tia) or end of follow - up in thin (death, transfer out, or the practice 's last data - collection date). patients with ait must have been first diagnosed with ait during active follow - up in thin and have been prescribed thyroxine during follow - up. individuals with other causes for hypothyroidism (e.g., previous thyroidectomy) were excluded from analyses (figure 1). diagnoses for past or ongoing conditions (including ait) are sometimes recorded retrospectively in the first few months after a patient registers with a practice. to ensure that we enrolled incident cases, we excluded patients with ait who were diagnosed during their first year of follow - up in thin. all individuals (exposed and unexposed) who had a code for stroke or tia before start of follow - up, or who were younger than 18 years, were not considered for inclusion. also, because the unexposed (non - ait) individuals had no other autoimmune disorders, individuals with ait who had a preexisting or who developed a second autoimmune disorder were also excluded. unexposed individuals had to be actively enrolled in thin at the index date of their matched individual with ait; to ensure that they were truly active, they had to have a consultation with the practice in the 6 months before or in the year after the index date of the patient with ait. aid = autoimmune disease; ait = autoimmune thyroiditis. in the conceptual framework for this study (figure 2), cofactors were categorized as follows: a priori confounders (sex, current age, general practice); other potential confounders (smoking, alcohol consumption, calendar year, time in study); and factors that could be either confounders or on the causal pathway from ait to stroke, depending on whether they occurred before or after ait diagnosis (figure 2b). missing data for smoking, alcohol, and body mass index (bmi) were treated using the missing indicator method. an individually matched analysis is not needed in matched cohort studies, but inclusion of the matched variables in a multivariable analysis is advisable; this also addressed any imbalances between exposed and unexposed patients regarding these variables after the application of exclusion criteria. (b) differentiation between confounding and mediating effects of factors hypothesized to be on the causal pathway. presence of factors at the index date was assumed to be attributable to confounding in the primary analysis. ait = autoimmune thyroiditis; bmi = body mass index; chd = coronary heart disease; chf = congestive heart failure. statistical analyses were conducted using stata 11 (statacorp, college station, tx). baseline characteristics were compared using cross - tabulation, means or medians as appropriate, and tests, 2-sided t tests, and wilcoxon rank - sum tests. main analyses were performed using random - effects multivariable poisson regression models allowing for clustering within general practice. , we assessed potential confounding by smoking, alcohol consumption, calendar year, and time in study, introducing these variables sequentially into the model and retaining them if they changed the effect estimate of ait on stroke incidence appreciably. the third model additionally assessed risk factors (hypertension, af, hyperlipidemia, diabetes, chd, congestive heart failure , and bmi) that were present at the index date and could therefore also confound the association between ait and stroke. in the final model, we examined time - updated values of these factors for individuals who developed these conditions during follow - up to assess potential causal pathways between diagnosed ait and stroke. we applied this analysis strategy to 2 main outcomes, first stroke, then stroke or tia. in all models, standard errors were examined for evidence of colinearity, and p values were obtained using likelihood ratio tests. we investigated effect modification by ait duration as a proxy for the effects of hypothyroidism (likely to be evident in the early stages of disease, before adequate thyroxine replacement) vs the possible cumulative effect of autoimmunity (long - term increased risk, likely to occur later during follow - up). we also investigated effect modification by age (< 60, 6080, > 80 years) and performed several sensitivity analyses. first, analyses were repeated starting follow - up at the date of first ait code instead of first thyroxine script. second, cardiovascular conditions present among patients with ait at diagnosis could be early mediating factors for stroke rather than a confounding variable; to assess their importance, we performed subgroup analyses for individuals without a history of any cardiovascular disease ethics approval was obtained from the south - east multicentre research ethics committee and from the london school of hygiene and tropical medicine ethics committee. this was a historical cohort study using data from the health improvement network (thin) database. thin is a population - based database of electronic health records of approximately 6 million patients from more than 300 general practices in the united kingdom. thin has been validated for a wide range of medical conditions, including stroke, and individuals contributing data to thin are representative of the uk population. data available include prescribed medications, medical diagnoses, lifestyle conditions, demographic / personal information, and feedback from specialist appointments and hospital admissions. the present study population was a subset of a larger thin study, in which patients with an autoimmune disease enrolled in thin between 1987 and 2007 had each been matched by age, sex, and general practice to up to 6 individuals without any autoimmune disease. ait was the exposure of interest; unexposed individuals comprised patients without ait (or any other autoimmune disease) who had been matched on age, sex, and general practice to the patients with ait in the original thin dataset. the outcome of interest was first - ever stroke (including ischemic and hemorrhagic) or tia during follow - up. exposure and outcome were defined using prespecified read code lists (appendix e-1 on the neurology web site at neurology.org). start of follow - up for individuals with ait and their unexposed counterparts was the date of the first thyroxine prescription in the patients with ait (the index date). end of follow - up was defined as the day of the outcome (stroke or tia) or end of follow - up in thin (death, transfer out, or the practice 's last data - collection date). patients with ait must have been first diagnosed with ait during active follow - up in thin and have been prescribed thyroxine during follow - up. individuals with other causes for hypothyroidism (e.g., previous thyroidectomy) were excluded from analyses (figure 1). diagnoses for past or ongoing conditions (including ait) are sometimes recorded retrospectively in the first few months after a patient registers with a practice. to ensure that we enrolled incident cases, we excluded patients with ait who were diagnosed during their first year of follow - up in thin. all individuals (exposed and unexposed) who had a code for stroke or tia before start of follow - up, or who were younger than 18 years, were not considered for inclusion. also, because the unexposed (non - ait) individuals had no other autoimmune disorders, individuals with ait who had a preexisting or who developed a second autoimmune disorder were also excluded. unexposed individuals had to be actively enrolled in thin at the index date of their matched individual with ait; to ensure that they were truly active, they had to have a consultation with the practice in the 6 months before or in the year after the index date of the patient with ait. in the conceptual framework for this study (figure 2), cofactors were categorized as follows: a priori confounders (sex, current age, general practice); other potential confounders (smoking, alcohol consumption, calendar year, time in study); and factors that could be either confounders or on the causal pathway from ait to stroke, depending on whether they occurred before or after ait diagnosis (figure 2b). missing data for smoking, alcohol, and body mass index (bmi) were treated using the missing indicator method. an individually matched analysis is not needed in matched cohort studies, but inclusion of the matched variables in a multivariable analysis is advisable; this also addressed any imbalances between exposed and unexposed patients regarding these variables after the application of exclusion criteria. (b) differentiation between confounding and mediating effects of factors hypothesized to be on the causal pathway. presence of factors at the index date was assumed to be attributable to confounding in the primary analysis. ait = autoimmune thyroiditis; bmi = body mass index; chd = coronary heart disease; chf = congestive heart failure. statistical analyses were conducted using stata 11 (statacorp, college station, tx). baseline characteristics were compared using cross - tabulation, means or medians as appropriate, and tests, 2-sided t tests, and wilcoxon rank - sum tests. main analyses were performed using random - effects multivariable poisson regression models allowing for clustering within general practice. , we assessed potential confounding by smoking, alcohol consumption, calendar year, and time in study, introducing these variables sequentially into the model and retaining them if they changed the effect estimate of ait on stroke incidence appreciably. the third model additionally assessed risk factors (hypertension, af, hyperlipidemia, diabetes, chd, congestive heart failure , and bmi) that were present at the index date and could therefore also confound the association between ait and stroke. in the final model , we examined time - updated values of these factors for individuals who developed these conditions during follow - up to assess potential causal pathways between diagnosed ait and stroke. we applied this analysis strategy to 2 main outcomes, first stroke, then stroke or tia. in all models, standard errors were examined for evidence of colinearity, and p values were obtained using likelihood ratio tests. we investigated effect modification by ait duration as a proxy for the effects of hypothyroidism (likely to be evident in the early stages of disease, before adequate thyroxine replacement) vs the possible cumulative effect of autoimmunity (long - term increased risk, likely to occur later during follow - up). we also investigated effect modification by age (< 60, 6080, > 80 years) and performed several sensitivity analyses. first, analyses were repeated starting follow - up at the date of first ait code instead of first thyroxine script. second, cardiovascular conditions present among patients with ait at diagnosis could be early mediating factors for stroke rather than a confounding variable; to assess their importance, we performed subgroup analyses for individuals without a history of any cardiovascular disease ethics approval was obtained from the south - east multicentre research ethics committee and from the london school of hygiene and tropical medicine ethics committee. in total, 184,539 eligible individuals from 287 general practices were included in this study, of whom 34,907 had ait (figure 2) and 149,632 had no evidence of ait. individuals with ait were slightly older than individuals without ait (median 59.8 vs 57.0 years), were followed up for longer (median 3.2 vs 3.0 years), and a slightly higher proportion was female (81.5% vs 80.0% ; table 1). for cardiovascular risk factors, individuals with ait were more likely to have been diagnosed with hypertension, diabetes, hyperlipidemia, chd, or chf at baseline (all p < 0.001). this pattern remained after adjusting for the slight age and sex imbalances (data not shown). baseline characteristics of individuals with and without ait individuals with ait had a 13% increased rate of stroke compared with individuals without ait after adjusting for current age and sex and allowing for clustering in practice (model 1, table 2). the increased risk was very similar (rate ratio = 1.14, 95% confidence interval : 1.041.24, p = 0.003) after adjusting for alcohol and smoking (model 2) and was not affected by the duration of follow - up or calendar year. the rr decreased slightly to 1.10 (95% ci : 1.011.20, model 3) after adjusting additionally for cardiovascular risk factors present at baseline (hypertension, hyperlipidemia, af, and bmi) and was not further changed by consideration of existing diabetes, chd, and chf. multivariable analysis of the effect of ait on stroke and tia adjusted for covariates (and allowing for clustering in practice) individuals with ait had an increased risk of developing chd, hyperlipidemia, chf, and diabetes during follow - up compared with unexposed individuals (table e-1), but not hypertension or af. after adjusting for these potential mediating factors between ait and stroke, the rr for stroke was further reduced to 1.06 (95% ci : 0.971.15, model 4). effect sizes were slightly higher for all models when the outcome definition was expanded to both stroke and tia (table 2). there was evidence that the effect of ait on stroke varied with duration of disease (pinteraction = 0.078, figure 3). the effect of ait on stroke was largest in the first year after diagnosis (rr = 1.33, 95% ci : 1.141.56) and was increased both in the first 6 months (rr = 1.44, 95% ci : 1.171.78) and 7 to 12 months after start of treatment (rr = 1.22, 95% ci : 1.021.55). similar were obtained for analyses using tia or stroke and tia alone as the outcome. there was no evidence that the effect of ait varied with age (pinteraction = 0.516). * adjusted for current age, sex, alcohol, smoking, hypertension, atrial fibrillation, hyperlipidemia, and body mass index (all at baseline) and allowing for clustering in practice. * * using a likelihood ratio test to compare a model with against a model without an interaction term for time since diagnosis. all sensitivity analyses showed compatible to those obtained from the main analyses (data not shown). our study provides strong evidence for a slightly increased risk of stroke in patients with ait, particularly in the first year after ait diagnosis. our analyses indicated that people with ait were more likely to develop hyperlipidemia, chd, and chf, adding evidence to previous discussions about whether ait is associated with these factors, and that some, but not all, of the increased risk of stroke among patients with ait was mediated via these classic cardiovascular risk factors. our systematic review identified 7 studies, which showed effect sizes ranging from 0.8 to 1.6 (table e-2). all of these studies except for the scottish study from 2006 were small and had effect estimates with wide cis that overlapped with those from the present study. other methodologic limitations of previous studies included overadjustment for variables that could be mediators of increased stroke risk, and residual confounding (for example, the scottish study lacked information on smoking, alcohol use, and medication history). this could explain their slightly higher effect estimates, because thyroid hormone status is often assessed at stroke units, ing in better ait ascertainment in individuals with a stroke history (who are at higher risk of a subsequent stroke). three of the studies were conceptually different from this study because they used asymptomatic patients identified as hypothyroid by laboratory tests who were mostly untreated with thyroxine. we found differences between individuals with and without ait in the prevalence of traditional cardiovascular risk factors at baseline. individuals with ait were more likely to have af and hypertension at baseline compared to those without ait; however, they were not at increased risk of developing these conditions during follow - up. this finding is consistent with that from a recent population - based study from germany, which reported that hypothyroidism was associated with prevalent but not incident hypertension. hypertension and af may not be on the causal pathway between ait and stroke, although the differences we observed at baseline could represent early changes induced by undiagnosed ait. differences in af (and possibly hypertension) at baseline between individuals with ait and those without could also be explained by differences in ascertainment of ait, if individuals with af were more likely to have thyroid - stimulating hormone levels measured. our main model of interest, adjusted for these baseline variables, is based on the assumption that pathologic mechanisms on the causal pathway to stroke did not start until after ait diagnosis and initiation of thyroxine treatment. however, it is likely that the date of ait diagnosis / start of treatment did not accurately capture start of disease. given that patients with ait often present with nonspecific symptoms, diagnosis of ait can be delayed. thus, it is plausible that cardiovascular causal processes could have started before formal diagnosis of ait. if so, model 3 (which adjusted for these variables) may have provided a conservative estimate of relative stroke risk, and model 2 might provide an estimate closer to the true effect. however, the effect estimates produced by these 2 models were similar; also, the sensitivity analysis excluding all individuals with cardiovascular disease related conditions at baseline showed similar to the main analysis. our finding of a higher effect of ait on stroke risk in the first year after diagnosis is compatible with the hypothesis of increased cerebrovascular risk in patients with ait due to prediagnosis hypothyroidism and the time taken to reduce this risk after thyroid hormone replacement. there was no evidence for an increasing effect with increased length of follow - up (a possible proxy for a long - term effect mediated by autoimmune processes). an alternative explanation is that the observed effect of ait on stroke was caused by thyroxine treatment because thyroxine can have a procoagulant effect. however, if the observed effect was due to thyroxine, we might expect to have seen the excess stroke risk restricted to the period soon after start of treatment, whereas the risk remained increased 6 to 12 months after diagnosis in our study. moreover, a thyroxine - mediated effect might also be expected to generate short - term differences in incidence of af between patients with ait and individuals without ait because thyroxine in high doses can induce af; this was not the case. nevertheless, we can not exclude that some of the effect attributed to hypothyroidism could have been due to thyroxine, and we were unable to examine this formally because all patients with ait received thyroxine. we used a considerably larger study population than all previous studies, and thus had greater power to ascertain effect estimates. we had information on a variety of potential confounding factors that could be considered in the analysis. unlike previous studies, we excluded individuals with a history of stroke, avoiding introduction of bias. moreover, excluding people with other autoimmune diseases from our study population enabled us to observe the effect of ait unmodified by other autoimmune disorders. this might not have been the case in other studies, because patients with ait are at higher risk of acquiring other autoimmune disorders, some of which (e.g., rheumatoid arthritis, type 1 diabetes) are associated with considerably increased risk of cardio- and cerebrovascular disease. almost all noniatrogenic hypothyroidism in the united kingdom is due to ait, and we excluded individuals who had other reasons for hypothyroidism. however, our read code list included nonspecific hypothyroidism codes, which increased sensitivity of ait diagnosis but could have included a few non - ait cases. because hypothyroidism can remain undiagnosed for an unspecified time this is likely to have happened largely independently of subsequent stroke risk, and thus may have driven the rr toward 1.0. those with preexisting af could have been more likely to have their ait diagnosed, which could have ed in some overestimation of the effect size of ait on the risk of stroke. however, our sensitivity analyses excluding individuals with af at baseline indicate that this is unlikely to have been a major problem. differential misclassification of the outcome was unlikely in this study because stroke is a serious condition and should have been recorded equally in both groups independent of ait status, or health - seeking behavior. however, we could not differentiate between cases of ischemic and hemorrhagic stroke, because 85% of stroke codes did not specify the stroke subtype, consistent with previous studies of stroke using electronic health data. we hypothesized that ait might be associated specifically with ischemic stroke because hypothyroidism is linked with classic risk factors for ischemia. moreover, carotid artery intima - media thickness and af, 2 large risk factors for ischemic stroke, have been linked to ait. ischemic stroke causes approximately 80% of stroke cases in adults, and effect sizes for tia (an ischemic process) were comparable to those shown for stroke in our study, further supporting that ait is associated with ischemic stroke. nevertheless, ait could also affect the risk of hemorrhagic stroke, because hypothyroidism has been shown to be associated with the development of von willebrand syndrome and bleeding events. it is therefore possible that they were more likely to be diagnosed and treated for conditions such as hyperlipidemia or hypertension. monitoring and prompt treatment of these conditions could have decreased stroke risk associated with these conditions and might partly explain the relatively small increase in stroke risk associated with ait. nevertheless, this study provides a realistic picture of the residual effect of ait after standard treatment and surveillance in the united kingdom. data concerning the potential confounders of ethnicity and socioeconomic status were not available. however, socioeconomic status was partly accounted for by allowing for clustering by practice. although thin has been shown to be representative for the uk population, the of this study might not be generalizable to all patients with ait. if hypothyroidism is on the causal pathway between ait and stroke, the observed effect relates to treated rather than all patients with ait. excluding individuals with ait who had a second autoimmune disorder allowed us to pinpoint the specific effect of ait, but may have excluded some patients with ait who had stronger autoimmune processes and thus might not have captured all of the long - term risk of stroke mediated by autoimmune pathology. our study has demonstrated a 10% to 14% increased risk of stroke among patients with hypothyroid ait after extensive consideration of confounding and potential biases. given the relatively high prevalence of ait and the morbidity and mortality associated with stroke, even small increases in stroke risk might be of high clinical relevance. the of our study highlight the potential for regular screening for cardiovascular risk factors and preventive treatment (e.g., with statins) in patients with ait. both authors developed the study design. a.k. conducted the data management, analyzed the data, and wrote the manuscript, with contributions from s.t. both authors interpreted the findings, contributed to critical revision of the manuscript for important intellectual content, and approved the final version. supported by a project grant from the wellcome trust (079482/z/06/z) and by a postgraduate fellowship of the daad (german academic exchange service, for a.k .).

objective: to investigate the effect of autoimmune thyroiditis (ait) on risk of stroke and to assess whether any increased risk varied by ait duration, and was independent of classic cardiovascular risk factors.methods:this was a large historical cohort study using data from the health improvement network database. rates of first stroke during follow - up in thyroxine - treated patients with ait (n = 34,907) were compared with those in matched individuals without ait (n = 149,632) using random - effects poisson regression models.:there was strong evidence for a slightly increased risk of stroke in patients with ait (adjusted rate ratio = 1.10, 95% confidence interval : 1.011.20). the observed increase was partly independent of cardiovascular risk factors. higher effect sizes were identified in the first year after ait diagnosis (rate ratio = 1.33, 95% confidence interval : 1.141.56) but not in the long - term, consistent with a residual effect of hypothyroidism.:our support the hypothesis of a slightly increased risk of stroke in patients with ait. the higher effect size found soon after ait diagnosis suggests an increased cardiovascular risk due to thyroid - hormone deficiency rather than a cumulative effect of autoimmune pathology. better screening and early treatment of patients with asymptomatic hypothyroid ait could help reduce excess risk of stroke in the first year after diagnosis.

nano - sized metal particles embedded in glass are of great interest because of their potential application as non - linear material for photonic devices. the non - linear properties of nanocomposite glasses equipped with such particles are induced by the surface plasmon resonance at the interface between particles and glass matrix. this means that the optical effects in the spectral region around the surface plasmon resonance from an electric field enhancement or a quantum confinement. thus, applications are possible as in integrated photonic networks, in nanoelectronics, for surface enhanced raman scattering, for up - conversion processes and laser materials. recently, the preparation of specific bimetallic nanoparticles like core - shell structures has been intensively investigated because of the far - reaching possibilities to modify the macroscopic properties. a special way to extend the range of manipulating the optical properties of such nanocomposite glasses there are known some first examples for hollow nanoparticles in glass that were prepared by sequential implantation of two different metal ions. a further degree of freedom introducing anisotropic optical properties is the method of femtosecond laser pulse - induced shape transformation of the nanoparticles which has been studied intensively in recent years. depending on the actual irradiation parameters, uniformly oriented prolate or oblate nanoparticles can be prepared, whose orientation is controlled by the laser polarization. so far these effects have been demonstrated for ag nanoparticles at low concentration. in this letter, we will demonstrate that a similar shape modification of initially spherical bimetallic and hollow ag / au nanoparticles in soda - lime glasses is possible by irradiation with femtosecond laser pulses at high intensity, but below damage threshold; in particular, it will be shown that anisotropic particle shapes or nearly linear arrangements of nanoparticles with preferential orientation along the direction of laser polarization can be fabricated with the help of this irradiation technique. the samples used for this study were sheets of soda - lime glass containing (in mol%) 72.4% sio2 and 14.4% na2o as main components, which were exposed subsequently to au (150 kev) and ag (100 kev) ion implantation at room temperature. by this sequential high - dose ion implantation of ag and au, metal particles have been formed in a surface - near region of the soda - lime silicate glass. the dose of implanted ions was 4 10 ions / cm for each type of ions (for further details see). to characterize the surface plasmon resonance due to the metal nanoparticles formed in the implanted areas the optical density of glass samples was recorded by means of a perkin - elmer spectrometer in the wavelength range of 250900 nm. these samples were irradiated by linearly polarized laser pulses of 150 fs temporal width at a wavelength = 550 nm. this wavelength is the sum frequency of a 1 khz repetition rate ti: sapphire laser at = 800 nm and the idler (= 1,760 nm) of a travelling - wave optical parametric amplifier of superfluorescence (topas). the laser beam was focused on the sample to a spot size of ~100 m. moving the sample continuously on a motorized x y translation stage, several parallel lines of ~1.5 mm length and 150 m lateral distance have been inscribed in the glass at a velocity of 0.5 mm / s, corresponding to, on average, 200 laser pulses hitting each spot within the lines. the polarization direction of the laser was parallel to the lines (writing direction). the effect of irradiating the sample in the described way with average single pulse energy of 20 j is shown in figs. the lines prepared by high intensity fs laser irradiation exhibit a certain subdivision into a few parallel traces of decoloration as well as, at a few positions along the line center, some damage at the very glass surface. in the central region there is still a color change visible, but the dichroism decreases continuously to that of the original glass region. optical micrographs of the au / ag nanoparticles containing glass after laser irradiation recorded usinganon - polarized, andb, cpolarized light, respectively optical density within the central region of irradiated glass measured before (original glass, solid line) and after laser irradiation with polarized light, parallel (dotted) or perpendicular (dashed) with respect to laser pulse polarization and line direction (see fig . 1). the spectra of non - irradiated samples are identical for parallel and perpendicular polarization to evaluate the nanoscopic of the observed optical changes, the parameters mean size, size distribution, shape, and penetration depth of the metal particles have been examined by transmission electron microscopy (tem) using a jem 1010 operating at 100 kv and a jem 4010 operating at 400 kv. for this purpose, planar and cross - section preparation were applied including mechanical grinding, polishing and argon ion - beam etching followed by deposition of a thin carbon film on both sides. for the samples of this study, prepared by applying an ion dose of 4 10/cm for both of the metals, a particle - containing region has been obtained that extends from the very glass surface to a depth of about 135 nm. tem imaging of cross - section samples reveals that this particle layer exhibits a non - uniform spatial distribution of particles as already reported earlier. in the middle of the particle layer mainly larger particles are situated whose anomalous image contrasts point to the presence of voids in their interior as may be recognized from the tem image of a planar preparation sample shown in fig. while for the entire set of particles present in the layer a mean size of 5.34 3.89 nm has been determined, the mean outer diameter of the void - containing particles amounts to 16.2 nm. altogether the particle sizes range from about 1.524 nm. the frequency of the surface plasmon resonance of the non - irradiated glass (see fig . 2) appearing between those of pure ag and au particles demonstrates the formation of ag au alloy nanoparticles. the relation of concentrations of both elements incorporated into the nanoparticles should be ~1:1 corresponding to calculations of theoretical spectra and to experiments by anomalous small angle x - ray scattering. 4 10/cmauion implanted sample the above - described ultrashort laser pulse processing of this sample ed in a totally different appearance of the structural characteristics of this particles - in - glass composite material which, however, is restricted to those regions where the laser lines have been inscribed into the particle layer. by careful target preparation we achieved to place the position of the hole at the edge of one of these lines; this allowed us to image within one specimen regions without laser irradiation as well as regions where the maximum laser pulse intensity had been applied. the tem examination reveals that ultrashort laser pulse processing causes fundamental changes in size, shape, arrangement and configuration of the metal nanoparticles in the irradiated regions (see fig . 4), whereas no changes can be observed in non - irradiated regions. in the center of the irradiated regions most of the larger particles are elongated nearly along a common direction parallel to the laser lines inscribed (and thus along the laser polarization vector). the elongated particle shapes may be described as spheroidal, but with a certain degree of irregularity. altogether the particle dimensions range from about 4.4 to 52.6 nm for the minor axis (mean value 16.68 8.69) and from 5.6 to 79.6 nm for the major axis (mean value 20.98 13.47). the aspect ratio (i.e., the ratio of major to minor axes lengths) of the particles increases nearly linearly from a value of 1 for particles of about 4.7 nm diameter to a value of 1.5 for particles of about 60.4 nm diameter (these diameters refer to spheres of the same volume as the spheroids have). the total increase in particle size compared to the initial situation in the sample before irradiation corresponds to a more than tenfold volume increase for the individual particle. so, the laser processing ed in a totally different appearance, not only the particle dimensions have increased by a factor of 3, but also the size distribution has become a little bit narrower and lost part of its asymmetry, and, what is the important issue in this , shape and arrangement of the particles deviate from the previous isotropic appearance. on the other hand the optical absorption remains fairly constant or even decreases indicating that the total amount of silver and gold does not grow. so it can be concluded that the volume increase of the particles is compensated by a simultaneous decrease of their number. the optical spectra also show that the composition of alloy nanoparticles should be similar to that before the irradiation. there are only slight shifts due to variations in sizes of particles and concentrations of elements within the particles. tem micrograph of the above sample upon ultrashort laser pulse irradiation finally, we want to discuss briefly which physical mechanisms may lead to the observed shape changes of bimetallic nanoparticles upon intense fs laser irradiation, in particular explaining the preferential orientation of elongated particles more or less arranged parallel to the laser polarization. the shape changes observed here can be compared to similar previous obtained on two quite different types of metal - dielectric nanocomposites. the first type of material is glass containing low concentration of ag nanoparticles. for such systems it has been found that only the individual particle and its immediate surroundings are affected by laser - induced modifications. the sequence of processes there starts with field - driven electron emission from the particle, followed by electron trapping in the glass matrix, ion emission, their local recombination with trapped electrons, and diffusion and precipitation of ag atoms at the poles of the particle in the transiently heated nearest shell of the matrix. the second type of material studied previously is plasma polymer embedding a quasi 2-dimensional metal island film. irradiating these systems with similar parameters as in this work, a grating - like superstructure oriented along the laser polarization with a typical period of 2/3 of the laser wavelength has been observed, where stripes of unchanged metal nanostructure (percolation region) are alternating with stripes of coagulated larger, spherical particles. the explanation for these self - organized structures comprises spatially modulated energy input in the metal layer by interference of the incoming laser light with the scattered surface wave, where statistical inhomogeneities of the sample provide a feedback, so that the structures are becoming more pronounced and regular shot by shot. comparing the situation with the plasma polymer samples, we here also observe a large increase of particle sizes, but no regular superstructure. comparing with isolated ag nanoparticles in glass, we also find individual non - spherical shapes with the long axes oriented preferentially along the laser polarization; but, in addition, particles are growing and sometimes merging, preferentially in situations where together they form a coagulated particle with longer axis along the laser polarization. from these considerations we conclude that the laser - induced shape changes of bimetallic nanoparticles are initiated by the same mechanisms as in the case of low - concentrated ag nanoparticles, i.e., directional electron emission and capture in the glass matrix, followed by the processes listed above. the main difference appears to come from the higher metal concentration leading to spatially and temporally more extended regions of high temperature around metal particles in the matrix, enabling much larger diffusion distances of electrons and metal ions or atoms. it can not be decided at present if, in addition to the basic mechanism of particle reshaping during laser irradiation (migration of individual atoms or ions), also processes like migration and coalescence of very small particles contribute to the observed particle growth. the reason is that all processes are started by an ~100 fs laser pulse; then the surrounding glass is heated within a few ps and cools down again by heat conduction within a few ns. particle formation or growth under such strongly non - equilibrium conditions has, to the best of our knowledge, so far not been modeled theoretically. still, however, the local trapping sites for electrons in glass are obviously a necessary prerequisite for shape anisotropy of the particles. this is confirmed by the lack of similar shape anisotropy of the metal particles after fs irradiation in plasma polymers. furthermore, the temperature increase during laser irradiation within the particle regions explains the transformation of hollow particles into solid ones because of their thermal instability. the vacancies leave the central void toward the outer surface and the hollow region disappears at elevated temperatures. the laser - induced changes of shape and configuration of nanoparticles described above can also explain the slight blue shift of the surface plasmon resonance observed for perpendicularly polarized light as well as the lacking red - shift for parallel polarization (as shown in fig . , one can conclude a reduced concentration of precipitated particles compared with the nanocomposites before irradiation . that is, obviously the recombination of emitted ions with trapped electrons is not completed . while, however, the probability for emission during interaction with an ultrashort laser pulse is the same for au and ag ions, the mobility of au in the glass matrix is considerably less than that of ag species . this difference should also affect the amount of both elements being incorporated into the particles again ; so in the end the concentration ratio in the particles will be shifted toward ag atoms, and this will shift their plasmon resonances toward shorter wavelengths . in , we have shown that spherical, bimetallic au / ag nanoparticles in glass at high concentration can be transformed to anisotropic shapes ( accompanied by size increase) preferentially oriented along the direction of the linear laser polarization. the mechanism appears to be similar to that observed for silver nanoparticles in glass at low concentration, but with additional effects like coalescence caused by the close proximity of the particles. overall, the demonstrated high - intensity laser processing is a promising and flexible technique to design the linear and non - linear optical properties of metal - glass nanocomposites. the authors would like to thank the institute of solid state physics of the friedrich schiller university of jena for implantation of glass samples.

bimetallic, initially spherical ag / au nanoparticles in glass prepared by ion implantation have been irradiated with intense femtosecond laser pulses at intensities still below the damage threshold of the material surface. this high - intensity laser processing produces dichroism in the irradiated region, which can be assigned to the observed anisotropic nanoparticle shapes with preferential orientation of the longer particle axis along the direction of laser polarization. in addition, the particle sizes have considerably been increased upon processing.

the institutional review board of gyeongsang national university hospital approved the study protocol (no . 2015 - 06 - 031), and the protocol complied with the tenets of the declaration of helsinki. all consecutive patients in a tertiary referral hospital, gyeongsang national university hospital in jinju, korea, who underwent tsf for aphakia due to various causes, including iol dislocation, retinal detachment, trauma, zonulysis, endophthalmitis, phacomorphic, and acute angle closure glaucoma, between january 2012 and december 2014 were identified by a review of the medical records. before surgery and 1 day and 6 months after surgery all patients underwent examinations to measure refraction, best - corrected visual acuity (bcva), intraocular pressure, and specular microscopy, as well as slit lamp and fundus examinations. the power of iol was calculated using the sanders - retzlaff - kraff ii formula. was conducted by one surgeon (ksj), and the surgical procedures were as follows: at the limbus, two points 180 from each other were marked (fig . to make a 2-mm - sized pocket, lamellar dissection without conjunctival dissection was performed with a crescent blade, then the blade was advanced about 1 to 1.5 mm ( fig . polypropylene was passed through the transconjunctival transcleral passage, which was located 1.5 mm posterior from the limbus . anterior to the limbus, a clear corneal incision ( cci) was made using a keratome, and the prolene sutures were exteriorized through the cci pocket. an ophthalmic viscosurgical device was inserted into the anterior chamber. a three - piece foldable acrylic iol (sensar soft acrylic iol ; abbott medical optics, santa ana, ca, usa) was injected through the cci, and the prolene sutures for each haptic were tied (fig . the iol was placed in the anterior chamber, and its orientation was adjusted by pulling the prolene sutures . the iol was centralized, and the ends of the prolene were sutured in the intrascleral pocket ( fig . the cci and other paracentesis wounds were then hydrated, and the conjunctival edges were joined without suture . the scleral flap with conjunctival division technique was as follows : after conjunctival dissection, a triangular - shaped scleral flap was made, a 2.8-mm cci was generated at the anterior limbus using a keratome, and a three - piece foldable acrylic iol was injected . the haptic ends were exteriorized through the cci, after which each haptic was tied . continuous data were presented as mean standard deviation, and categorical variables were presented as number ( %). the two surgical groups were compared in terms of bvca, endothelial cell count (ecc), spherical equivalent, and astigmatism using the paired t - test. 19.0 (ibm co., armonk, ny, usa). a p - value less than 0.05 the institutional review board of gyeongsang national university hospital approved the study protocol (no . 2015 - 06 - 031), and the protocol complied with the tenets of the declaration of helsinki. all consecutive patients in a tertiary referral hospital, gyeongsang national university hospital in jinju, korea, who underwent tsf for aphakia due to various causes, including iol dislocation, retinal detachment, trauma, zonulysis, endophthalmitis, phacomorphic, and acute angle closure glaucoma, between january 2012 and december 2014 were identified by a review of the medical records. before surgery and 1 day and 6 months after surgery all patients underwent examinations to measure refraction, best - corrected visual acuity (bcva), intraocular pressure, and specular microscopy, as well as slit lamp and fundus examinations. the power of iol was calculated using the sanders - retzlaff - kraff ii formula. the intrascleral pocket technique for tsf was conducted by one surgeon (ksj), and the surgical procedures were as follows: at the limbus, two points 180 from each other were marked (fig . to make a 2-mm - sized pocket, lamellar dissection without conjunctival dissection was performed with a crescent blade, then the blade was advanced about 1 to 1.5 mm ( fig . polypropylene was passed through the transconjunctival transcleral passage, which was located 1.5 mm posterior from the limbus . anterior to the limbus, a clear corneal incision ( cci) was made using a keratome, and the prolene sutures were exteriorized through the cci pocket. an ophthalmic viscosurgical device was inserted into the anterior chamber. a three - piece foldable acrylic iol (sensar soft acrylic iol ; abbott medical optics, santa ana, ca, usa) was injected through the cci, and the prolene sutures for each haptic were tied (fig . the iol was placed in the anterior chamber, and its orientation was adjusted by pulling the prolene sutures . the iol was centralized, and the ends of the prolene were sutured in the intrascleral pocket ( fig . knots were buried under the sclera flaps ( fig . 1 g and 1h). the cci and other paracentesis wounds were then hydrated, and the conjunctival edges were joined without suture. the scleral flap with conjunctival division technique was as follows: after conjunctival dissection, a triangular - shaped scleral flap was made, a 2.8-mm cci was generated at the anterior limbus using a keratome, and a three - piece foldable acrylic iol was injected. the haptic ends were exteriorized through the cci, after which each haptic was tied. continuous data were presented as mean standard deviation, and categorical variables were presented as number (%). the two surgical groups were compared in terms of bvca, endothelial cell count (ecc), spherical equivalent, and astigmatism using the paired t - test. 19.0 (ibm co., armonk, ny, usa). a p - value less than 0.05 in total, 40 consecutive patients with aphakia underwent tsf in our hospital between january 2012 and december 2014. the mean age of the cohort was 59.8 4.1 years, and there were 28 males and 12 females. the mean age of the intrascleral pocket and conventional - flap groups was 59.7 9.2 and 59.8 9.7 years, respectively. in both groups, the main cause of aphakia was iol dislocation. other causes were phacoemulsification, lens dislocation, zonulysis, and vitrectomy with lensectomy, which occurred in the two groups with similar frequencies (table 1). the two groups did not differ significantly in terms of preoperative bcva and ecc or ecc at 6 months after surgery (table 2). however, the intrascleral pocket group had significantly better bcva at 1 day and 6 months after surgery compared to the conventional - flap group (p = 0.03 and 0.04, respectively). in contrast, five of the 20 patients in the conventional - flap group complained of irritation after surgery (table 2). one day and 6 months after surgery, the intrascleral pocket group exhibited significant decrease in spherical diopter (p = 0.018 and 0.005 compared to preoperative values, respectively) and astigmatism (p = 0.027 and 0.028, respectively). this was also observed for the conventional - flap group for spherical diopter (p = 0.000 and 0.007, respectively) and astigmatism (p = 0.009 and 0.007, respectively) (fig . tsf of an iol is widely used for aphakic patients with poor support of the posterior chamber or ciliary body . many recent studies have reported modifications of the tsf technique that was first introduced by malbran et al . in 1986 . however, all of these methods can cause suture - related complications, including knot exposure or iol dislocation due to suture decomposition or breakage . of these complications, knot exposure is one of the most common and can have deleterious outcomes as it can act as a pathway for bacterial invasion into the eye, thereby causing endophthalmitis . when szurman et al . retrospectively analyzed the of 45 eyes that underwent tsf of pciol using z - sutures and 22 eyes that underwent tsf of an iris prosthesis, they found one case of ciliary body hemorrhage during the 22.4 months of follow - up, but no complications such as knot exposure, suture looseness, conjunctival atrophy, or chronic inflammation . ma et al . also analyzed the clinical outcomes of a knotless external fixation technique of pciol tsf in five patients who had to undergo this technique because of adhesion between the conjunctiva and cornea or because the location of the conjunctival flap was near a corneal wound caused by trauma . therefore, this study introduced the sutureless intrascleral pocket technique and compared the effects and complications including astigmatism or postoperative visual prognosis . compared with conventional techniques, this technique ed in less irritation and astigmatism since knots were buried in the intrascleral pocket, and vertical and regular wounds were formed without a gap of wound . in addition, this technique led to rapid visual recovery and return to daily life, since the visual acuity the day after the operation was comparable to the bcva before the surgery . during the follow - up period, there were no complications except for mild ciliary body hemorrhage in one case and iol tilting immediately after the operation due to excessive knot tightness in another case . the bcva in 16 of 20 cases ( 80%) improved, while the remaining case did not exhibit a change in bcva. thus, the outcomes of the intrascleral pocket technique in the present study were generally as successful as those of the knotless external fixation technique of pciol in a previous study. the intrascleral pocket technique of tsf is especially useful for patients who previously underwent vitrectomy. patients after vitrectomy, especially those who underwent surgery with a 20-gauge needle, usually have a relatively thin conjunctiva or conjunctiva - tenon - scleral complex due to previous conjunctival dissection. this makes conjunctival dissection more difficult and increases the possibility of conjunctival defects and scleral injury. in addition, since this technique does not involve conjunctival dissection, the operation time and bleeding risk are reduced. moreover, subsequent glaucoma surgery can improve the success rate of the intrascleral pocket technique because the conjunctiva and tenon's space maintain their native integrity. the intrascleral pocket technique also allows for rapid visual acuity recovery and return to daily life because the associated irritation is low. in addition, the vertical and regular wound without a gap helps to reduce astigmatism. in a previous study using the sutureless intrascleral pocket technique this prompted us to reassess patients at 6 months after the surgery in this study, and the wounds were found to be clean without dehiscence or knot exposure ( fig. unlike the reverse pocket technique, the intrascleral pocket technique leaves the corneal limbus intact, which reduces the chance of injury to the corneal nerve. moreover, it is possible to perform the intrascleral pocket technique with topical anesthesia because it is associated with less pain than other techniques. in addition, the intrascleral pocket technique is quite similar to glaucoma or flap - making procedures, which means that surgeons can rapidly become proficient in this technique. , the present study showed that the intrascleral pocket technique of tsf had better clinical than the conventional scleral flap with conjunctival division technique, as determined by ophthalmological examinations before and 1 day and 6 months after the operation. the improvement in bcva was more marked in the intrascleral pocket group, and no postoperative complications were observed. these observations, together with the fact that this technique is relatively easy, suggest that this technique is a good method of tsf.. however, the simplicity and good outcomes of this technique suggest that it is a good method of tsf.

purposeto compare the two transscleral fixation (tsf) techniques of intrascleral pocket and conventional scleral flap with conjunctival division techniques in terms of short - term clinical effects.methodsthis retrospective cohort study included all consecutive patients with aphakia in gyeongsang national university hospital in jinju, korea, who underwent tsf between january 2012 and december 2014. the medical records of all patients were retrospectively reviewed, and the endothelial cell count (ecc), refraction, best - corrected visual acuity (bcva), intraocular pressure, slit lamp, and fundus examination before and 1 day and 6 months after surgery were recorded. the postoperative complications and visual outcomes were also recorded.the intrascleral pocket and conventional - flap groups did not differ significantly in terms of demographics, presurgical bcva, or ecc. however, the intrascleral pocket group had a significantly lower bcva at 1 day and 6 months after surgery compared to the conventional - flap group. the two groups did not differ in terms of ecc 6 months after surgery. the intrascleral pocket group had no postoperative complications, but five patients in the conventional - flap group complained of irritation. in both groups, the intraocular lens was well positioned without tilting or subluxation, and astigmatism was significantly reduced at 1 day and 6 months after surgery.the intrascleral pocket technique of tsf does not involve conjunctival dissection and is a successful method of sulcus fixation. it stably corrects the intraocular lens and is easy to perform, which helps to reduce operation time. it also reliably yields rapid visual acuity recovery without complications.